Composition containing lycium barbarum exosome as well as preparation method and application of composition

Abstract

The invention relates to a method for preparing a Chinese wolfberry exosome, the Chinese wolfberry exosome prepared by the method and application of the Chinese wolfberry exosome. Specifically, the preparation method of the lycium barbarum plant exosome disclosed by the invention comprises a layer-by-layer filtration step, and the layer-by-layer filtration step sequentially comprises deep filtration and optional membrane filtration.

Description

Technical Field

[0001] The present disclosure relates to a method for extracting exosomes from plants such as wolfberries, the plant exosomes extracted by this method, compositions containing the same, and their applications in the field of skin. Background Art

[0002] Exosomes are a type of nanoparticle with a phospholipid bilayer secreted by cells, usually with a diameter of 30 - 200 nm, containing abundant proteins, nucleic acid substances, etc. Exosomes can transmit active substances such as proteins, mRNA, and microRNA between cells and participate in many important physiological and pathological processes. Their unique functions have received increasing attention in the industry.

[0003] Currently, exosomes are mainly obtained from materials such as milk-derived samples, various cell culture supernatants, and exudates from tumor tissues. However, obtaining exosomes from sources such as cell culture supernatants has limited material sources, high costs, low exosome content, and even higher costs after purification, which restricts the large-scale industrial production of exosomes.

[0004] Various plants are also important sources of exosomes, and the extraction rate of plant-derived exosome-like nanoparticles (PEN) is much higher than that of mammalian cell cultures, indicating their economic effectiveness as nanomachines. [1] Most importantly, recent studies have shown that plant exosomes can be absorbed by intestinal macrophages and play roles in cell-to-cell communication and immune regulation. [2-3]

[0005] Correct extraction and purification methods are crucial for exosome analysis because they are usually present in overly complex matrices. The research on exosome extraction methods not only needs to examine the specificity and purity of the extracts but also needs to consider the simplicity of operation and scale efficiency. Currently, methods commonly used for separating exosomes include ultracentrifugation, polymer precipitation, gel exclusion, antibody affinity precipitation, density gradient centrifugation, membrane filtration, etc. However, each method has its drawbacks. For example, ultracentrifugation has complex operations, high technical difficulties, and is time-consuming. The separated exosomes may be damaged by high-speed centrifugation and contain a large amount of miscellaneous proteins; the exosomes extracted by the polymer precipitation method lack specificity and selectivity and have low purity; the gel exclusion method has relatively high technical difficulties, but it is difficult to ensure the sterility of the separated exosomes, is time-consuming, and is difficult to scale up; immunoaflinity chromatography usually improves the purity of the sample but reduces the efficiency and recovery rate; while membrane filtration improves the efficiency and recovery rate but reduces the sample specificity and purity. [4]

[0006] ​​Generally, exosomes of mammalian origin are isolated from biological fluids, while exosomes of plant origin are isolated from apoplastic washings. For mammalian-derived exosomes, current isolation and purification techniques mainly include microfluidic techniques, ultracentrifugation methods, size-based separation techniques for exosomes, precipitation techniques, and immunoaffinity capture techniques. The available methods for isolating and purifying plant-derived exosomes are mainly based on the established techniques for mammalian-derived exosomes, but have varying degrees of drawbacks. For example, in differential centrifugation, due to physiological differences between plants and animals, plant-derived exosomes obtained by this method are often contaminated with proteins, nucleic acid aggregates, and other vesicles, and thus further purification using density gradient ultracentrifugation is required to separate the contaminants. Another example is that due to the presence of high molecular weight components in plant sap, such as cellulose and starch, which usually cause difficulties in centrifugation, a combination of various centrifugation methods (such as a combination of differential centrifugation and sucrose density gradient centrifugation) is needed to address the above drawbacks of traditional centrifugation techniques. In addition, although several other separation techniques, such as immunoaffinity capture, ultrafiltration or size exclusion chromatography (SEC), co-precipitation methods, and microfluidic techniques, have been successfully applied to mammalian-derived exosomes, they are not ideal for the isolation of plant-derived exosomes. For example, although immunoaffinity capture based on the formation of immune complexes against extracellular vesicle surface antigens is an ideal method for purifying mammalian extracellular exosomes, for plant-derived exosomes, the lack of marker proteins and specific antibodies for plant-derived exosomes limits the application of this technique. Therefore, methods suitable for the extraction of mammalian exosomes are not applicable to the extraction of plant exosomes.

[0007] There is a lack of a general method for the isolation of plant exosomes. Currently, the commonly used method in research is ultracentrifugation, or a combination of multiple means such as extraction, membrane filtration for impurity removal, microfiltration for sterilization, nanofiltration for concentration, and even supplemented with complexation reactions to obtain plant exosomes. [7] As mentioned above, although ultracentrifugation is generally considered the gold standard for exosome isolation and can isolate relatively pure exosomes. [5] However, the ultracentrifugation method has many drawbacks and relies on ultra-high centrifugal force and duration to separate particles by sedimenting them to the bottom, so it is time-consuming and the extraction scale is limited by the processing volume of the ultracentrifuge.

[0008] The skin is an important protective barrier against external stimuli such as bacteria and ultraviolet rays. The skin barrier is mainly composed of the stratum corneum and keratinocytes, and its main function is to protect internal tissues and fluids from the external environment. [8] Exposure to ultraviolet rays can damage the stratum corneum of the skin and reduce the expression of intracellular barrier proteins, thereby impairing the skin barrier function. [9]Meanwhile, the skin is also a target organ for UV-induced oxidative damage. The energy carried by UVB can activate endogenous photosensitive substances in the skin, generating various reactive oxygen species such as singlet oxygen, oxygen free radicals, and hydrogen peroxide.

[10] Therefore, inhibiting the intracellular reactive oxygen species response induced by ultraviolet rays and the disruption of the permeable skin barrier is one of the key factors in combating skin photoaging and skin diseases.

[0009] Goji berry is a commonly used traditional Chinese medicine ingredient.

[11] It contains rich nutrients, including carotenoids, vitamin A, calcium, and iron. Usually, exosomes are mainly responsible for intercellular material transfer, thereby further regulating various physiological mechanisms, including immune responses and inflammatory responses.

[12] –

[14] However, no literature has reported the extraction of exosomes from goji berries, and there is no research on its beneficial effects on skin barrier damage and antioxidant aspects.

[0010] Therefore, a simple, efficient method suitable for large-scale separation and extraction of high-purity plant exosomes at the industrial level is needed to reduce the production cost and increase the yield of exosomes, and at the same time, the exosomes have beneficial effects on the skin. Summary of the Invention

[0011] To overcome the deficiencies and shortcomings of the existing methods for extracting plant exosomes, the inventors first proposed a method for separating and extracting plant exosomes using layer-by-layer filtration. This method is simple, efficient, suitable for industrial scale (as Figure 1 shown), and the separated and purified exosomes have a high purity.

[0012] Specifically, the present disclosure uses two methods, layer-by-layer filtration and ultracentrifugation, to isolate plant exosomes from goji berries. By analyzing the physical properties and efficacy of the isolated plant exosomes, and combining with plant metabolomics analysis, the differences between the plant exosomes extracted by the two methods are compared, and thus a method suitable for the separation and extraction of plant exosomes is screened out.

[0013] The research results show that compared with the ultracentrifugation method, the time for layer-by-layer filtration to process the same mass of plant raw materials is shortened by 3 times, the yield is increased by at least 40 times, and metabolomics analysis shows that the plant exosomes separated and extracted by layer-by-layer filtration contain more active metabolites. When the plant raw materials are magnified 10 times or even 20 times, the advantages of the method for separating and extracting plant exosomes by layer-by-layer filtration are more obvious. In addition, the plant exosomes separated and extracted by layer-by-layer filtration have a typical exosome-like saucer shape, rich in protein content, and have anti-inflammatory and immunomodulatory functional activities in vitro.

[0014] Specifically, the layer-by-layer filtration method of the present disclosure has a lower time consumption compared to ultracentrifugation, has a higher yield of exosomes or particle concentration, can prevent the filter membrane from being blocked due to excessive impurities, and can ensure the sterility of the product. The plant exosomes extracted using the layer-by-layer filtration method of the present disclosure have a typical exosome-like saucer shape, with a particle size in the range of 30-200 nm, and exhibit excellent anti-inflammatory and immunomodulatory activities.

[0015] The method for extracting plant exosomes of the present disclosure is suitable for large-scale production; preferably, the method can handle the amount of plant material corresponding to large-scale production by increasing the filtration membrane area, such as plant materials with a mass of at least about 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9000 g, 10000 g, 50000 g, or even 100000 g.

[0016] Therefore, on the one hand, the present disclosure provides a method for separating and extracting plant exosomes, specifically a method for separating and extracting plant exosomes by depth filtration, the method comprising the following steps:

[0017] (a) Obtaining a plant material;

[0018] (b) Pretreating the obtained plant material to obtain a crude plant extract; and

[0019] (c) Subjecting the obtained crude plant extract to layer-by-layer filtration treatment,

[0020] wherein, in step (c), the layer-by-layer filtration treatment includes a depth filtration step and an optional membrane filtration step.

[0021] In an embodiment of the method for separating and extracting plant exosomes of the present disclosure, in step (c), the layer-by-layer filtration step includes using depth filtration, preferably, the depth filtration uses depth filtration with a pore size of about 1-50 μm, more preferably depth filtration with a pore size of about 2-30 μm. In a specific embodiment, in step (c), the layer-by-layer filtration step includes a depth filtration step, and the depth filtration step uses depth capsule filtration (preferably having a pore size of about 1-50 μm), more preferably the depth capsule filtration is depth capsule filtration with a pore size of about 2-30 μm.

[0022] In a specific embodiment, in step (c), the depth filtration step comprises using a depth filter, preferably, the depth filter is a depth filter having a pore size of about 1-50 μm, more preferably a depth filter having a pore size of 2-30 μm. In a specific embodiment, the depth filter is a depth capsule filter having a pore size of 1-50 μm, more preferably a depth capsule filter having a pore size of about 2-30 μm, for example, suitable depth capsule filters include depth filters produced by Pall Corporation (NY), such as Supracap TM Depth Filter Capsules.

[0023] In an embodiment of the method for separating and extracting plant exosomes disclosed in the present invention, the layer-by-layer filtration also includes at least one membrane filtration step performed after the deep filtration step. Preferably, the membrane filtration includes anti-clogging membrane filtration, or further includes sterilizing membrane filtration, for example, anti-clogging membrane filtration with a pore size of about 0.4-0.8 μm and / or sterilizing membrane filtration with a pore size of about 0.1-0.3 μm. More preferably, the membrane filtration includes membrane filtration of about 0.45-0.8 μm and membrane filtration of about 0.1-0.22 μm. Most preferably, the membrane filtration consists of anti-clogging membrane filtration of about 0.45 μm and sterilizing membrane filtration of about 0.22 μm performed sequentially.

[0024] In one embodiment of the method for separating and extracting plant exosomes disclosed herein, the flow rate of the depth filtration, 0.45 μm membrane filtration and 0.22 μm membrane filtration is about 20-1000 mL / min, for example, about 20-500 mL / min, about 20-200 mL / min, about 30-100 mL / min.

[0025] In one embodiment of the method for separating and extracting plant exosomes disclosed in the present invention, the layer-by-layer filtration in step (c) consists of depth filtration as generally, specifically or preferably defined in the present invention and membrane filtration as generally, specifically or preferably defined in the present invention.

[0026] In one embodiment of the method for separating and extracting plant exosomes disclosed in the present invention, in step (b), the pretreatment includes one or more of mixing, crushing, grinding, stirring, filtering, and centrifugation to obtain a crude plant extract. In a specific embodiment, the pretreatment includes mixing, crushing, filtering, and centrifugation.

[0027] In a specific embodiment, the mixing in step (b) uses an aqueous solution, such as an aqueous solution; preferably, the aqueous solution is an isotonic aqueous solution; more preferably, the isotonic aqueous solution is an isotonic sodium chloride aqueous solution, such as sodium chloride injection.

[0028] In a specific embodiment, the step (b) can be carried out at any suitable temperature. For example, the step (b) can be carried out at a temperature of about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C or 40°C, or any sub-range within about 0°C to about 40°C (e.g., any range between any two of the above temperatures), preferably about 2-30°C, more preferably 2-20°C, more preferably 2-8°C, and most preferably 4°C. In some embodiments, the step (b) of the present disclosure can be carried out at ambient temperature (e.g., 25°C).

[0029] In the method embodiments of the present disclosure, the plant material may be terrestrial or aquatic. Preferably, the plant material is selected from wolfberry, ginseng, dendrobium, and / or angelica.

[0030] On the other hand, the present disclosure provides a plant exosome (preferably wolfberry exosome), which is prepared by the above-mentioned general or preferred method embodiments, or is derived from wolfberry, ginseng, dendrobium, and / or angelica; preferably, the exosome has a diameter of about 30-450nm, preferably a diameter of about 30-250nm, more preferably a diameter of about 30-200nm; and / or the exosome has a saucer shape; and / or the exosome has more metabolites in one or more of the following aspects compared to the plant exosome prepared by the ultracentrifugation method: lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, organic heterocyclic compounds, organic acids and their derivatives and / or benzene compounds.

[0031] On the other hand, the present disclosure provides a plant exosome (preferably wolfberry exosome) composition, such as an exosome solution, comprising plant exosomes prepared by the method generally or preferably defined in the present disclosure, and optionally a suitable excipient or carrier, which is acceptable in terms of nutrition, health, pharmacy, food, dermatology and / or cosmetics.

[0032] In a specific embodiment, the exosome composition is a pharmaceutical composition. In a specific embodiment, the exosome composition comprises at least one of Lycium barbarum exosomes, ginseng exosomes, Dendrobium officinale exosomes and Angelica sinensis exosomes prepared by the method of the present disclosure; and an optional pharmaceutically acceptable carrier.

[0033] On the other hand, the present disclosure provides a composition comprising Lycium barbarum exosomes; optionally, the composition further comprises ginseng exosomes, and / or Angelica sinensis exosomes; preferably, the composition does not contain Dendrobium officinale exosomes.

[0034] On the other hand, the present disclosure provides a composition comprising any one, any two, any three, or all four of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes.

[0035] In a specific embodiment, the composition comprises wolfberry exosomes and ginseng exosomes.

[0036] In a specific embodiment, the composition comprises wolfberry exosomes and angelica exosomes.

[0037] In a specific embodiment, the composition comprises wolfberry exosomes, angelica exosomes, and ginseng exosomes.

[0038] In a specific embodiment, at least one of the wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes is prepared by the method of the present disclosure or is respectively derived from wolfberry, ginseng, dendrobium, and angelica.

[0039] In a specific embodiment, the composition comprises wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes, wherein the particle number ratio between any two exosomes ranges from about 1:10 10 -10 10 :1, such as about 1:10 9 -10 9 :1, 1:10 8 -10 8 :1, 1:10 7 -10 7 :1, 1:10 6 -10 6 :1, 1:10 5 -10 5 :1, 1:10 4 -10 4 :1, 1:10 3 -10 3 :1, 1:10 2 -10 2 :1, 1:10 - 10:1, 1:9 - 9:1, 1:8 - 8:1, 1:7 - 7:1, 1:6 - 6:1, 1:5 - 5:1, 1:4 - 4:1, 1:3 - 3:1, 1:2 - 2:1 or 1:1.

[0040] In a specific embodiment, the composition comprises wolfberry exosomes and angelica exosomes, wherein the ratio of the particle number of wolfberry exosomes to the particle number of angelica exosomes is about 10 10 :1, 10 9 :1, 10 8 :1, 10 7 :1, 106 : 1, 10 5 : 1, 10 4 : 1, 10 3 : 1, 10 2 : 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1: 10, 1: 10 2 , 1: 10 3 , 1: 10 4 , 1: 10 5 , 1: 10 6 , 1: 10 7 , 1: 10 8 , 1: 10 9 , 1: 10 10 。

[0041] In a specific embodiment, the ratio range of the number of particles of wolfberry exosomes to the number of particles of angelica exosomes is about at least 1:1, and preferably the ratio of the number of particles of wolfberry exosomes to the number of particles of angelica exosomes is about 1:1.

[0042] In a specific embodiment, the composition comprises wolfberry exosomes and ginseng exosomes, wherein the ratio of the number of particles of wolfberry exosomes to the number of particles of ginseng exosomes is about 10 10 : 1, 10 9 : 1, 10 8 : 1, 10 7 : 1, 10 6 : 1, 10 5 : 1, 10 4 : 1, 10 3 : 1, 10 2 : 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, 1: 10, 1: 10 2 , 1: 10 3 , 1: 10 4 , 1: 10 5 , 1: 10 6 , 1: 10 7 , 1: 10 8 , 1: 10 9 , 1: 10 10 。

[0043] In a specific embodiment, the ratio range of the number of particles of wolfberry exosomes to the number of particles of ginseng exosomes is about at least 1:1.

[0044] In a specific embodiment, the ratio of the number of particles of Lycium barbarum exosomes to the number of particles of Panax ginseng exosomes is about 1:1.

[0045] In a specific embodiment, the composition comprises Lycium barbarum exosomes and Panax ginseng exosomes, wherein the composition has improved skin repair effect or antioxidant effect compared to the exosomes of each component. Preferably, the particle number ratio of Lycium barbarum exosomes to Panax ginseng exosomes is about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, such as 1:1.

[0046] In a specific embodiment, the composition comprises Lycium barbarum exosomes and Angelica sinensis exosomes, wherein the composition has improved skin repair effect or antioxidant effect compared to the exosomes of each component. Preferably, the particle number ratio of Lycium barbarum exosomes to Angelica sinensis exosomes is about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, such as 1:1.

[0047] In a specific embodiment, the composition comprises Lycium barbarum exosomes, Angelica sinensis exosomes and Panax ginseng exosomes, and the particle number ratio among Lycium barbarum exosomes, Angelica sinensis exosomes and Panax ginseng exosomes is about 1:1:1.

[0048] On the other hand, the present disclosure provides a plant exosome composition comprising plant exosomes or a composition prepared according to the method of the present disclosure, and optionally suitable excipients or carriers.

[0049] In a specific embodiment, the plant exosome composition is a pharmaceutical composition, a food composition, a beauty or cosmetic composition or a nutraceutical composition.

[0050] In a specific embodiment, the exosome composition is a food composition, which comprises plant exosomes prepared by the methods generally or preferably defined in the present disclosure, and optionally other food ingredients and / or edible excipients and / or carriers. In a specific embodiment, the food composition comprises at least one of Lycium barbarum exosomes, Panax ginseng exosomes, Dendrobium officinale exosomes and Angelica sinensis exosomes prepared by the method of the present disclosure, and optionally other food ingredients and / or edible excipients and / or carriers. In a specific embodiment, the exosome composition is a beauty (or cosmetic) composition, which comprises plant exosomes prepared by the methods generally or preferably defined in the present disclosure, and optionally beauty / cosmetic acceptable excipients or carriers. In a specific embodiment, the beauty composition comprises at least one of Lycium barbarum exosomes, Panax ginseng exosomes, Dendrobium officinale exosomes and Angelica sinensis exosomes prepared by the method of the present disclosure, and at least one beauty / cosmetic acceptable excipients or carriers.

[0051] In a specific embodiment, the exosome composition is a nutraceutical composition, which comprises plant exosomes prepared by the methods generally or preferably defined in the present disclosure, and optionally nutraceutically acceptable excipients or carriers. In a specific embodiment, the nutraceutical composition comprises at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes prepared by the methods of the present disclosure, and at least one nutraceutically acceptable excipient or carrier. On the other hand, the present disclosure also provides the use of plant exosomes (preferably wolfberry exosomes) prepared by the methods of the present disclosure or compositions containing the same (including but not limited to pharmaceutical compositions / food compositions / beauty or cosmetic compositions / nutraceutical compositions) for treating or preventing—or contributing to (or assisting in) improving, alleviating, or controlling—inflammation, preferably skin inflammation, or for use in—or assisting in—the field of dermatology for—repairing, anti-inflammation, antioxidant, anti-aging, whitening, or moisturizing.

[0052] On the other hand, the present disclosure also provides the use of plant exosomes (preferably wolfberry exosomes) prepared by the methods of the present disclosure or compositions containing the same (including but not limited to pharmaceutical compositions / food compositions / beauty compositions / nutraceutical compositions) in the preparation of products (drugs, foods, nutraceuticals, beauty / cosmetics, etc.) for treating or preventing—or contributing to (or assisting in) improving, alleviating, or controlling—inflammation, preferably skin inflammation, or for use in or assisting in the field of dermatology for repairing, anti-inflammation, antioxidant, anti-aging, whitening, or moisturizing.

[0053] On the other hand, the present disclosure provides a method for treating or preventing inflammation, preferably skin inflammation, or treating or preventing dermatological symptoms, or assisting in improving, alleviating, or controlling dermatological symptoms, which comprises administering to a subject in need a plant exosome (preferably wolfberry exosome) prepared by the methods generally or preferably defined above or a composition containing the same (pharmaceutical composition / food composition / beauty or cosmetic composition / nutraceutical composition).

[0054] On the other hand, the present disclosure provides the use of the above-mentioned plant exosomes (preferably wolfberry exosomes) or plant exosome compositions in the biomedical or drug delivery system.

[0055] On the other hand, the present disclosure also provides a method for preparing pharmaceutical compositions, food compositions, nutraceutical compositions, beauty / cosmetic compositions, which comprises preparing plant exosomes (preferably wolfberry exosomes) according to the methods generally or preferably defined in the present disclosure, and incorporating the plant exosomes into the pharmaceutical compositions, food compositions, nutraceutical compositions, beauty / cosmetic compositions. Description of the Drawings

[0056] The following drawings illustrate preferred embodiments of the present disclosure. For the purpose of illustrating the present disclosure, the currently preferred embodiments are shown in the drawings. However, it should be understood that the present disclosure is not limited to the specific embodiments shown in the drawings.

[0057] Figure 1 An exemplary step flow showing the method for isolating and extracting plant exosomes of the present disclosure.

[0058] Figure 2A -F shows the compound classification of six plant exosomes extracted by the layer-by-layer filtration method of the present disclosure; A: GQV01; B: RSV01; C: SHV01; D: HJV01; E: DGV01; F: DHV01.

[0059] Figure 3A -F shows the compound classification of six plant exosomes extracted by the ultracentrifugation method; A: LyE01; B: GsE01; C: DeE01; D: PoE01; E: AnE01; F: ReE01.

[0060] Figure 4 Shows the volcano plot of differential metabolites of six plant exosomes extracted by layer-by-layer filtration and ultracentrifugation; A: GQV01 vs LyE01; B: RSV01 vs GsE01; C: SHV01 vs DeE01; D: HJV01 vs PoE01; E: DGV01 vs AnE01; F: DHV01 vs ReE01. The abscissa is the fold change value of the metabolite expression difference between the two groups, that is, log2FC; the ordinate is the statistical test value of the metabolite expression amount change difference, that is, -log10(p value), and the numerical values of the abscissa and ordinate are logarithmically processed.

[0061] Figure 5A -F shows the KEGG pathway enrichment analysis diagram of six plant exosomes extracted by layer-by-layer filtration and ultracentrifugation; A: GQV01 vs LyE01; B: RSV01 vs GsE01; C: SHV01 vs DeE01; D: HJV01 vs PoE01; E: DGV01 vs AnE01; F: DHV01 vs ReE01. The abscissa represents the pathway name, and the ordinate represents the enrichment rate, which represents the ratio of the number of metabolites enriched in the pathway (Metabolite number) to the number of metabolites annotated to the pathway (Background number). The larger the ratio, the higher the degree of enrichment. The color gradient of the column represents the significance of enrichment. By default, the darker the color, the more significantly enriched the KEGG term. Among them, the KEGG terms with P value < 0.001 are marked as ***, those with P value < 0.01 are marked as **, and those with P value < 0.05 are marked as *.

[0062] Figure 6 TEM analysis diagrams showing exosomes isolated by layer-by-layer filtration; A: GQV01; B: RSV01; C: SHV01; D: HJV01; E: DGV01; F: DHV01.

[0063] Figure 7A -B shows the analysis diagram for GQV01, A: TEM analysis diagram, B: Particle size and concentration of GQV01 analyzed by nanoparticle tracking analysis.

[0064] Figure 8A Flow cytometry detection of PKH67 staining in vitro of exosomes isolated by layer-by-layer filtration; A: GQV01; B: RSV01; C: SHV01; D: HJV01; E: DGV01; F: DHV01. Figure 8B Flow cytometry detection of PKH67 staining in vitro of the analysis diagram for GQV01.

[0065] Figure 9 Activity detection of six plant exosomes isolated by layer-by-layer filtration; A: Protein concentration of six plant exosomes. B: Six plant exosomes inhibit LPS-induced TNF-α release in RAW264.7. C: Inhibition rate of TNF-α release by wolfberry exosomes. The ordinate is the concentration of TNF-α detected by ELISA kit. Among them, those with P value < 0.001 are marked as ***, and those with P value < 0.01 are marked as **.

[0066] Figure 10 Shows the repair effect of wolfberry on 3D skin model irradiated with UV. A. Representative tissue sections of 3D skin model tissue morphology (H&E staining), arrows indicate sunburn cells after UV irradiation; B. Quantitative analysis of sunburn cells in tissue morphology sections (H&E staining) of 3D skin model; C. Quantitative analysis of tissue viability detection results; D. TEWL detection results. When statistical analysis is performed using the t-test method, compared with the BC group, significance is indicated by #, #: P-value < 0.05, ##: P-value < 0.01; compared with the NC group, significance is indicated by *, *: P-value < 0.05, **: P-value < 0.01. Among them, Con is the normal group without UV irradiation, and WY14643 is the positive control (purchased from Sigma-Aldrich, catalog number C7081).

[0067] Figure 11Show the effect of wolfberry on the expression of skin barrier-related proteins in a 3D skin model irradiated with UV. A. Representative immunohistochemical images of filaggrin (FLG), loricrin (LOR), and claudin-1 (CLDN1); B. Quantitative analysis of the relative integrated optical density (IOD) values of FLG, LOR, and CLDN1. When statistical analysis was performed using the t-test method, compared with the BC group, significance was indicated by #, #: P-value < 0.05, ##: P-value < 0.01; compared with the NC group, significance was indicated by *, *: P-value < 0.05, **: P-value < 0.01. Among them, the normal group (con), the UV modeling group (UV), the positive control group (WY14643), and the drug treatment group (GQ).

[0068] Figure 12 Show the effect of wolfberry on the moisturizing effect of a 3D skin model irradiated with UV. A. Representative immunofluorescence image of aquaporin 3 (AQP3); B. Quantitative analysis of the relative integrated optical density (IOD) value of AQP3; C. Quantitative analysis of the detection results of the content of natural moisturizing factor PCA. When statistical analysis was performed using the t-test method, compared with the BC group, significance was indicated by #, #: P-value < 0.05, ##: P-value < 0.01; compared with the NC group, significance was indicated by *, *: P-value < 0.05, **: P-value < 0.01. Among them, the normal group (con), the UV modeling group (UV), the positive control group (WY14643), and the drug treatment group (GQ).

[0069] Figure 13 Show the detection results of lipid peroxidation (malondialdehyde) in a 3D skin model irradiated with UV. When statistical analysis was performed using the t-test method, compared with the BC group, significance was indicated by #, #: P-value < 0.05, ##: P-value < 0.01; compared with the NC group, significance was indicated by *, *: P-value < 0.05, **: P-value < 0.01. Among them, the normal group (con), the UV modeling group (UV), the positive control group (WY14643), and the drug treatment group (GQ).

[0070] Figure 14Expression and semi - quantitative results of four plant exosomes and their mixtures on UVB - induced barrier protein Claudin 1. A. WB was used to detect the level difference of Claudin 1 expression in HaCaT cells. The specific treatment groups and corresponding mixed plants are marked in the figure (con: blank control group; UV: ultraviolet irradiation group; Cer: ceramide group; GQ: Lycium barbarum; DG: Angelica sinensis; RS: Panax ginseng; SH: Dendrobium officinale; mix1: GQ:DG:RS:SH = 1:1:1:1; mix2: GQ:DG:RS:SH = 3:1:1:3; mix3: GQ:DG:RS:SH = 1:1:3:3, where the total concentration of exosomes in the GQ, DG, RS, SH, mix1, mix2 or mix3 group is 1x10 8 cells / ml, and the concentration of each component in each mix is converted according to the shown ratio); B. Semi - quantitative analysis of the WB results in Figure A.

[0071] Figure 15 Expression and semi - quantitative results of three plant exosomes and their mixtures on UVB - induced barrier protein Claudin 1. A. WB was used to detect the level difference of Claudin 1 expression in HaCaT cells. The specific treatment groups and corresponding mixed plants are marked in the figure (con: blank control group; UV: ultraviolet irradiation group; Cer: ceramide group; GQ: Lycium barbarum; DG: Angelica sinensis; RS: Panax ginseng; SH: Dendrobium officinale; mix1: GQ:DG:RS = 1:1:1; mix2: GQ:DG:SH = 1:1:1; mix3: GQ:RS:SH = 1:1:1; mix4: DG:RS:SH = 1:1:1, where the total concentration of exosomes in the GQ, DG, RS, SH, mix1, mix2, mix3 and mix4 group is 1x10 8 cells / ml, and the concentration of each component in each mix is converted according to the shown ratio); B. Semi - quantitative analysis of the WB results in Figure A.

[0072] Figure 16 Expression and semi - quantitative results of two plant exosomes and their mixtures on UVB - induced barrier protein Claudin 1. A. WB was used to detect the level difference of Claudin 1 expression in HaCaT cells. The specific treatment groups and corresponding mixed plants are marked in the figure (con: blank control group; UV: ultraviolet irradiation group; Cer: ceramide group; GQ: Lycium barbarum; DG: Angelica sinensis; RS: Panax ginseng; mix1: GQ:DG = 1:1; mix2: GQ:RS = 1:1; mix3: DG:RS = 1:1, where the total concentration of exosomes in the GQ, DG, RS, mix1, mix2, mix3 group is 1x10 8 cells / ml, and the concentration of each component in each mix is converted according to the shown ratio); B. Semi - quantitative analysis of the WB results in Figure A.

[0073] Figure 17 Exosomes of Lycium barbarum and Angelica sinensis and their mixtures, as well as exosomes of Lycium barbarum and Panax ginseng and their mixtures on the expression and semi-quantification results of the barrier protein Claudin 1 induced by UVB. A. Exosomes of Lycium barbarum and Angelica sinensis and their mixtures on the expression and semi-quantification results of the barrier protein Claudin 1 induced by UVB, WB was used to detect the level difference of Claudin 1 expression in HaCaT cells. The specific treatment groups and corresponding mixed plants have been marked in the figure (con: blank control group; UV: ultraviolet irradiation group; Cer: ceramide group; GQ: Lycium barbarum; DG: Angelica sinensis; 1:1: GQ:DG = 1:1; 1:3: GQ:DG = 1:3; 3:1: GQ:DG = 3:1, where the total concentration of exosomes in GQ, DG, and each mixture is 1x10 8 cells / ml, and the concentration of each component in each combination was converted according to the displayed ratio); B. Semi-quantitative analysis of the WB results in Figure A; C. Exosomes of Lycium barbarum and Panax ginseng and their mixtures on the expression and semi-quantification results of the barrier protein Claudin 1 induced by UVB, WB was used to detect the level difference of Claudin 1 expression in HaCaT cells. The specific treatment groups and corresponding mixed plants have been marked in the figure (con: blank control group; UV: ultraviolet irradiation group; Cer: ceramide group; GQ: Lycium barbarum; RS: Panax ginseng; 1:1: GQ:RS = 1:1; 1:3: GQ:RS = 1:3; 3:1: GQ:RS = 3:1, where the total concentration of exosomes in GQ, RS, and each mixture is 1x10 8 cells / ml, and the concentration of each component in each combination was converted according to the displayed ratio); D. Semi-quantitative analysis of the WB results in Figure C.

[0074] Figure 18 Antioxidant efficacy results and quantitative statistics of exosomes of Panax ginseng (RS) and Lycium barbarum (GQ) and their different compounding ratios. A. The kit was used to detect the level difference of ROS in HaCaT cells and fluorescence microscopy imaging was performed; B. Quantitative analysis of the fluorescence imaging results in Figure A, the difference analysis used two-tailed Student’s T test, # indicates the difference between the data group and the con (control) group, #: P-value < 0.0001, * indicates the difference between the data group and the UVB group, ***: P-value < 0.001; ****: P-value < 0.0001, where the total concentration of exosomes in GQ, RS, and each composition is 1×10 6 cells / mL, and the concentration of each component in each composition was converted according to the displayed ratio.

[0075] Figure 19Antioxidant efficacy results and quantitative statistics of wolfberry (GQ) and angelica (DG) exosomes and their different compounding ratios. A. Detection of intracellular ROS levels in HaCaT cells using a kit and fluorescence microscopy imaging; B. Quantitative analysis of the fluorescence imaging results in Figure A, with differential analysis using a two-tailed Student's T-test. # indicates the difference between the data group and the con (control) group, #: P-value < 0.0001, * indicates the difference between the data group and the UVB group, ****: P-value < 0.0001. The total concentration of exosomes in GQ, DG, and each composition is 1×10 6 per mL, and the concentration of each component in each composition is converted according to the displayed ratio. DETAILED DESCRIPTION OF THE INVENTION

[0077] I. Definitions and Terms

[0078] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0079] As used in the context of this disclosure, a "vesicle", also known as an extracellular vesicle, is a membrane-enclosed structure that is released by cells into the extracellular space, both in vitro and in vivo. Extracellular vesicles can contain proteins, lipids, and nucleic acids and can mediate intercellular communication between different cells (including different cell types). Two types of extracellular vesicles are exosomes and microvesicles.

[0080] As used in the context of this disclosure, "exosomes" refers to lipid-bound, cell-secreted small vesicles that are released from cells by the fusion of multivesicular endosomes (MVE) with the plasma membrane and mediate intercellular communication through the intercellular transport of proteins and RNA. Generally, exosomes range in size from about 30 nm to about 200 nm, sometimes also 30 - 150 nm or 30 - 100 nm, and have a bilayer membrane structure and a saucer-like morphology.

[0081] As used in the context of this disclosure, "plant material" has the meaning commonly understood in the art. Plant raw materials can be in a fresh state or dried. Generally, except for individual plants (such as wolfberry), it is more preferred to produce plant exosomes using fresh plant materials.

[0082] As used in the context of this disclosure, "depth filtration (DF)" means the removal of particles (e.g., impurities) from a liquid such as a solution using a depth filtration medium, by a combination of size (screening / filtration) and intermolecular interactions (e.g., electrostatic attraction and hydrophobic interactions between oppositely charged surfaces), to retain particles and impurities throughout the deep portion of the porous structure of the filtration medium. Depth filtration has been successfully used for the initial clarification of bacterial, yeast, insect, and mammalian cell suspensions, either alone or in combination with centrifugation. Depth filtration media can include cellulose (e.g., cellulose fibers) and / or polypropylene (e.g., polypropylene fibers), and / or filter aids (such as activated carbon, diatomaceous earth (DE), and / or perlite), and / or resins (e.g., polymeric resins). In some embodiments, medium-sized particles are removed less by depth filtration; very small particles are effectively removed by Brownian diffusion (plus adsorption) (e.g., particles smaller than the voids of the medium can enter into the interior of the medium), while very large particles are captured by physical screening or interception (e.g., retained and attached to the medium).

[0083] The term "depth filter" or "depth capsule filter" as used herein effects filtration within the depth of the filter material. Such filters are those that comprise a random fiber matrix that binds to form a complex maze of tortuous flow channels. Particle separation in these filters is caused by entrapment or adsorption onto the fiber matrix. The most frequently used depth filter media for bioprocesses of cell culture broths and other feeds include cellulose fibers, filter aids such as DE, and positively charged resin binders. Unlike absolute filters, depth filter media retain particles throughout the porous medium, thus allowing retention of particles larger than the pore size. Companies in the industry that currently have depth filtration media products include Sartorius, Merck Millipore, PALL, 3M, and others. Commercially available depth filters include, but are not limited to, the Millistak+Pod depth filter system, XOHC media (Millipore), Zeta Plus TM depth filters (3MPurification). In the present disclosure, depth filtration can be performed using two or more depth filters arranged in parallel. In such cases, commercially available depth filters can include, for example, Millistak+mini DOHC (Millipore) and XOHC filters (Millipore) or filters from Pall corporation NY, such as Supracap TM Depth Filter Capsules.

[0084] In the context of this disclosure, the terms "cosmetic", "cosmetic composition", "beauty product" or "beauty composition" refer to any chemical industrial product or fine chemical product that is applied, sprayed or otherwise distributed on any part of the human body surface, such as the skin, hair, fingernails, toenails, lips, teeth, etc., for the purpose of cleaning, maintaining, beautifying, modifying and changing the appearance, or correcting body odor and maintaining a good state. In some embodiments, the cosmetics, cosmetic compositions, beauty products or beauty compositions of the present disclosure are for non-therapeutic purposes.

[0085] In the context of this disclosure, the term "nutraceutical composition" refers to a product for supplementing the nutrient requirements of the human body, improving physical health and physical functions, and can be administered through the gastrointestinal tract or parenterally, usually through the gastrointestinal tract.

[0086] In the context of this disclosure, "skin" should be understood to include the layers from the outermost or stratum corneum to the innermost or subcutaneous layer (including both the outermost or stratum corneum and the innermost or subcutaneous layer). These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, mast cells, neurons, and / or adipocytes, etc. The term "skin" also includes the scalp. The term "skin" encompasses the skin of mammals and includes human skin. Similarly, the terms "hair, nails and mucous membranes" encompass the hair, nails and mucous membranes of mammals (such as humans).

[0087] In the context of this disclosure, the term "treatment" encompasses treatment methods, including methods involving the administration of an extract according to the present disclosure to alleviate or eliminate a disease or disorder or reduce or eliminate one or more symptoms associated with the disease or disorder. The term "treatment" also encompasses treatment methods involving alleviating or eliminating the physiological consequences of a disease or disorder.

[0088] In the context of this disclosure, the term "care" refers to maintaining the properties of the skin, hair, nails and / or mucous membranes. The properties are improved or maintained by performing cosmetic treatments and / or care in healthy subjects, subjects with sensitive skin, and subjects showing symptoms of the skin, hair, nails and / or mucous membranes (such as, but not limited to, ulcers and skin lesions, psoriasis, dermatitis, acne or rosacea, etc.).

[0089] In the context of the present disclosure, when the terms "treatment" and "care" are qualified by the modifiers "cosmetic" and / or "non-therapeutic", it means that the purpose of the treatment or care is to contribute to or assist in improving, alleviating, or controlling the condition involved, for example, to contribute to or assist in improving or maintaining the cosmetic properties of the skin, hair, nails, and / or mucous membranes, which affect the aesthetic appearance of the skin, hair, nails, and / or mucous membranes. Specifically, for example, to contribute to or assist in repair, anti-inflammation, antioxidant, anti-aging, whitening, or moisturizing, etc., to improve the hydration, elasticity, firmness, gloss, tone, or texture of the skin, hair, nails, and / or mucous membranes.

[0090] In the context of the present disclosure, the term "prevention" refers to the ability of the extracts of the present disclosure to prevent, delay, or hinder the occurrence or development of a disease or disorder, or to prevent, delay, or hinder changes in the cosmetic properties of the skin, mucous membranes, and / or hair. As used in the present disclosure, the term "prevention" may be used interchangeably with the term "inhibition of occurrence", that is, it refers to the ability of the extracts of the present disclosure to inhibit the occurrence or development of a disease or disorder, or to inhibit changes in the cosmetic properties of the skin, hair, nails, and / or mucous membranes.

[0091] In the context of the present disclosure, when used for a specific numerical value or range of values, the term "about" means that the numerical value associated with it fluctuates by ±10%, for example, by ±5%, ±2%, or ±1%. For example, as used herein, the expression "about 100" includes 90 and 110 and all values therebetween (e.g., 90.5, 95, 101, 105, 109.95... etc.). For ratios, the term "about" is used to define each number of the given ratio. For example, the ratio "about 1:1" means a ratio of (0.9 - 1.1):(0.9 - 1.1). For another example, the range of "about n - m" or "about n - about m" means 90%n - 110%n to 90%m - 110%m.

[0092] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. The methods and materials described below are only exemplary.

[0093] All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0094] II. Preparation of Exosomes

[0095] Raw materials

[0096] The raw materials involved in the methods of the present disclosure are plant materials. Preferably, the plant materials can be terrestrial (such as wolfberry, ginseng, dendrobium, and / or angelica). More preferably, the plant material is wolfberry.

[0097] The plant material can be part or all of a plant, and can be selected, for example, from wood, roots, rhizomes, barks, tree trunks, flowers, petals, sepals, seeds, fruits, stems, leaves, and / or germ, and mixtures of one or more thereof. In some embodiments, the plant material includes wolfberry, ginseng, dendrobium, polygonatum, angelica, rehmannia, or mixtures of one or more thereof.

[0098] In some embodiments, the plant material includes wolfberry (Lycium chinense Miller), which is a perennial woody plant of the genus Lycium in the Solanaceae family. Forms of wolfberry that can be used in the methods of the present disclosure include dried wolfberry, wolfberry powder, wolfberry extract, its physiologically acceptable salts, and its derivatives. Wolfberries that can be used in the methods of the present disclosure include, but are not limited to, Lycium barbarum, Lycium chinense, Lycium ruthenicum, Lycium dasystemum, Lycium truncatum, Lycium cylindricum, Lycium yunnanense, and Lycium changjiense. In addition, wolfberries also include wolfberry varieties, such as Lycium barbarum var. auranticarpum, Lycium chinense var. potaninii, or Lycium chinense var. rubricaulis. The methods of the present disclosure can use a mixture of one or more wolfberries as the plant material for exosome extraction.

[0099] In some embodiments, the plant material is or includes ginseng (Panax ginseng C.A.Mey.), which is a perennial herbaceous plant of the family Araliaceae and the genus Panax. Ginseng that can be used in the methods of the present disclosure can be one or more of the seeds, roots, stems, leaves, and fruits of Panax plants, preferably roots, preferably dried ginseng roots, or various processed traditional Chinese medicine pieces of ginseng. Specifically, the plants in the genus Panax can be Korean ginseng (Panax ginseng), American ginseng (Panax quinquefolium), Panax notoginseng, Panax japonicus, Panax trifolius, Panax pseudoginseng, Panax vietnamensis, etc. Specifically, the ginseng that can be used in the methods of the present disclosure can be garden ginseng (cultivated ginseng) or wild ginseng, and specifically includes sun-dried ginseng, sugar ginseng, sun-dried wild ginseng, red ginseng, white ginseng, etc. The methods of the present disclosure can use a mixture of one or more ginsengs or a mixture of one or more parts of ginseng as the plant material for exosome extraction.

[0100] In some embodiments, the plant material is or includes Lycium chinense Miller, a perennial woody plant of the genus Lycium in the Solanaceae family. Forms of Lycium that can be used in the methods of the present disclosure include dried Lycium, Lycium powder, Lycium extract, its physiologically acceptable salts, and its derivatives. Lycium that can be used in the methods of the present disclosure includes, but is not limited to, Lycium barbarum, Lycium chinense, Lycium ruthenicum, Lycium dasystemum, Lycium truncatum, Lycium cylindricum, Lycium yunnanense, and Lycium changjiense. In addition, Lycium also includes Lycium varieties, such as Lycium barbarum var. auranticarpum, Lycium chinense var. potaninii, or Lycium chinense var. rubricaulium. The methods of the present disclosure can use a mixture of more than one type of Lycium as the plant material for exosome extraction.

[0101] In some embodiments, the plant material is or includes Angelica sinensis (Oliv.) Diels, a perennial herbaceous plant of the genus Angelica in the Apiaceae family. The Angelica sinensis that can be used in the methods of the present disclosure is particularly its Angelica root, such as the dried root, and its forms include, but are not limited to, powder, flakes, or blocks. The types of Angelica sinensis that can be used in the methods of the present disclosure include, but are not limited to, Qin Angelica, Chuan Angelica, Xi Angelica, Yun Angelica, Dong Angelica, Taiwan Angelica, Chuan Angelica, Minxian Angelica, Beihai Angelica, Dahe Angelica, Korean Angelica, etc. The methods of the present disclosure can use a mixture of more than one type of Angelica sinensis as the plant material for exosome extraction.

[0102] Pretreatment

[0103] In some embodiments, the extraction method includes pretreating the plant material (preferably Lycium). Pretreatment techniques are well known in the art and include physical, chemical, and biological pretreatments, or any combination thereof.

[0104] In some embodiments, the pretreatment includes, but is not limited to, one or more of washing, mixing with an isotonic solution, crushing, grinding, stirring, filtering, and centrifuging. Preferably, the pretreatment is carried out in a sterile environment.

[0105] In some embodiments, the washing of the pretreatment can be carried out with an aqueous carrier or other edible or medicinal solvents (including organic solvents or inorganic solvents). Preferably, the cleaning agent is an aqueous carrier. Aqueous carriers are known in the art and include, but are not limited to, sterile water, water for injection, or isotonic solutions. Optionally, the cleaning treatment also contains a certain amount of surfactant in the cleaning solvent. More preferably, the cleaning agent is water or water for injection.

[0106] In some embodiments, the isotonic solution used in the pretreatment comprises a solution of an isotonic agent. Isotonic solutions include, but are not limited to, sodium chloride solution, phosphate buffer, Ringer's injection, isotonic glucose injection, Ringer's injection of glucose and lactate. Isotonic agents include isotonic agents selected from sodium chloride, mannitol, lactose, glucose (hydrous or anhydrous), sucrose, glycerol, and sorbitol or a solution of any one of the above. In certain embodiments, sodium chloride is present in an isotonic amount. Preferably, the isotonic solution is a sterile solution or sodium chloride injection containing 9 mg / ml (or 0.9%) sodium chloride.

[0107] In some embodiments, plant material (preferably wolfberry) is mixed with an isotonic solution in a certain ratio. For example, the plant material is mixed with the isotonic solution according to the following mass-to-volume ratios (w / v, g / mL, plant mass: isotonic solution volume): about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:20, about 1:30, about 1:50, about 1:100, about 1:200, or about 1:500. Preferably, the mass-to-volume ratio of the plant material to the isotonic solution ranges from about 1:2 - 1:50, about 1:3 - 1:20, or about 1:5 - 1:20.

[0108] In some embodiments, the comminution in the pretreatment includes any suitable manner known in the art, including but not limited to dry grinding, wet grinding, and vibratory ball milling. For example, the comminution includes pulverizing the plant or part thereof using powdering technology or cell wall breaking technology. The cell wall breaking technology uses a cell wall breaker.

[0109] In some embodiments, the pretreatment can be carried out at any suitable temperature. For example, the pretreatment can be carried out at the following temperatures: about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, or 40°C, or any sub-range within about 0°C to about 40°C (for example, any range between any two of the above temperatures), preferably about 2 - 30°C, more preferably about 2 - 20°C, even more preferably about 2 - 8°C, and most preferably about 4°C. In some embodiments, step (b) of the present disclosure can be carried out at ambient temperature (e.g., about 25°C).

[0110] In some embodiments, the stirring, filtration, or centrifugation operations in the pretreatment can be selected and adjusted by those skilled in the art according to specific needs.

[0111] Layer-by-layer filtration

[0112] The method for extracting plant exosomes of the present disclosure includes a step of filtering layer by layer.

[0113] The layer-by-layer filtration of the method of the present disclosure includes at least one filtration step, preferably multiple filtration steps, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more filtration steps. In some embodiments, for example, the layer-by-layer filtration may include 3 filtration steps. In some embodiments, the 3 filtration steps may be carried out independently or continuously. In some embodiments, the layer-by-layer filtration step includes a depth filtration step and an anti-clogging filtration. Preferably, the anti-clogging filtration may include membrane filtration, such as 0.4 - 0.8 μm membrane filtration; more preferably, the layer-by-layer filtration step includes a depth filtration step, an anti-clogging filtration and a sterilization filtration. Preferably, the anti-clogging filtration may include 0.4 - 0.8 μm membrane filtration and the sterilization filtration includes 0.1 - 0.3 μm membrane filtration.

[0114] The method of the present disclosure can be used to process the plant (preferably wolfberry) at a suitable rate. For example, for one production batch, the rate range for processing the plant by the method of the present disclosure is about 30 g / hour, 50 g / hour, 60 g / hour, 70 g / hour, 80 g / hour, 90 g / hour, 100 g / hour, 200 g / hour, 300 g / hour, 400 g / hour, 500 g / hour, 600 g / hour, 700 g / hour, 800 g / hour, 900 g / hour, 1,000 g / hour, or any sub-range within about 10 g / hour to about 1,000 g / hour (such as any range between any two of the above production rates).

[0115] Each filtration step of the layer-by-layer filtration of the disclosed method may be the same or different. For example, the filtration pore sizes are different between different filtration steps. In some embodiments, the layer-by-layer filtration includes 1 filtration step, wherein two filtration units with different pore sizes are used in this filtration step, preferably the two pore sizes decrease successively along the filtrate fluid direction. For example, the first filtration pore size is about 0.45 μm and the second filtration pore size is about 0.22 μm. In some embodiments, the layer-by-layer filtration includes 2 filtration steps, wherein the pore size in the first filtration step is different from the pore size in the second filtration step. For example, the two pore sizes decrease successively along the filtrate fluid direction. For example, the pore size in the first filtration step is about 0.45 μm and the pore size in the second filtration step is about 0.22 μm; or for another example, the pore size in the first filtration step is about 2 - 30 μm (such as 30 μm) and the pore size in the second filtration step is about 0.45 μm. In some embodiments, the layer-by-layer filtration includes 3 filtration steps, wherein the pore size in the first filtration step, the pore size in the second filtration step and the pore size in the third filtration step are not completely the same or at least partially different. Preferably, the three pore sizes decrease successively along the filtrate fluid direction. For example, the pore size in the first filtration step is about 2 - 30 μm, the pore size in the second filtration step is about 0.45 μm, and the pore size in the third filtration step is about 0.22 μm.

[0116] In the method of the present disclosure, the size of the pore diameter in the filtration step can be about 1000 μm, about 500 μm, about 400 μm, about 300 μm, about 200 μm, about 150 μm, about 100 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, about 15 μm, about 10 μm, about 9 μm, about 8 μm, about 7 μm, about 6 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, about 1 μm, 0.9 μm, about 0.8 μm, about 0.7 μm, about 0.6 μm, about 0.5 μm, about 0.45 μm, about 0.4 μm, about 0.35 μm, about 0.3 μm, about 0.25 μm, about 0.22 μm, about 0.20 μm, about 0.15 μm, about 0.10 μm, about 0.05 μm, about 0.04 μm, about 0.03 μm, about 0.02 μm and about 0.01 μm; the range of the pore diameter in the filtration step can be the range composed of any two of the above pore diameters, such as but not limited to about 0.05 - 2 μm, 0.05 - 1 μm, 0.05 - 0.8 μm, 0.05 - 0.6 μm, 0.05 - 0.4 μm, 0.1 - 2 μm, 0.1 - 1 μm, 0.1 - 0.8 μm, 0.1 - 0.6 μm, 0.1 - 0.4 μm, 0.1 - 0.3 μm, 0.1 - 0.22 μm, 0.2 - 1 μm, 0.2 - 0.8 μm, 0.2 - 0.6 μm, 0.2 - 0.4 μm, 0.3 - 0.8 μm, 0.3 - 0.6 μm, 0.4 - 1.0 μm, 0.4 - 0.8 μm, 0.45 - 0.8 μm, 0.5 - 100 μm, 1 - 100 μm, 1 - 50 μm, 1 - 40 μm, 2 - 50 μm, 2 - 40 μm, 2 - 30 μm, 2 - 20 μm, 2 - 10 μm, 2 - 5 μm, 2 - 4 μm, 3 - 50 μm, 3 - 40 μm, 3 - 30 μm, 3 - 20 μm, 3 - 10 μm, 3 - 5 μm, 3 - 4 μm, 4 - 50 μm, 4 - 40 μm, 4 - 30 μm, 4 - 20 μm, 4 - 10 μm, 4 - 5 μm, 5 - 50 μm, 5 - 40 μm, 5 - 30 μm, 5 - 20 μm, 5 - 10 μm, 6 - 50 μm, 6 - 40 μm, 6 - 30 μm, 6 - 20 μm, 6 - 10 μm, 7 - 50 μm, 7 - 40 μm, 7 - 30 μm, 7 - 20 μm, 7 - 10 μm, 8 - 50 μm, 8 - 40 μm, 8 - 30 μm, 8 - 20 μm, 8 - 10 μm, 9 - 50 μm, 9 - 40 μm, 9 - 30 μm, 9 - 20 μm, 9 - 10 μm. Preferably, the pore diameters for layer-by-layer filtration can be respectively selected from about 1 - 50 μm, 2 - 30 μm, 2 - 20 μm, 6 - 30 μm, 0.05 - 1 μm, 0.1 - 0.8 μm, 0.1 - 0.22 μm, 0.1 - 0.3 μm, 0.2 - 0.8 μm, 0.2 - 0.6 μm, 0.2 - 0.4 μm, 0.3 - 0.8 μm, 0.4 - 1.0 μm, 0.4 - 0.8 μm, 0.45 - 0.8 μm. In some embodiments, more preferably, the pore size in the filtration step can be selected from about 1 - 50 μm, 0.4 - 0.8 μm, and 0.1 - 0.3 μm, or a combination thereof, such as about 2 - 30 μm, about 0.45 - 0.8 μm, or about 0.1 - 0.22 μm, such as about 6 - 30 μm, about 0.45 μm, or about 0.22 μm.

[0117] In the method of the present disclosure, the filtration step in the layer-by-layer filtration can be carried out in one device, where the device includes a plurality of individual units having a filtration function. In some embodiments, the layer-by-layer filtration can be carried out in two or more devices, where each device includes one or more individual units having a filtration function. In some embodiments, the layer-by-layer filtration can be carried out in one device, where the device includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, and 500 individual units having a filtration function.

[0118] In the disclosed method, the layer-by-layer filtration includes the use of solvents such as organic solvents and inorganic solvents used in conventional extraction, such as water, isotonic aqueous solutions of sodium chloride, ethanol, acetone, diethyl ether, petroleum ether, ethyl acetate, or mixtures thereof. In some embodiments, the solvents in the layer-by-layer filtration may have a suitable flow rate. For example, the solvents may have a flow rate in the range of about, for example, 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min, 45 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min, 90 mL / min, 100 mL / min, 200 mL / min, 300 mL / min, 400 mL / min, 500 mL / min, 600 mL / min, 700 mL / min, 800 mL / min, 900 mL / min, or 1,000 mL / min, or any sub-range within about 1 mL / min to about 1,000 mL / min, such as about 1 mL / min to about 800 mL / min, about 1 mL / min to about 500 mL / min, about 1 mL / min to about 200 mL / min, about 10 mL / min to about 1,000 mL / min, about 10 mL / min to about 800 mL / min, about 10 mL / min to about 500 mL / min, about 10 mL / min to about 200 mL / min, about 20 mL / min to about 1000 mL / min, about 20 mL / min to about 800 mL / min, about 20 mL / min to about 500 mL / min, about 20 mL / min to about 200 mL / min, about 50 mL / min to about 1000 mL / min, about 50 mL / min to about 500 mL / min, about 50 mL / min to about 200 mL / min. In embodiments for large-scale production, the flow rate of the solvent can be as high as about 1 L / min or even higher. In some embodiments, the flow rate of the solvent in the layer-by-layer filtration of the disclosed method is about 20 mL / min to about 1000 mL / min, such as about 20 mL / min to about 500 mL / min, about 20 to about 200 mL / min, about 30 to about 100 mL / min.

[0119] The layer-by-layer filtration of the present disclosure can be carried out at any suitable temperature. For example, the layer-by-layer filtration of the present disclosure can be carried out at a suitable temperature, such as about 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 25 °C, 30 °C, 35 °C or 40 °C, or any sub-range within about 0 °C to about 40 °C (for example, any range between any two of the above temperatures), such as about 0 °C to about 35 °C, about 0 °C to about 30 °C, preferably about 0 °C to about 25 °C. In some embodiments, the layer-by-layer filtration of the present disclosure can be carried out at ambient temperature (e.g., 25 °C). In other embodiments, the layer-by-layer filtration of the present disclosure can be carried out at about 4 °C, for example, the depth filtration of the layer-by-layer filtration of the present disclosure at about 4 °C, and the membrane filtration of the layer-by-layer filtration of the present disclosure at about 4 °C.

[0120] Generally, any suitable filtration material can be used for the layer-by-layer filtration of the present disclosure. For example, the material can be a polymer. In some embodiments, the polymer can be a hydrophobic polymer. In some embodiments, the polymer can be a hydrophilic polymer. In some specific embodiments, the polymer includes but is not limited to poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylysine or polyglutamic acid, polyethersulfone (PES) (unmodified), modified polyethersulfone (mPES), polyvinylidene fluoride (PVDF), cellulose acetate, nitrocellulose, MCE (mixed cellulose ester), ultra-high molecular weight polyethylene (UPE), polytetrafluoroethylene (PTFE), nylon, polysulfone, polyacrylonitrile, polypropylene, polyvinyl chloride, polycarbonate, ceramics, diatomaceous earth, glass fiber, resin-bonded glass fiber, and combinations thereof. The available forms of the filtration material include but are not limited to microporous membranes, homogeneous membranes, and asymmetric membranes.

[0121] In some embodiments, the layer-by-layer filtration includes using a filter membrane for filtration, and the filter membrane comprises the following polymers: nylon, copolymers of acrylic acid, polysulfone, polyvinylidene fluoride, fiber esters, and cellulose esters. The filter membrane is usually made of a polymeric support material, such as PTFE (polytetrafluoroethylene), PES (polyethersulfone), PVP (polyvinylpyrrolidone), PVDF (polyvinylidene fluoride), nylon (polyamide), PP (polypropylene), cellulose (including cellulose esters), PEEK (polyetheretherketone), nitrocellulose, glass fiber, resin-bonded glass fiber, etc. In some embodiments, the polymer can be a hydrophilic polymer. Hydrophilic membranes include but are not limited to Bioassure (from Cuno); EverLUX TM polyethersulfone; STyLUX TMPolyethersulfone (all from Meissner); Millex GV, Millex HP, Millipak 60, Millipak 200, and Durapore CVGL01TP3 membranes (from Millipore); Fluorodyne TM EX EDF membranes, Supor TM EAV, Supor TM EBV, Supor TM EKV (all from Pall); Sartopore TM (from Sartorius); Hydrophilic PES membranes from Sterlitech; and WFPES PES membranes from Wolftechnik.

[0122] In some other embodiments, the layer-by-layer filtration of the present disclosure, such as depth filtration, can also introduce adsorbents such as silica particles, diatomaceous earth, or carbon particles.

[0123] In some embodiments, the inherent properties of the membrane can be altered by treating the membrane surface. For example, it is known to prepare hydrophilic or hydrophobic membranes by treating with other materials (such as other polymers, graphite, silicon, etc.) to coat the membrane surface. For example, the membranes used in layer-by-layer filtration can be modified with a positive charge. For example, the filtration membrane in layer-by-layer filtration can be a charge-modified polyvinylidene fluoride (PVDF) membrane produced by Micropore, or a membrane obtained from Pall using nylon 66 or positively charged polyethersulfone sulfate. The filter membrane can be sterilized (e.g., autoclaved) before use to ensure its sterility.

[0124] Depth filtration

[0125] The present disclosure provides a method for preparing plant exosomes (preferably wolfberry exosomes), which includes a layer-by-layer filtration step having the function or effect of preventing or avoiding clogging. The method of the present disclosure can use any filtration having the function or effect of preventing or avoiding clogging, such as depth filtration (DF), microfiltration (MF), ultrafiltration (UF), sterile filtration, membrane chromatography (MC), and centrifugation.

[0126] In some embodiments, the present disclosure provides a method for preparing plant exosomes (preferably wolfberry exosomes), which includes a layer-by-layer filtration step, and the layer-by-layer filtration step includes depth filtration. Preferably, in some embodiments, the depth filtration is achieved by a depth filter, such as a depth capsule filter.

[0127] In some embodiments, the depth filter is a commercially available depth filter, such as a depth filter produced by Pall Corporation (NY), such as Supracap TM depth filter; it can also be an activated carbon filter such as MilliStak TM series produced by Millipore (Billerica, MA); it can also be other depth filters: Profile star 5μm depth filter (PALL, catalog number BYA050P6), Profile star 3μm depth filter (PALL, catalog number BYA030P6). In some embodiments, the depth filter used in the present disclosure can be a filtration product of Pall Corporation (NY), such as Supracap TM Depth Filter Capsules, and for another example, Supracap TM 50 depth filter capsules, or Supracap TM 100 Depth Filter Capsules.

[0128] The loading range for depth filtration that can be used in the methods of the present disclosure is about 50 - 200 L / m 2 , for example 50 - 150 L / m 2 , 40 - 100 L / m 2 .

[0129] For the present disclosure, a depth filter can be any filter with a depth filtration function. In some embodiments, the depth filter can be a filter with any pore size. In some embodiments, the depth filters used in the present disclosure include depth filters having the following approximate pore size ranges: 0.5 - 100 μm, 1 - 100 μm, 1 - 50 μm, 1 - 40 μm, 2 - 50 μm, 2 - 40 μm, 2 - 30 μm, 2 - 20 μm, 3 - 50 μm, 3 - 40 μm, 3 - 30 μm, 3 - 20 μm, 4 - 50 μm, 4 - 40 μm, 4 - 30 μm, 4 - 20 μm, 5 - 50 μm, 5 - 40 μm, 5 - 30 μm, 5 - 20 μm, 6 - 50 μm, 6 - 40 μm, 6 - 30 μm, 7 - 50 μm, 7 - 40 μm, 7 - 30 μm, 7 - 20 μm, 8 - 50 μm, 8 - 40 μm, 8 - 30 μm, 8 - 20 μm, 9 - 50 μm, 9 - 40 μm, 9 - 30 μm, 9 - 20 μm. Preferably, the pore sizes for depth filtration can be respectively selected from approximately 1 - 50 μm, approximately 2 - 30 μm, approximately 2 - 20 μm, approximately 6 - 30 μm. More preferably, it is a depth filter having a pore size of approximately 2 - 30 μm, such as a depth capsule filter having a pore size of approximately 2 - 30 μm, such as Supracap TM Depth FilterCapsules filter.

[0130] For the present disclosure, a depth filter can be any form of depth filter, such as a filter plate filter, a filter disc filter, a filter element filter, a filter stack filter, a depth capsule filter, or a cartridge filter. In some embodiments, the depth filtration of the present disclosure is performed by a depth capsule filter. Depth capsule filters are generally used in the clarification filtration of fermentation broth and cell culture media, the filtration of serum and blood products, the filtration of enzyme preparations, the removal of impurities in chemical drugs, decarbonization filtration, or the filtration of colloidal or viscous materials. The inventors of the present disclosure unexpectedly found that depth capsule filters are also suitable for the extraction of plant materials, especially suitable for the method of extracting plant exosomes of the present disclosure.

[0131] A depth capsule filter generally can include a single-layer membrane structure or a double-layer membrane structure. The depth capsule filter used in the method of the present disclosure can be a single-layer membrane structure, which for example includes cellulose fibers, and / or filter aids (such as diatomaceous earth and perlite) and / or resins. In some embodiments, the depth capsule filter consists of two layers of membranes, where the upper filter plate has a larger pore size to intercept large-sized particles and protect the lower filter plate; the lower filter plate has a smaller pore size to further intercept small particles and ensure the clarity of the filtered liquid. For example, the pore size of the first layer of membrane can be approximately 11 - 30 μm, and the pore size of the second layer of membrane can be approximately 6 - 15 μm; or the pore size of the first layer of membrane can be approximately 8 - 20 μm, and the pore size of the second layer of membrane can be approximately 2 - 4 μm.

[0132] In some embodiments, the material of the deep capsule filter described herein includes any hydrophilic material with low protein adsorption, such as but not limited to polyethersulfone (PES), polyvinylidene fluoride (PVDF), and polypropylene (PP).

[0133] For the present disclosure, any suitable flow rate can be used for the deep filtration. For example, the filtration flow rate range for deep filtration in the methods of the present disclosure can be 100 - 1000 LMH (liquid volume passing through 1 m 2 membrane module per hour), such as about 100 LMH, about 200 LMH, about 500 LMH, about 800 LMH, about 120 - 500 LMH, about 500 - 1000 LMH, or any range formed by any two points between 100 - 1000 LMH. Alternatively, the flow rate of the deep filtration in the methods of the present disclosure can be expressed in mL / minute. For example, the deep filtration is carried out using a flow rate in the range of about 1 mL / minute to about 1,000 mL / minute, such as but not limited to about 1 mL / minute to about 800 mL / minute, about 1 mL / minute to about 500 mL / minute, about 1 mL / minute to about 200 mL / minute, about 10 mL / minute to about 1,000 mL / minute, about 10 mL / minute to about 800 mL / minute, about 10 mL / minute to about 500 mL / minute, about 10 mL / minute to about 200 mL / minute, about 20 mL / minute to about 1000 mL / minute, about 20 mL / minute to about 800 mL / minute, about 20 mL / minute to about 500 mL / minute, about 20 mL / minute to about 200 mL / minute, about 50 mL / minute to about 1000 mL / minute, about 50 mL / minute to about 500 mL / minute, about 50 mL / minute to about 200 mL / minute. Preferably, the feed rate of the present disclosure is about 20 mL / minute to about 1000 mL / minute, such as about 20 mL / minute to about 500 mL / minute, about 20 - 200 mL / minute.

[0134] For the present disclosure, any suitable pressure can be used for the deep filtration. For example, the deep filtration can be carried out using a pressure in the range of about 0.1 psi to about 100 psi, such as about 0.1 psi, 0.5 psi, 1 psi, 2 psi, 3 psi, 4 psi, 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, 10 psi, 20 psi, 30 psi, 40 psi, 50 psi, 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, or any sub - range within about 0.1 psi to about 100 psi (such as any range between any two of the above pressures).

[0135] For the present disclosure, the layer-by-layer filtration (e.g., depth filtration and optional other filtration steps) can be carried out for any suitable time period. For example, for one production batch, the layer-by-layer filtration can be carried out within a time range of about 10 minutes to about 10 hours, such as about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or any sub-range within about 10 minutes to about 10 hours (e.g., any range between any two of the above time points). Preferably, for example, for 500 ml of crude plant extract obtained by pretreatment, when using layer-by-layer filtration including depth filtration and membrane filtration (e.g., 0.45 μm membrane filtration and 0.22 μm membrane filtration) defined in the present disclosure, the treatment time is about 0.5 hours - about 2 hours, preferably about 1 hour, while the same crude plant extract takes about 3 hours by ultracentrifugation at 12,000 g for 70 minutes twice.

[0136] The method of the present disclosure can be used to process the plant (preferably Lycium barbarum) at a suitable rate. For example, for one production batch, the rate range of processing the plant such as crude plant extract by the method of the present disclosure is about 10 g / hour, 30 g / hour, 50 g / hour, 60 g / hour, 70 g / hour, 80 g / hour, 90 g / hour, 100 g / hour, 200 g / hour, 300 g / hour, 400 g / hour, 500 g / hour, 600 g / hour, 700 g / hour, 800 g / hour, 900 g / hour, 1,000 g / hour, or any sub-range within about 10 g / hour to about 1,000 g / hour (e.g., any range between any two of the above production rates).

[0137] The method for preparing plant exosomes by the layer-by-layer filtration (e.g., depth filtration and optional other filtration steps) of the present disclosure can obtain about the following number of plant exosomes per 100 g of plant: 1×10 10 individuals, 5×10 10 individuals, 10×10 10 individuals, 11×10 10 individuals, 12×10 10 individuals, 13×10 10 individuals, 14×10 10 individuals, 15×10 10 individuals, 16×10 10 individuals, 17×10 10 individuals, 18×10 10 individuals, 19×10 10 individuals, 20×10 10 individuals, 22×10 10 individuals, 24×10 10 individuals, 26×1010 pieces, 28×10 10 pieces, 30×10 10 pieces, 32×10 10 pieces, 34×10 10 pieces, 36×10 10 pieces, 38×10 10 pieces, 40×10 10 pieces, 42×10 10 pieces, 44×10 10 pieces, 46×10 10 pieces, 48×10 10 pieces, 50×10 10 pieces, 52×10 10 pieces, 54×10 10 pieces, 56×10 10 pieces, 58×10 10 pieces, 60×10 10 pieces, 66×10 10 pieces or more. In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 20×10 10 -50×10 10 pieces per 100 g of wolfberry, preferably about 30×10 10 -40×10 10 pieces, such as about 33×10 10 pieces of plant exosomes. In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 20×10 10 -60×10 10 pieces per 100 g of ginseng, preferably about 30×10 10 -50×10 10 pieces, such as about 39×10 10 pieces of plant exosomes. In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 10×10 10 -30×10 10 pieces per 100 g of dendrobium, preferably about 10×10 10 -20×10 10 pieces, such as about 12×10 10 pieces of plant exosomes. In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 30×10 10 -70×10 10 pieces per 100 g of angelica, preferably about 40×10 10 -60×10 10 pieces, such as about 50×10 10 pieces of plant exosomes.

[0138] The deep filter described in the present disclosure includes a deep filtration medium. In some embodiments, the deep filtration medium may be a hydrophilic material with low protein adsorption, such as polyethersulfone (PES), polyvinylidene fluoride (PVDF), polypropylene (PP), etc. (preferably having a pore size range of 1-50 μm). In some embodiments, the deep filtration medium may be in the form of a filter plate. In some embodiments, the deep filtration medium layer is optionally selected from one or more of Supradur P filter plates, K300 filter plates, and BECOPADP 270 filter plates. Generally, common filter plates in the art are generally divided into two types: one is a filter plate made of synthetic fiber and plant fiber materials; the other is a filter plate made of asbestos and pulp. Specifically, the filter plates used in the present disclosure may be produced by companies such as 3M, PALL, and EATON in the United States, and their common models include the SP series filter plates of 3M, the P series filter plates of PALL, and the BECO series filter plates of EATON.

[0139] In some embodiments, the deep filtration medium may be in a gradient distribution or a mixed distribution. In some embodiments, the deep filtration medium may be in a gradient distribution. In some embodiments, the deep capsule filtration device has a double-layer membrane structure with a gradient distribution, and the pore sizes of the two-layer membrane structure gradually decrease along the direction of the filtrate flow. For example, the pore size of the first layer of the membrane can be selected to be about 11-30 μm, and the pore size of the second layer of the membrane can be about 6-15 μm; or the pore size of the first layer of the membrane is about 8-20 μm, and the pore size of the second layer of the membrane is about 2-4 μm.

[0140] In some embodiments, the deep filtration medium may further include a membrane filtration layer. For example, the filtration pore size of the membrane filtration layer is preferably about 0.1-10 μm. In some embodiments, the deep filtration medium of the present disclosure can be used in combination with a membrane filtration medium. In some embodiments, the membrane filtration layer is provided at the liquid outlet end of the deep filtration medium. In some embodiments, the membrane filtration layer is provided at the liquid inlet end of the deep filtration medium. In some embodiments, the membrane filtration material after deep capsule filtration generally selects a hydrophilic material with low protein adsorption, such as polyethersulfone (PES), polyvinylidene fluoride (PVDF), polypropylene (PP), etc.

[0141] In some embodiments, the deep filtration medium may further include a filter aid. In some embodiments, the filter aid may include diatomaceous earth, perlite, talcum powder, silica gel, activated carbon, asbestos, molecular sieve, clay, etc. In some embodiments, the filter aid may be a filter aid based on mineral sources such as silica, such as perlite, diatomaceous earth, or sand, or an activated carbon filter aid optionally derived from natural materials such as wood or coconut shell.

[0142] In some embodiments, a pump can be used for the depth filtration described in the present disclosure. In some embodiments, the pump can include a peristaltic pump, a diaphragm pump, a gear pump, and a centrifugal drive pump. In some embodiments, a peristaltic pump can be used for the depth filtration.

[0143] In some embodiments, the flow rate of the pump used for the depth filtration can be in the following approximate ranges: 1 - 1000 mL / min, 10 - 1000 mL / min, 10 - 800 mL / min, 10 - 500 mL / min, 15 - 700 mL / min, 16 - 600 mL / min, 17 - 500 mL / min, 18 - 300 mL / min, 19 - 400 mL / min, 20 - 1000 mL / min, 20 - 500 mL / min, 20 - 300 mL / min, 20 - 200 mL / min, 30 - 100 mL / min, 40 - 100 mL / min, or 50 - 100 mL / min. Preferably, the pump used for the depth filtration in the present disclosure is a peristaltic pump, and the flow rate is, for example, about 20 - 1000 mL / min, such as about 20 - 500 mL / min, about 20 - 200 mL / min, about 30 - 100 mL / min.

[0144] In some embodiments, an aqueous solution is used to rinse the depth filter in the depth filtration described in the present disclosure. Preferably, an isotonic solution is used, more preferably an isotonic sodium chloride solution, and most preferably sodium chloride injection is used to rinse the depth capsule filter. For example, in the depth filtration described in the present disclosure, preferably before the start of filtration, sodium chloride injection is used to rinse the depth capsule filter at a flow rate of, for example, about 20 - 1000 mL / min, such as about 20 - 500 mL / min, about 20 - 200 mL / min, about 30 - 100 mL / min.

[0145] In some embodiments, in the depth filtration, an aqueous solution is used to clean the depth filter. Preferably, an isotonic solution is used, more preferably an isotonic sodium chloride solution, and most preferably sodium chloride injection is used to clean the depth capsule filter. For example, in the depth filtration described in the present disclosure, preferably at the end of the depth filtration, sodium chloride injection is used to clean the depth capsule filter at a flow rate of, for example, about 20 - 1000 mL / min, such as about 20 - 500 mL / min, about 20 - 200 mL / min, about 30 - 100 mL / min.

[0146] Membrane filtration

[0147] In some embodiments, the layer-by-layer filtration further includes membrane filtration. For example, the layer-by-layer filtration of the present disclosure includes depth filtration and membrane filtration. For example, in the layer-by-layer filtration described above, the membrane filtration may include anti-clogging membrane filtration and sterilizing membrane filtration. In some embodiments, the anti-clogging membrane filtration may be performed first, and then optionally the sterilizing membrane filtration may be performed. In some embodiments, the anti-clogging membrane filtration may be performed before the sterilizing membrane filtration. In some embodiments, the anti-clogging membrane filtration may be performed one or more times, and then the sterilizing membrane filtration may be performed one or more times.

[0148] The pore size of the membrane filtration of the present disclosure may be about 2μm, about 1μm, 0.9μm, about 0.8μm, about 0.7μm, about 0.6μm, about 0.5μm, about 0.45μm, about 0.4μm, about 0.35μm, about 0.3μm, about 0.25μm, about 0.22μm, about 0.20μm, about 0.15μm, about 0.10μm, about 0.05μm, about 0.04μm, about 0.03μm, about 0.02μm and about 0.01μm; the pore size range may be a range composed of any two of the above pore sizes, such as but not limited to about the following ranges: 0.05 - 2μm, 0.05 - 1μm, 0.05 - 0.8μm, 0.05 - 0.6μm, 0.05 - 0.4μm, 0.1 - 2μm, 0.1 - 1μm, 0.1 - 0.8μm, 0.1 - 0.6μm, 0.1 - 0.4μm, 0.1 - 0.3μm, 0.1 - 0.22μm, 0.2 - 1μm, 0.2 - 0.8μm, 0.2 - 0.6μm, 0.2 - 0.4μm, 0.3 - 0.8μm, 0.3 - 0.6μm, 0.4 - 1.0μm, 0.4 - 0.8μm, 0.45 - 0.8μm. In some preferred embodiments, the pore size range of the membrane filtration may be selected from about 0.4 - 0.8μm and about 0.1 - 0.3μm or a combination thereof, such as about 0.45 - 0.8μm and about 0.1 - 0.22μm.

[0149] In some embodiments, the layer-by-layer filtration of the present disclosure includes membrane filtration after depth filtration. For example, the membrane filtration can include membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm) and membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm). In some embodiments, the membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm) can be carried out first, and then optionally the membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm). In some embodiments, the membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm) can be carried out before the membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm). In some embodiments, one or more membrane filtrations of about 0.4 - 0.8 μm (such as about 0.45 μm) can be carried out, and then one or more membrane filtrations of about 0.1 - 0.3 μm (such as about 0.22 μm). Preferably, one or more membrane filtrations can be carried out before the membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm), preferably 1 membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm), to reduce or prevent the clogging of the 0.22 μm membrane filtration.

[0150] In some embodiments, the membrane filtration includes passing through one or more membrane filters in series. In some embodiments, the number of membrane filters in series ranges from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 membrane filters in series, preferably 1 - 4, more preferably 2 - 3. The cut-off sizes of all the membrane filters in series can be the same or different. In some embodiments, at least 2 of the membrane filters in series have different cut-off sizes. In some embodiments, the size exclusion decreases from large to small along the series of membrane filters. For example, in 3 membrane filters in series, the first filter can be about 0.45 μm filter, the second filter can be 0.3 μm filter, and the last filter can be about 0.22 filter.

[0151] Exemplary membrane filters for the methods of the present disclosure include but are not limited to polyethersulfone membrane filters, polyvinylidene fluoride membrane filters, cellulose membrane filters, mixed cellulose ester membrane filters, cellulose acetate membrane filters, nitrocellulose membrane filters, polyamide membrane filters, polycarbonate membrane filters, polytetrafluoroethylene membrane filters, polypropylene membrane filters, nitrated cellulose membrane filters, glass fibers, resin-bonded glass fibers or bead filters.

[0152] For the method of the present disclosure, the material of the membrane used for membrane filtration can be any filtration material commonly used in the art, such as various filtration materials and membrane materials described in the above-mentioned layer-by-layer filtration section. In some embodiments, the anti-clogging membrane filtration is a resin-bonded fiberglass membrane, preferably with a pore size of about 0.45 - 0.8 μm. In some embodiments, the sterilizing membrane filtration is a polyethersulfone (PES) membrane, preferably with a pore size of about 0.1 - 0.22 μm.

[0153] In some embodiments, any suitable pressure can be used for the membrane filtration. For example, a feed pressure in the range of about 0.1 psi to about 100 psi can be used for the membrane filtration, such as about 0.1 psi, 0.5 psi, 1 psi, 2 psi, 3 psi, 4 psi, 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, 10 psi, 20 psi, 30 psi, 40 psi, 50 psi, 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, or any sub-range within about 0.1 psi to about 100 psi (e.g., any range between any two of the above feed pressures). Any suitable permeate-side pressure can be used for the membrane filtration. For example, a permeate pressure in the range of about 0.1 psi to about 100 psi can be used for the membrane filtration, such as about 0.1 psi, 0.5 psi, 1 psi, 2 psi, 3 psi, 4 psi, 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, 10 psi, 20 psi, 30 psi, 40 psi, 50 psi, 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, or any sub-range within about 0.1 psi to about 100 psi (e.g., any range between any two of the above pressures).

[0154] In some embodiments, the membrane filtration described in the present disclosure can use a pump. In some embodiments, the pump can include a peristaltic pump, a diaphragm pump, a gear pump, and a centrifugal drive pump. In some embodiments, the membrane filtration can use a peristaltic pump. In some embodiments, the flow rate of the pump used for membrane filtration can be in the following ranges: 1-1000 mL / min, 100-1000 mL / min, 200-1000 mL / min, 500-1000 mL / min, 10-800 mL / min, 10-500 mL / min, 20-1000 mL / min, 20-500 mL / min, 20-300 mL / min, 20-200 mL / min, 30-100 mL / min, 40-100 mL / min, or 50-100 mL / min. Preferably, the flow rate of the pump for the membrane filtration of the present disclosure is about 20-1000 mL / min, such as about 500-1000 mL / min, about 20-500 mL / min, about 20-200 mL / min, about 30-100 mL / min.

[0155] In some embodiments, an aqueous solution, preferably an aqueous isotonic solution, more preferably an isotonic sodium chloride solution, is used to rinse and / or clean the membrane filter in the membrane filtration described in the present disclosure. It is preferred to use sodium chloride injection solution to clean the membrane filter at a flow rate of about 20-1000 mL / min, such as about 20-500 mL / min, about 20-200 mL / min, about 30-100 mL / min.

[0156] In some embodiments, the membrane filtration can be carried out at any suitable flow rate. For example, the membrane filtration can be carried out at a flow rate in the range of about 1 mL / min to about 1,000 mL / min, such as about 1 mL / min to about 800 mL / min, about 1 mL / min to about 500 mL / min, about 1 mL / min to about 200 mL / min, about 10 mL / min to about 1,000 mL / min, about 10 mL / min to about 800 mL / min, about 10 mL / min to about 500 mL / min, about 10 mL / min to about 200 mL / min, about 20 mL / min to about 1,000 mL / min, about 20 mL / min to about 800 mL / min, about 20 mL / min to about 500 mL / min, about 20 mL / min to about 200 mL / min, about 50 mL / min to about 1,000 mL / min, about 50 mL / min to about 500 mL / min, about 50 mL / min to about 200 mL / min. In embodiments of large-scale production, the flow rate of membrane filtration can be as high as about 1 L / min or even higher. Preferably, the filtration flow rate of the present disclosure is about 20 - 1000 mL / min, such as about 20 - 200 mL / min, about 30 - 100 mL / min.

[0157] In some embodiments, the membrane filtration can be carried out for any suitable time limit. For example, for one production batch, the membrane filtration can be carried out within a time range of about 10 minutes to about 10 hours, such as about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or any sub-range within about 10 minutes to about 10 hours (e.g., any range between any two of the above time points). Preferably, for 500 mL of crude plant extract obtained by pretreatment, membrane filtration is carried out for about 1 hour. Preferably, when using layer-by-layer filtration including depth filtration, anti-clogging membrane filtration such as about 0.45 μm filtration and sterilizing membrane filtration such as about 0.22 μm filtration, the treatment time is about 0.5 hour - about 2 hours, preferably about 1 hour.

[0158] In some embodiments, the method of the present disclosure including depth filtration and membrane filtration can obtain the following yield of plant exosomes per 100 g of plant: 1×10 10 pieces, 5×10 10 pieces, 10×10 10 pieces, 11×10 10 pieces, 12×10 10 pieces, 13×10 10 pieces, 14×10 10 pieces, 15×1010 pieces, 16×10 10 pieces, 17×10 10 pieces, 18×10 10 pieces, 19×10 10 pieces, 20×10 10 pieces, 22×10 10 pieces, 24×10 10 pieces, 26,×10 10 pieces, 28×10 10 pieces, 30×10 10 pieces, 32×10 10 pieces, 34×10 10 pieces, 36×10 10 pieces, 38×10 10 pieces, 40×10 10 pieces, 42×10 10 pieces, 44×10 10 pieces, 46×10 10 pieces, 48×10 10 pieces, 50×10 10 pieces, 52×10 10 pieces, 54×10 10 pieces, 56×10 10 pieces, 58×10 10 pieces, 66×10 10 pieces or even more. In some embodiments, the membrane filtration of the present disclosure can obtain about 20×10 10 -50×10 10 pieces, preferably about 33×10 10 pieces of plant exosomes per 100 g of wolfberry. In some embodiments, the membrane filtration of the present disclosure can obtain about 30×10 10 -50×10 10 pieces, preferably about 39×10 10 pieces of plant exosomes per 100 g of ginseng. In some embodiments, the membrane filtration of the present disclosure can obtain about 10×10 10 -20×10 10 pieces, preferably about 12×10 10 pieces of plant exosomes per 100 g of dendrobium. In some embodiments, the membrane filtration of the present disclosure can obtain about 45×10 10 -60×10 10 pieces, preferably about 50×10 10 pieces of plant exosomes.

[0159] Preferred embodiments

[0160] In one embodiment, the method for preparing plant exosomes of the present disclosure includes the following steps:

[0161] (a) Obtain plant raw materials;

[0162] (b) Pretreat the obtained plant raw materials to obtain a crude plant extract;

[0163] (c) Perform layer-by-layer filtration on the crude plant extract;

[0164] Among them, the layer-by-layer filtration treatment includes a deep filtration step and an optional membrane filtration step.

[0165] In one embodiment, the method for preparing plant exosomes of the present disclosure includes the following steps:

[0166] (a) Obtain plant raw materials;

[0167] (b) Pretreat the obtained plant raw materials, preferably perform mixing, pulverization, filtration and centrifugation to obtain a crude plant extract; (c) Perform layer-by-layer filtration on the crude plant extract to obtain a filtrate;

[0168] Among them, the layer-by-layer filtration treatment includes a deep filtration step. Preferably, the deep filtration step uses deep filtration with a pore size of about 1-50 μm, more preferably about 2-30 μm; and

[0169] (d) Perform membrane filtration on the filtrate in step (c), preferably perform anti-blocking membrane filtration and / or sterilizing membrane filtration, such as anti-blocking membrane filtration with a pore size of about 0.4-0.8 μm and / or sterilizing membrane filtration with a pore size of about 0.1-0.3 μm. More preferably, membrane filtration with a pore size of about 0.45-0.8 μm and membrane filtration with a pore size of about 0.1-0.22 μm. Most preferably, the membrane filtration consists of anti-blocking membrane filtration with 0.45 μm and sterilizing membrane filtration with 0.22 μm performed in sequence to obtain plant exosomes.

[0170] In the above embodiment, in step (b), the obtained plant material is pretreated, and the pretreatment includes mixing the plant material with an isotonic solution (preferably sodium chloride injection solution), pulverizing the plant raw materials using a wall breaker to obtain a plant residue solution, filtering the plant residue solution with a filter screen to obtain a plant juice, and centrifuging the plant juice and collecting the supernatant to obtain a crude plant extract.

[0171] In the above embodiment, large-scale mixing and pulverization of the plant material can be performed in step (b). In some specific embodiments, in step (b), plants with a mass of about 1000 g or even more, such as about 500-1000 g, about 50-200 g, such as about 100 g, are mixed and pulverized. In some embodiments, step (b) is performed at about 4 °C.

[0172] In the above embodiments, in step (b), the pulverized slag liquid is subjected to stirring treatment. Preferably, the stirring speed is about 50 - 500 rpm, more preferably about 100 - 250 rpm, and even more preferably about 200 ± 20 rpm. In some embodiments, the stirring time is about 10 - 200 min, preferably about 2 - 100 min, and even more preferably about 30 min.

[0173] In some preferred embodiments, the centrifugation in step (b) is carried out at about 1000 - 10000 g. In some preferred embodiments, the centrifugation in step (b) is carried out at least at about 3000 - 6000 g. In some preferred embodiments, the centrifugation time in step (b) is about 5 - 60 min. In some preferred embodiments, the ultracentrifugation time in step (b) is about 15 min, and / or is carried out at about 4 °C.

[0174] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes wolfberry.

[0175] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes ginseng.

[0176] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes dendrobium, preferably Dendrobium officinale.

[0177] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes angelica.

[0178] In a specific embodiment of the above method for preparing plant exosomes, the step of layer-by-layer filtration treatment includes a deep filtration step, wherein the deep filter is a deep capsule filter with a pore size of about 1 - 50 μm, preferably about 2 - 30 μm, and the loading range is about 50 - 200 L / m 2 , such as 50 - 150 L / m 2 , 40 - 100 L / m 2 ; preferably, the deep filter is a deep filter produced by Pall Corporation (NY), which is Supracap TM Depth Filter Capsules.

[0179] In a specific embodiment of the above method for preparing plant exosomes, in step (d), the filtrate in step (c) is successively filtered through a filter of about 0.45 μm and a filter of about 0.22 μm, and the final filtrate is collected to obtain plant exosomes.

[0180] Technical effects

[0181] The layer-by-layer filtration method of the present disclosure has the following advantages compared to the ultracentrifugation method:

[0182] (1) Lower time consumption;

[0183] (2) Higher exosome yield or particle concentration;

[0184] (3) It can prevent the filter membrane from being blocked due to excessive impurities and can ensure the sterility of the product;

[0185] (4) The extracted plant exosomes have excellent metabolomic performance;

[0186] (5) The extracted plant exosomes have a typical saucer shape similar to exosomes, and the particle size is in the range of 30 - 200 nm;

[0187] (6) The extracted plant exosomes have anti-inflammatory and immunomodulatory activities.

[0188] The method for preparing plant exosomes (preferably wolfberry exosomes) of the present disclosure is suitable for small-scale, pilot-scale, and large-scale production. In some embodiments, the exosome preparation method of the present disclosure is particularly used for large-scale production, and the amount of plant material that can be processed is, for example, at least about 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9000 g, 10000 g, 50000 g, or even up to 100000 g. Preferably, the exosome preparation method of the present disclosure can process plant material within about 1000 g, more preferably within about 10000 g, and most preferably within about 100000 g of plant raw material.

[0189] In particular, the layer-by-layer filtration treatment of the present disclosure is particularly suitable for large-scale production. For example, based on the deep filtration and membrane filtration preferably shown in the examples of the present disclosure, large-scale production is achieved by increasing the membrane area of the filter. Through appropriately increasing the membrane area in the layer-by-layer filtration of the present disclosure, at least about 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9000 g, 10000 g, 50000 g, or even 100000 g of plant material or its corresponding pretreatment solution can be filtered.

[0190] III. Exosomes

[0191] The plant exosomes (preferably wolfberry exosomes, angelica exosomes, ginseng exosomes, dendrobium exosomes) prepared according to the method of the present disclosure can have any suitable hydrodynamic particle size or diameter. For example, the plant exosomes can have a hydrodynamic particle size or diameter of about 10 nm to about 10 μm, such as about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm and 10 μm, or any sub-range within about 10 nm to about 10 μm (for example, any range between any two of the above particle sizes). Preferably, the plant exosomes prepared according to the method of the present disclosure have a diameter of about 30 - 450 nm, preferably about 30 - 250 nm, and more preferably have a diameter of about 30 - 200 nm.

[0192] The plant exosomes can have any suitable shape, including but not limited to saucer shape, spherical shape, discoid shape. Preferably, the plant exosomes prepared according to the method of the present disclosure have a saucer shape.

[0193] The plant exosomes (preferably wolfberry exosomes) prepared according to the method of the present disclosure have more metabolites than the plant exosomes prepared by the ultracentrifugation method in one or more of the following aspects: lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzenoids. In some embodiments, the number of lipid species of the plant exosomes prepared according to the method of the present disclosure is greater than the number of lipid species of the plant exosomes prepared by the ultracentrifugation method. As shown in Table 4 of the examples below, the lipid classification mainly includes fatty acyl (FA), glycerolipids (GL), glycerophospholipids (GP), prenol lipids (PR), sphingolipids (SP), steroids (ST), and saccharolipids (SL). In some embodiments, the number of fatty acyl compounds of the plant exosomes prepared according to the method of the present disclosure is 190 - 300, preferably 200 - 250. In some embodiments, the number of glycerolipid compounds of the plant exosomes prepared according to the method of the present disclosure is 2 - 10, preferably 3 - 9, more preferably 4 - 8. In some embodiments, the number of glycerophospholipid compounds of the plant exosomes prepared according to the method of the present disclosure is 2 - 10, preferably 3 - 8, more preferably 4 - 7. In some embodiments, the number of prenol lipid compounds of the plant exosomes prepared according to the method of the present disclosure is 190 - 400, preferably 200 - 350, more preferably 200 - 300. In some embodiments, the number of sphingolipid compounds of the plant exosomes prepared according to the method of the present disclosure is 2 - 9, preferably 2 - 5, more preferably 3. In some embodiments, the number of steroid compounds of the plant exosomes prepared according to the method of the present disclosure is 10 - 100, preferably 20 - 50, more preferably 20 - 40. In some embodiments, the number of saccharolipid compounds of the plant exosomes prepared according to the method of the present disclosure is 0 - 5, preferably 2 - 5, most preferably 2 - 3.

[0194] The plant exosomes (preferably wolfberry exosomes) prepared according to the method of the present disclosure have metabolic upregulation (Figure 5) compared to the plant exosomes prepared by the ultracentrifugation method, such as tryptophan metabolism, arachidonic acid metabolism, phenylpropanoid biosynthesis, starch and sucrose metabolism, alanine, aspartate and glutamate metabolism, galactose metabolism, ABC transporters, valine, leucine and isoleucine biosynthesis, biosynthesis of cutin, suberin and wax, linoleic acid metabolism, β-alanine metabolism, nucleotide metabolism, histidine metabolism, aminoacyl-tRNA biosynthesis, arginine biosynthesis, biosynthesis of various secondary metabolites.

[0195] Specifically, there are 73 metabolic pathways related to differential metabolites between the wolfberry exosomes prepared by the method according to the present disclosure and those prepared by the ultracentrifugation method, and a total of 11 pathways are significantly enriched.

[0196] In some embodiments, there are 617 significantly differential metabolites between the wolfberry exosomes prepared by the method according to the present disclosure and those prepared by the ultracentrifugation method, with 401 up-regulated metabolites and 216 down-regulated metabolites. There are 705 significantly differential metabolites between the ginseng exosomes prepared by the method according to the present disclosure and those prepared by the ultracentrifugation method, with 527 up-regulated metabolites and 178 down-regulated metabolites; there are 770 significantly differential metabolites between the dendrobium exosomes prepared by the method according to the present disclosure and those prepared by the ultracentrifugation method, with 537 up-regulated metabolites and 233 down-regulated metabolites; there are 747 significantly differential metabolites between the angelica exosomes prepared by the method according to the present disclosure and those prepared by the ultracentrifugation method, with 659 up-regulated metabolites and 88 down-regulated metabolites.

[0197] In some embodiments, in the analysis of staining plant exosomes with the lipophilic dye PKH67 in vitro, the positive rates of the wolfberry exosomes prepared by the method according to the present disclosure are 64.5% or 68.8% respectively. In the detection of the protein content of plant exosomes, the protein contents of the wolfberry exosomes, angelica exosomes, ginseng exosomes, and dendrobium exosomes prepared by the method according to the present disclosure are 47434.90 μg / mL, 5862.76 μg / mL, 2521.15 μg / mL, and 875.89 μg / mL respectively.

[0198] In some embodiments, the wolfberry exosomes prepared by the method of the present disclosure show good anti-inflammatory effects, and the TNF-α inhibition rate shown in the examples is above about 60%, and even close to 100%.

[0199] IV. Compositions

[0200] The present disclosure provides a plant exosome composition, comprising at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes, preferably at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes prepared by the method for preparing plant exosomes as generally or preferably or specifically defined in the present disclosure. Specifically, the plant exosome composition provided by the present disclosure is a pharmaceutical composition, which, in addition to comprising wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes (preferably at least one or more of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes prepared by the method for preparing plant exosomes as generally or preferably or specifically defined in the present disclosure), further optionally comprises a pharmaceutically acceptable carrier, or other active ingredients. The carriers are usually used in pharmaceutical preparations, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil.

[0201] In some embodiments, the present disclosure provides a composition, which comprises at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes. In some embodiments, the present disclosure provides a composition, which comprises wolfberry exosomes, and preferably the composition does not contain dendrobium exosomes. In some embodiments, the present disclosure provides a composition, which comprises at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes prepared by the method for preparing plant exosomes as generally or preferably or specifically defined. In some embodiments, the present disclosure provides a composition, which comprises any two, any three, or all four of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes, and preferably at least one of the wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure.

[0202] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and ginseng exosomes, preferably at least one of the wolfberry exosomes and ginseng exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and dendrobium exosomes, preferably at least one of the wolfberry exosomes and dendrobium exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and angelica exosomes, preferably at least one of the wolfberry exosomes and angelica exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure. In some embodiments, the present disclosure provides a composition comprising ginseng exosomes and dendrobium exosomes, preferably at least one of the ginseng exosomes and dendrobium exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure. In some embodiments, the present disclosure provides a composition comprising ginseng exosomes and angelica exosomes, preferably at least one of the ginseng exosomes and angelica exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure. In some embodiments, the present disclosure provides a composition comprising dendrobium exosomes and angelica exosomes, preferably at least one of the dendrobium exosomes and angelica exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure.

[0203] In some embodiments, the present disclosure provides a composition comprising ginseng exosomes, dendrobium exosomes and angelica exosomes, preferably at least one of the ginseng exosomes, dendrobium exosomes and angelica exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes, dendrobium exosomes and angelica exosomes, preferably at least one of the wolfberry exosomes, dendrobium exosomes and angelica exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes, ginseng exosomes and angelica exosomes, preferably at least one of the wolfberry exosomes, ginseng exosomes and angelica exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes, ginseng exosomes, dendrobium exosomes, preferably at least one of the wolfberry exosomes, ginseng exosomes, dendrobium exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure.

[0204] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes, ginseng exosomes, dendrobium exosomes and angelica exosomes, preferably at least one of the wolfberry exosomes, ginseng exosomes, dendrobium exosomes and angelica exosomes is prepared by the method for preparing plant exosomes as defined in the present disclosure.

[0205] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes, and the particle number ratio between any two exosomes ranges from about 1:10 10 -10 10 :1, for example, about 1:10 9 -10 9 :1, 1:10 8 -10 8 :1, 1:10 7 -10 7 :1, 1:10 6 -10 6 :1, 1:10 5 -10 5 :1, 1:10 4 -10 4 :1, 1:10 3 -10 3 :1, 1:10 2 -10 2 :1, 1:10 - 10:1, 1:9 - 9:1, 1:8 - 8:1, 1:7 - 7:1, 1:6 - 6:1, 1:5 - 5:1, 1:4 - 4:1, 1:3 - 3:1, 1:2 - 2:1, 1:1. Preferably, at least one of the wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes is prepared by the method for preparing plant exosomes defined in the present disclosure.

[0206] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and angelica exosomes, wherein the ratio of the number of particles of wolfberry exosomes to the number of particles of angelica exosomes can be any ratio, for example, about 10 10 :1, 10 9 :1, 10 8 :1, 10 7 :1, 10 6 :1, 10 5 :1, 10 4 :1, 10 3 :1, 10 2 :1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:10 2 、1:10 3 、1:10 4 、1:10 5 、1:10 6 、1:10 7 、1:108 、1:10 9 、1:10 10 , preferably, the ratio range of the number of particles of wolfberry exosomes to the number of particles of angelica exosomes is about at least 1:1. For example, the ratio range of the number of particles of wolfberry exosomes to the number of particles of angelica exosomes is about 1:1, and preferably it can be understood that the ratio range of the number of particles of exosomes is that the number of particles of wolfberry exosomes is equal to or more than 1 part compared to the number of particles of 1 part of angelica exosomes.

[0207] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and ginseng exosomes, wherein the ratio range of the number of particles of wolfberry exosomes to the number of particles of ginseng exosomes can be any ratio, such as about 10 10 :1, 10 9 :1, 10 8 :1, 10 7 :1, 10 6 :1, 10 5 :1, 10 4 :1, 10 3 :1, 10 2 :1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:10 2 、1:10 3 、1:10 4 、1:10 5 、1:10 6 、1:10 7 、1:10 8 、1:10 9 、1:10 10 , preferably, the ratio range of the number of particles of wolfberry exosomes to the number of particles of ginseng exosomes is about at least 1:1. For example, the ratio range of the number of particles of wolfberry exosomes to the number of particles of ginseng exosomes is about 1:1, and preferably it can be understood that the ratio range of the number of particles of exosomes is that the number of particles of wolfberry exosomes is equal to or more than 1 part compared to the number of particles of 1 part of ginseng exosomes.

[0208] In some embodiments, the present disclosure provides a composition comprising any two, any three, or four of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes, and the composition has improved skin repair effect or antioxidant effect compared to the exosomes of each component.

[0209] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and ginseng exosomes. Optionally, at least one of the wolfberry exosomes and ginseng exosomes is prepared by the method for preparing plant exosomes defined in the present disclosure. Preferably, the composition has enhanced skin repair effect or antioxidant effect compared to the exosomes of each component. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and angelica exosomes. Optionally, at least one of the wolfberry exosomes and angelica exosomes is prepared by the method for preparing plant exosomes defined in the present disclosure. Preferably, the composition has enhanced skin repair effect or antioxidant effect compared to the exosomes of each component.

[0210] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes; optionally, the composition further comprises ginseng exosomes and / or angelica exosomes; preferably, the composition does not contain dendrobium exosomes. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and the composition does not contain dendrobium exosomes. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes, angelica exosomes and ginseng exosomes. The composition has enhanced skin repair effect or antioxidant effect compared to the exosomes of each component, wherein the wolfberry exosomes, angelica exosomes and ginseng exosomes are the main active ingredients, or the only active ingredients.

[0211] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and ginseng exosomes. The composition has enhanced skin repair effect or antioxidant effect compared to the exosomes of each component. Preferably, the particle number ratio of wolfberry exosomes to ginseng exosomes is about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, such as 1:1. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and ginseng exosomes. The composition has enhanced skin repair effect or antioxidant effect compared to the exosomes of each component, wherein the wolfberry exosomes and ginseng exosomes are the main active ingredients, or the only active ingredients.

[0212] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and angelica exosomes. The composition has enhanced skin repair effect or antioxidant effect compared to the exosomes of each component. Preferably, the particle number ratio of wolfberry exosomes to angelica exosomes is about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, such as 1:1. In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes and angelica exosomes. The composition has enhanced skin repair effect or antioxidant effect compared to the exosomes of each component, wherein the wolfberry exosomes and angelica exosomes are the main active ingredients, or the only active ingredients.

[0213] In some embodiments, the present disclosure provides a composition comprising wolfberry exosomes, angelica exosomes, and ginseng exosomes, and the particle number ratio among the wolfberry exosomes, angelica exosomes, and ginseng exosomes can be any value, such as about 1:1:1.

[0214] Specifically, the plant exosome composition provided by the present disclosure is a food composition or a nutraceutical composition, which comprises at least one or more of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes, preferably at least one or more of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes prepared by the method for preparing plant exosomes generally or preferably or specifically defined by the present disclosure. When the food composition or nutraceutical composition of the present disclosure is used as a food additive, the composition can be directly added and used together with other foods or food ingredients according to conventional methods. Generally, when preparing foods or beverages, the food composition or nutraceutical composition of the present disclosure in an amount of about 15 parts by weight or less, preferably about 10 parts by weight or less, based on the total weight of the composition, can be added.

[0215] The nutraceutical composition of the present disclosure itself can be in the form of a nutritional supplement or a health care product. In addition to the wolfberry exosomes of the present disclosure, it can also contain other nutrients and / or nutraceutically acceptable excipients or carriers, such as various proteins, fats, vitamins, prebiotics, probiotics, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, carbonates used in carbonated beverages, etc.

[0216] Foods to which the above food composition or nutraceutical composition can be added include but are not limited to meats, sausages, breads, chocolates, sugars, fast foods, cookies, pizzas, ramen, other noodles, chewing gums, dairy products including ice creams, various soups, beverages, tea beverages, alcoholic beverages, and vitamin complexes, etc. In some embodiments, the food composition of the present disclosure can be prepared into a functional food.

[0217] The plant exosome composition (drug composition / nutraceutical composition / food composition) of the present disclosure can be in different forms for oral administration, such as capsules (including gelatin capsules, soft capsules, hard capsules), tablets (including sugar-coated tablets, tablets, pills, powders, granules, chewing gums), solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, gels or gelatin, and any other forms known to those skilled in the art; it can also be administered by topical or transdermal routes, or by any other suitable route (such as the parenteral route).

[0218] Specifically, the exosome composition provided by the present disclosure is a cosmetic / cosmetic composition, which contains at least one or more of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes, preferably at least one or more of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes prepared by the method for preparing plant exosomes generally or preferably or specifically defined in the present disclosure. The cosmetic / cosmetic composition of the present disclosure usually contains at least one excipient or adjuvant acceptable in cosmetics / cosmetics. The "excipient or adjuvant acceptable in cosmetics / cosmetics" can be selected from: solvents, solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, humectants, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, propellants / propellants, fragrances, pigments, and other functional additives. The forms of the cosmetic composition are, for example, but not limited to, soaps, soaps, facial cleansers, cleansing foams, cleansing lotions, cleansing creams, body washes, softening lotions, skin care gels, skin care lotions, skin care creams, essence, eye creams, facial masks, aerosols or sprays, skin toners, skin softeners, toners, astringents, emulsions, milk emulsions, moisturizing emulsions, nourishing emulsions, massage creams, nourishing creams, moisturizing creams, hand creams, foundations, essence, nourishing essence, powder compacts, body lotions, and skin cleansing lotions, etc. These forms of the cosmetic composition can be prepared by methods well-known to those skilled in the art.

[0219] When the cosmetic composition of the present disclosure is a paste, cream or gel, animal fibers, plant fibers, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silica gels, bentonites, silica, talc, zinc oxide, etc. can be used as carrier components. When the cosmetic composition of the present disclosure is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. can be used as carrier components. In particular, when the preparation is a spray, a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether can also be included. When the cosmetic composition of the present disclosure is a solution or emulsion, a solvent, solubilizer or emulsifier can be used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol fatty acid esters, polyethylene glycol or sorbitan fatty acid esters can be used. When the cosmetic composition of the present disclosure is a suspension, a liquid diluent such as water, ethanol or propylene glycol, and a suspending agent such as ethoxyisostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan anhydride esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc. can be used as carrier components.

[0220] V. Uses

[0221] Exosomes or compositions for any use of the present disclosure may include the exosomes or compositions generally or specifically disclosed in the present disclosure, preferably the exosomes of Part III or the compositions of Part IV of the present disclosure. Those skilled in the art will understand that exosomes for any use of the present disclosure can freely combine all the features disclosed herein. For example, the exosomes of Part III of the present disclosure can be exosomes with any combination of features such as different particle densities, different metabolite species, and different percentages of their metabolite species as defined.

[0222] Many diseases are accompanied by inflammatory responses, including skin-related inflammations (such as dermatitis, acne, pimples, melasma, etc.). Therefore, reducing the inflammation level will be beneficial to disease treatment and care. Accordingly, in some embodiments, the present disclosure provides the use of plant exosomes (preferably wolfberry exosomes) or a composition containing the same for treating or preventing, or contributing to / assisting in improving, alleviating or controlling inflammation, such as skin inflammation. In some other embodiments, the present disclosure provides the use of plant exosomes (preferably wolfberry exosomes) or a composition containing the same for or contributing to / assisting in the cosmetic, non-therapeutic treatment and / or care of the skin, hair, nails and / or mucous membranes, preferably for or contributing to / assisting in the cosmetic, non-therapeutic treatment and / or care of the inflammation of the skin, hair, nails and / or mucous membranes.

[0223] In some embodiments, the plant exosomes (preferably wolfberry exosomes) or a composition containing the same of the present disclosure can be used in the skin field for repair, anti-inflammation, antioxidant, anti-aging, whitening, moisturizing and other uses. For example, the plant exosomes or a composition containing the same can be used for anti-inflammatory use, preferably for anti-inflammatory use in the skin field. For another example, the plant exosomes or a composition containing the same can be used for antioxidant use. For another example, the plant exosomes or a composition containing the same can be used for repair use in the skin field. For another example, the plant exosomes or a composition containing the same can be used for anti-aging use. For another example, the plant exosomes or a composition containing the same can be used for whitening use. For another example, the plant exosomes or a composition containing the same can be used for moisturizing use.

[0224] Therefore, the present disclosure also provides the use of plant exosomes (preferably wolfberry exosomes) or a composition containing the same in the preparation of products (drugs, foods, nutritional health products, beauty / cosmetics). In some embodiments, the drug is used for treating or preventing inflammation, such as dermatitis, acne, pimples, melasma, etc. In some embodiments, the nutritional health product contributes to or assists in enhancing the skin barrier function, antioxidant, repair, anti-aging, whitening and / or moisturizing.

[0225] The present disclosure also provides a method for treating or preventing inflammation, preferably skin inflammation, or treating or preventing dermatological symptoms, or assisting in improving, alleviating, or controlling dermatological symptoms (specifically for repair, anti-inflammation, antioxidant, anti-aging, whitening, or moisturizing), which includes administering to a subject in need a plant exosome (preferably wolfberry exosome) or a composition containing the same (pharmaceutical composition / food composition / beauty or cosmetic composition / nutritional and health composition) prepared by the aforementioned general or preferred defined method.

[0226] In most cases, skin diseases start from the deterioration or breakdown of the skin barrier function, and filaggrin, loricrin (LOR), and Claudins proteins play important roles in the skin barrier function. Due to the reduction of filaggrin, loricrin (LOR), and Claudins proteins, the skin barrier and moisturizing ability become poor, and as a result, skin diseases such as atopic dermatitis can be caused. Therefore, substances that promote the synthesis of these proteins play an important role in improving the skin condition. In some embodiments, the wolfberry exosome or the composition containing the same of the present disclosure has the function of enhancing the skin barrier; or treating, preventing, or assisting (or being used in an auxiliary manner) in improving, alleviating, and controlling skin barrier damage. In some embodiments, the wolfberry exosome or the composition containing the same of the present disclosure can promote the synthesis of filaggrin. In some embodiments, the wolfberry exosome or the composition containing the same of the present disclosure can promote the synthesis of filaggrin, loricrin (LOR), and / or Claudins proteins. The wolfberry exosome or the composition containing the same of the present disclosure can promote the synthesis of filaggrin. For example, the promotion rate of profilaggrin synthesis is 30.00%-150.00%, preferably 50.00%-100.00%, and most preferably 75.00%-80.00%. The wolfberry exosome or the composition containing the same of the present disclosure can promote the synthesis of loricrin. For example, the promotion rate of loricrin synthesis is 50.00%-200.00%, preferably 80.00%-150.00%, and most preferably 125.00%-135.00%. In some embodiments, the wolfberry exosome or the composition containing the same of the present disclosure can promote the synthesis of Claudins proteins (such as CLDN1). For example, the promotion rate of Claudins protein synthesis is 50.00%-140.00%, preferably 60.00%-100.00%, and most preferably 75.00%-85.00%.

[0227] In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same have skin repair and moisturizing effects. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same can treat, prevent or contribute to (or be used in an auxiliary manner for) improving, alleviating, or controlling skin photo-damage, such as skin damage caused by ultraviolet rays. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same can reduce the production of sunburn cells. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same protect the skin and mucous membranes against all types of external assaults, such as ultraviolet radiation. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same prevent damage to the skin caused by exposure to sunlight (such as ultraviolet rays) or a dry environment. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same can improve skin tissue vitality. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same can reduce the transepidermal water loss rate of the skin after ultraviolet irradiation, or prevent the epidermal water loss rate after ultraviolet irradiation. Aquaporins are a class of transmembrane proteins that transport water and small molecules in solutions (such as glycerol and urea), and they promote water transport in epithelial and endothelial cells. The discovery of aquaporin 3, aquaporin-3 or AQP3 in human skin, especially in the plasma membrane of keratinocytes in the epidermal proliferative layer, highlights the importance of regulated water flow into the skin. AQP3 is capable of transporting water and glycerol, and the latter plays an important role in the formation of the surface water-lipid membrane, the elasticity of the stratum corneum, and the maintenance of sensory quality. It has been found that AQP3 hydration is closely related to its content in keratinocytes. Therefore, an increase in skin AQP3 improves epidermal hydration. The aquaporin described in the present disclosure is aquaporin 3 or AQP3, which generally refers to being present in the membrane of keratinocytes. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same prevent or treat or contribute to (or be used in an auxiliary manner for) improving, alleviating, or controlling diseases caused by aquaporin dysfunction in the skin and mucous membranes, such as eczema, xerosis, atopic dermatitis, or dryness of the oral cavity, eyes, or vagina. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same improve skin hydration and prevent or control dryness of the skin and mucous membranes. In some embodiments, the wolfberry exosomes of the present disclosure or a composition containing the same promote the expression of aquaporin, preferably aquaporin 3.

[0228] In some embodiments, the goji berry exosomes of the present disclosure or a composition comprising the same have a moisturizing effect. Natural Moisturizing Factor (NMF) is a hydrophilic and water-retaining substance present in skin keratinocytes, which plays an important role in skin moisturization. Natural Moisturizing Factor is a broad group composed of multiple components, including amino acids, ceramides, hyaluronic acid, cholesterol, fatty acids, triglycerides, phospholipids, glycosphingolipids, urea, linoleic acid, glycosaminoglycans, mucopolysaccharides, and sodium PCA (pyrrolidone carboxylic acid). Pyrrolidone carboxylic acid (PCA) or sodium PCA (pyrrolidone carboxylic acid) is a natural moisturizer with excellent moisture absorption properties and is present in large amounts in the stratum corneum of the skin as the most important component of natural moisturizing factor. PCA is formed by the hydrolysis of FLG by Caspase-14 and can effectively adsorb and lock in moisture, strengthening the moisture barrier. In some embodiments, the goji berry exosomes of the present disclosure or a composition comprising the same can treat, prevent, or contribute to (or be used as an adjunct for) improving, alleviating, or controlling skin dryness symptoms or related diseases thereof, wherein the skin dryness symptoms are selected from, for example, skin dryness, skin aging, skin roughness, seborrheic dermatitis, reduced skin tension and elasticity, erythema, sclerosis, keratosis, and chapping; and the diseases related to skin dryness symptoms are selected from, for example, xeroderma, senile xeroderma, ichthyosis vulgaris, infantile dry skin, atopic dermatitis, allergic dermatitis, seborrheic eczema, sensitive skin, seasonal xeroderma, aquagenic pruritus, and eczema in women. In some embodiments, the goji berry exosomes of the present disclosure or a composition comprising the same promote skin humidification or hydration. In some embodiments, the goji berry exosomes of the present disclosure or a composition comprising the same are used for skin moisturization. In some embodiments, the goji berry exosomes of the present disclosure or a composition comprising the same can increase the content of PCA.

[0229] Malondialdehyde (MDA) is produced due to the peroxidation reaction of membrane lipids in tissues or organs, and its content is closely related to aging and stress damage. UV radiation generates a large amount of reactive oxygen species (ROS), exceeding the normal defense ability of the body. While consuming superoxide dismutase (SOD) and catalase (CAT) in the skin, it attacks the polyunsaturated fatty acids in phospholipids on the biological membrane, triggering a chain reaction of membrane lipid peroxidation, ultimately leading to an increase in MDA content. Ultraviolet irradiation can induce membrane lipid peroxidation reaction, causing damage to biological membranes and DNA. Therefore, the levels of SOD and MDA in the skin determine the degree of skin damage. In some embodiments, the Lycium barbarum exosomes or compositions containing the same of the present disclosure can treat, prevent or contribute to (or be adjuvantly used for) improving, alleviating, and controlling oxidative damage of skin cells. In some embodiments, the Lycium barbarum exosomes or compositions containing the same of the present disclosure are used for skin antioxidant. In some embodiments, the Lycium barbarum exosomes or compositions containing the same of the present disclosure can reduce or alleviate ROS generated by ultraviolet irradiation. In some embodiments, the Lycium barbarum exosomes or compositions containing the same of the present disclosure can alleviate the content of lipid peroxides in the skin, such as reducing the content of MDA.

[0230] For the above methods and uses of the present disclosure, the plant exosomes or compositions containing the same can be administered orally, topically, or parenterally. In particular, topical or transdermal application can be implemented by iontophoresis, sonophoresis, electroporation, mechanical pressure, osmotic gradient, occlusion therapy, microinjection, microneedles (or microneedle arrays), needle-free injection by pressure, through a microelectrical patch, a facial mask, or any combination thereof. Among them, some techniques for creating channels in the skin (such as microneedles or lasers) themselves as a certain treatment means can be used in combination with the therapy of plant exosomes or compositions containing the same, which helps the plant exosomes or compositions containing the same to be absorbed and reach deeper skin layers, thereby enhancing their active ability. The local area of such combination treatment will be determined by the nature of the condition, disorder, and / or disease to be treated and / or addressed.

[0231] The daily dosage and administration frequency of the plant exosomes (preferably Lycium barbarum exosomes) or compositions containing the same can vary according to various factors, such as the stage of the disease to be treated, age, health status, the presence of complications, etc. On the one hand, the composition can be administered at a daily dosage of, for example, 1 μg / kg to 200 mg / kg, more specifically 50 μg / kg to 50 mg / kg, 1 - 3 times a day. However, the dosage does not limit the scope of the present disclosure in any way.

[0232] The plant exosomes of the present disclosure also have uses for treating various diseases in mammals (such as humans). In some embodiments, the plant exosomes have uses for treating or preventing diseases related to the following systems: respiratory system, digestive system, circulatory system, nervous system, endocrine system, urogenital system or musculoskeletal system. In some embodiments, the treatment or prevention of diseases related to the respiratory system includes treating COVID-19, inhibiting lung inflammation, treating pulmonary fibrosis or preventing pulmonary fibrosis and lung inflammation. In some embodiments, the treatment or prevention of diseases related to the digestive system includes promoting the recovery of colitis, treating colon cancer, treating acute and chronic colitis, treating ulcerative colitis, treating chronic periodontitis, preventing alcohol-induced liver damage, treating IBD and inhibiting the progression of CAC, preventing colitis, preventing dextran sulfate sodium-induced colitis, treating DSS-induced colitis, inhibiting liver metastasis of colon cancer, inhibiting Clostridium difficile infection, treating liver inflammation, preventing GalN / LPS-induced acute liver injury, inhibiting the proliferation of hepatocellular carcinoma cells or inhibiting the growth of tumor cells. In some embodiments, the treatment or prevention of diseases related to the circulatory system includes improving doxorubicin-induced myocardial injury, preventing damage to the vascular system by various stressors, or preventing oxidative stress of human mesenchymal stromal cells. In some embodiments, the treatment or prevention of diseases related to the nervous system includes treating glioma, treating glioblastoma, inhibiting the development of glioma or stimulating the neural differentiation of stem cells. In some embodiments, the treatment or prevention of diseases related to the endocrine system includes preventing insulin resistance and obesity, or preventing or inhibiting obesity-related gastrointestinal inflammation caused by a high-fat and high-sugar diet. In some embodiments, the treatment or prevention of diseases related to the urogenital system includes inhibiting the occurrence and development of breast cancer in mammals, or treating cervical cancer. In some embodiments, the treatment or prevention of diseases related to the musculoskeletal system includes promoting wound healing. In addition, the plant exosomes of the present disclosure can also be used as drug delivery tools. In some embodiments, the plant exosomes can be used as drug delivery tools to transport various drug molecules for treating the above diseases, such as MTX for treating colitis, PTX for treating colon cancer, and DOX for treating glioblastoma. In some embodiments, the plant exosomes can be used as delivery tools for treating plant diseases caused by fungal infections. In some embodiments, different plant exosomes have different properties, such as changes in size, charge and stability. These properties will inevitably affect the drug loading in the plant exosomes. For example, in certain embodiments, the plant exosomes used as drug delivery tools are plant exosomes with a smaller size, such as 30-80um, 30-60um or 30-50um.Due to the higher surface area to volume ratio, the plant exosomes with smaller sizes have a larger drug-loading capacity, a faster release rate, and higher stability. In some other embodiments, plant exosomes can be used as drug delivery tools to transport positively charged drug molecules. Due to the presence of phosphate, plant exosomes are usually negatively charged, and thus, under the influence of electrostatic attraction, positively charged molecules are more effectively absorbed and encapsulated by these negatively or neutrally charged plant exosomes. Examples

[0233] The following further describes the technical solutions of the present disclosure in conjunction with specific examples, but the protection scope of the present disclosure is not limited to these examples. Any changes or equivalent substitutions that do not deviate from the concept of the present disclosure are included in the protection scope of the present disclosure.

[0234] For the experimental methods and means without specific conditions noted in the following examples, they are generally carried out according to the conventional conditions of such experimental operations in the art. For the experimental equipment without specific sources and models marked in the following examples, they are all conventional equipment in the art well-known to those skilled in the art and can be routinely determined and operated by those skilled in the art. The experimental materials and reagents used in the following examples can be obtained from commercial channels without further purification and can be used directly unless otherwise specified. Unless otherwise stated, the percentages and parts are weight percentages and weight parts respectively. Unless otherwise stated, the ratio of liquids is volume ratio, and the temperatures used in the present disclosure are all in degrees Celsius (°C).

[0235] 1. Experimental methods

[0236] 1.1 Preparation of crude plant extract

[0237] Wash the plant (such as wolfberry, ginseng, dendrobium, or angelica sinensis) clean and dry it with a clean cloth. Weigh the plant to be processed. According to the mass-volume ratio (m / v, g / mL), plant mass: sodium chloride injection volume = 1:3 - 1:20, mix the weighed plant with sodium chloride injection. Use a wall breaker to crush the plant, and collect the crushed plant residue liquid into a measuring cylinder. Place the measuring cylinder on an ice medium and stir it at a stirrer speed of 200 ± 20 rpm for 30 min. Use a filter screen to pour the plant residue liquid to separate the plant residue from the juice, and collect the plant juice. Centrifuge the collected plant juice at 2,000 - 5,000 g for 10 - 35 min at 4°C. In a biosafety cabinet, discard the precipitate and collect the supernatant P1 as the crude plant extract.

[0238] 1.2 Separation and extraction of plant exosomes using the method of layer-by-layer filtration

[0239] S1 Depth filtration: In a biosafety cabinet, connect a peristaltic pump, pump tubing, and a depth capsule filter (Supracap) of about 2 - 30 μm TMDepth Filter Capsules, Pall, filtration capacity is about 50 - 200 L / m 2 ) Connect them together in combination. Rinse the depth filter capsule with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid. Filter P1 at a flow rate of 20 - 200 mL / min and collect the filtrate P2. Remove the filter, clean the pipeline with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid and remove the pump tube.

[0240] S2 0.45μm filtration: In the biosafety cabinet, connect the peristaltic pump, pump tube and 0.45μm membrane filter (PreFlow membrane filter capsules, Pall) to the peristaltic pump. Rinse the 0.45μm membrane filter with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid. Filter P2 at a flow rate of 20 - 200 mL / min and collect the filtrate P3. Remove the filter, clean the pipeline with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid and remove the pump tube.

[0241] S3 0.22μm filtration: In the biosafety cabinet, connect the peristaltic pump, pump tube and 0.22μm membrane filter (Supor TM EXGrade ECV in Mini Kleenpak TM capsules, Pall) to the peristaltic pump. Rinse the 0.22μm membrane filter with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid. Filter P3 at a flow rate of 20 - 200 mL / min to obtain plant exosomes. Remove the filter, clean the pipeline with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid and remove the pump tube.

[0242] 1.3 Separate and extract plant exosomes by ultracentrifugation

[0243] Centrifuge the collected crude plant extract P1 at 120,000 g for 70 min at 4°C using an ultracentrifuge. After centrifugation, discard the supernatant and resuspend the precipitate with sodium chloride injection. After centrifuging again at 120,000 g for 70 min at 4°C, resuspend the precipitate with sodium chloride injection to obtain plant exosomes.

[0244] 1.4 Transmission electron microscopy detection

[0245] Take 5 μL of each of the prepared plant exosome samples and drop them on a copper mesh, and incubate at room temperature for 5 min; after incubation, absorb the excess liquid on one side with absorbent paper; drop one drop of 2% uranyl acetate on the copper mesh and incubate at room temperature for 1 min; after incubation, absorb the excess liquid on one side with absorbent paper; dry at room temperature for about 20 min. Use a nano transmission electron microscope (FEI, Tecnai G2 Spirit BioTwin) to perform transmission electron microscopy (TEM) detection on the instrument.

[0246] 1.5 Nanoparticle size tracking analysis

[0247] Use nanoparticle tracking analysis technology (Nanoparticle Tracking Analysis, NTA) to measure the particle size and concentration of each of the prepared plant exosomes: Dilute the standard stock solution to a calibration mother solution 1000 times (1 μL of the standard stock solution can be prepared into 1 mL of the calibration mother solution according to the ratio). Take 100 μL of the above-prepared standard mother solution and add it to 25 mL of pure water to dilute it into a calibration solution 250,000 times. When testing the sample, generally dilute the sample 1000 times first. After calibrating the instrument (Particle Metrix, PMX120) with the calibration mother solution, inject the test sample into the sample cell with a syringe, measure the sample concentration, and repeat it three times. 1.6 In vitro labeling of plant exosomes with PKH67

[0248] Dilute 1 mM of PKH67 (green fluorescent tracer dye) 50 times to 20 μM with Diluent C (universal membrane labeling diluent). Subsequently, take 5 μL of 20 μM PKH67 and 5 μL of the above-prepared plant exosomes and mix them, and incubate at room temperature for 15 min. Use a nano flow cytometer (Flow NanoAnalyzer) to detect the positive rate of PKH67 in the plant exosomes.

[0249] 1.7 Trace protein detection

[0250] After filtering 40% SDS prepared with pure water, dilute it to 2% SDS with pure water as the standard curve diluent. According to Micro BCA TMInstructions for Protein Detection Kit (Thermo Fisher Scientific). Prepare the working reagent WR (Working Reagent, WR) by mixing reagents MA:MB:MC at a ratio of 25:24:1. Prepare standard curves of 200, 40, 20, 10, 5, 2.5, 1, 0.5, 0 μg / mL by diluting the 2.0 mg / mL BSA stock solution with a diluent. Dilute plant exosomes with a diluent, add an equal volume of WR, and incubate at 100 rpm and 37 °C with constant shaking for 2 hours. Measure the absorbance of standards and samples at 570 nm using a microplate reader, and calculate the protein concentration of the samples.

[0251] 1.8 Detection of TNF-α Inhibition Rate

[0252] RAW264.7 cells (mouse monocyte macrophage leukemia cells) were cultured in DMEM basal medium supplemented with 10% FBS for 48 h, and then seeded in a 96-well plate at a density of 1.875×10 4 / cm 2 . The cells were cultured in an incubator at 37.0 °C and 5% CO2 for 24 h. After pretreatment with plant exosomes (1×10 9 particles / mL) and dexamethasone (final concentration 1 μg / mL) for 24 h, LPS (final concentration 5 ng / mL) was added for co-treatment for 4 h. The cell supernatant was collected and centrifuged at 500 g for 5 min at 4 °C. The concentration of TNF-α was detected using a Mouse TNF-α ELISA kit (R&D systems), and the inhibition rate was calculated as follows:

[0253] Inhibition rate (%) = 1 - (average concentration of experimental group / average concentration of LPS-treated group).

[0254] 1.9 Sample Preparation for Untargeted Metabolomics Analysis of Plant Exosomes

[0255] Absorb 100 μL of the plant exosome liquid sample into a 1.5 mL centrifuge tube, add 400 μL of the extraction solution (acetonitrile:methanol = 1:1, containing 0.02 mg / mL of the internal standard L-2-chlorophenylalanine), vortex for 30 s, then perform low-temperature ultrasonic extraction for 30 min (5 °C, 40 KHz), let the sample stand at -20 °C for 30 min. Centrifuge at 4 °C, 13000 g for 15 min, transfer the supernatant, dry it with nitrogen, re-dissolve it with 100 μL of the reconstitution solution (acetonitrile:water = 1:1), perform low-temperature ultrasonic extraction for 5 min (5 °C, 40 KHz), centrifuge at 4 °C, 13000 g for 10 min, transfer the supernatant to an injection vial with an inner insert for on-machine analysis. Mix equal volumes of the metabolites of all samples to prepare a quality control sample (Quality control, QC). During the instrument analysis process, insert one QC sample into every 5 - 15 samples to examine the repeatability of the entire analysis process.

[0256] 1.10 LC-MS / MS analysis

[0257] Mix equal volumes of the metabolites of all samples to prepare a quality control sample. During the instrument analysis process, insert one QC sample into every 5 - 15 samples to examine the repeatability of the entire analysis process. The sample mass spectrometry signal acquisition adopts positive and negative ion scanning modes, and the mass scanning range is 70 - 1050 m / z. The sheath gas flow rate is 50 psi, the auxiliary gas flow rate is 13 psi, the auxiliary gas heating temperature is 425 °C, the positive mode ion spray voltage is set to 3500 V, the negative mode ion spray voltage is set to -3500 V, the ion transfer tube temperature is 325 °C, and the normalized collision energy is 20 - 40 - 60 V cyclic collision energy. The resolution of the first-stage mass spectrometry is 60000, the resolution of the second-stage mass spectrometry is 7500, and data is collected using the DDA mode.

[0258] 1.11 Substance identification and analysis

[0259] After the on-machine analysis is completed, import the LC-MS raw data into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Finally, obtain a data matrix of retention time, mass-to-charge ratio, and peak intensity. At the same time, match the MS and MSMS mass spectrometry information with the metabolic public databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) and the self-built database of Meiji to obtain metabolite information.

[0260] Upload the data matrix after database search to the Majorbio Cloud Platform (cloud.majorbio.com) for analysis. First, preprocess the data matrix as follows: The data matrix uses the 80% rule to remove missing values, that is, retain variables with non-zero values of more than 80% in at least one group of samples, and then fill in the missing values (fill in the missing values with the minimum value in the original matrix). To reduce the errors caused by sample preparation and instrument instability, use the sum normalization method to normalize the response intensity of the sample mass spectrometry peaks to obtain the normalized data matrix. At the same time, delete variables with a relative standard deviation (RSD) of > 30% in the QC samples and perform log10 logarithmic transformation to obtain the final data matrix for subsequent analysis.

[0261] Secondly, use the ropls package (Version 1.6.2) in R language to perform principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) on the preprocessed data matrix, and use 7-fold cross-validation to evaluate the stability of the model. The selection of significantly different metabolites is determined based on the variable importance in the projection (VIP) values obtained from the OPLS-DA model and the p-values of the student's t-test. Metabolites with VIP > 1 and p < 0.05 are significantly different metabolites.

[0262] Annotate the metabolic pathways of the different metabolites through the KEGG database (Kyoto Encyclopedia of Genes and Genomes, https: / / www.kegg.jp / kegg / pathway.html) to obtain the pathways involved in the different metabolites. Use the Python package scipy.stats for pathway enrichment analysis and obtain the biological pathways most relevant to the experimental treatment through Fisher's exact test.

[0263] 2. Experimental Results and Analysis

[0264] 2.1 Screening of Extraction Methods

[0265] For ginseng, wolfberry, and dendrobium, based on the above Example 1.1, perform the following different filtration treatment combinations and evaluate the final results. The specific extraction methods and corresponding results are shown in the following table:

[0266]

[0267]

[0268] Among them, "+" represents the inclusion of relevant treatments, and "-" represents the exclusion of relevant treatments.

[0269] 2.2 Extract plant exosomes (PEN) using the methods of layer-by-layer filtration and ultracentrifugation respectively

[0270] Use the layer-by-layer filtration method in Examples 1.1 - 1.2 to isolate and extract exosomes from plants (Lycium barbarum, Panax ginseng, Dendrobium officinale, Polygonatum sibiricum, Angelica sinensis, and Rehmannia glutinosa). Specifically, first, the plants (up to 1000 g) are mixed (treated with sodium chloride injection as the solvent according to the mass - volume ratio (g / mL) shown in Table 1), crushed, and stirred to promote the release of plant exosomes into the sodium chloride injection solvent. The plant residues are removed by centrifugation at 4000 g for 15 min to obtain the crude plant extract P1. Larger impurities in P1 are removed through S1 (depth filtration) to obtain P2, larger particles in P2 are pre - filtered through S2 (0.45 μm membrane filtration) to obtain P3, and finally, sterile - grade exosomes are harvested through S3 (0.22 μm membrane filtration). On average, it takes about 1 h for 500 mL of the crude plant extract P1.

[0271] Use the ultra - centrifugation method in Example 1.3 to isolate and extract exosomes from the above six plants. Specifically, the crude plant extract P1 is centrifuged twice at 12000 g for 70 min at 4°C. On average, it takes about 3 h for 500 mL.

[0272] Table 1 Name labels of six plants for separating PEN using ultra - centrifugation and layer - by - layer filtration

[0273]

[0274]

[0275] The following various tests are carried out on the exosome samples of each plant obtained from the above operations. The results show that they have the various characteristics and effects of exosomes described in the detailed description part of the above invention. As a representative example, the following gives the test results of exosomes obtained from 100 g of each type of the above - mentioned plants after the above treatment (the name labels of each group are shown in Table 1).

[0276] 2.3 Physical properties of plant exosomes

[0277] After performing NTA detection according to Example 1.5, the particle concentration, particle size, and final volume of the plant exosomes obtained in Example 2.2 above are counted.

[0278] As shown in Table 2, the particle sizes of plant exosomes isolated and extracted by the layer - by - layer filtration and ultra - centrifugation methods are both in the range of 30 - 200 nm, but the yield of plant exosomes extracted by layer - by - layer filtration is significantly higher than that of exosomes extracted by ultra - centrifugation. The results show that, in terms of both the efficiency of the exosome separation method and the final yield obtained, the layer - by - layer filtration method is significantly better than ultra - centrifugation.

[0279] Table 2 Particle size and concentration of plant exosomes

[0280]

[0281] 2.4 Qualitative Metabolomics Analysis of Plant Exosomes

[0282] 2.4.1 Analysis of Specific Metabolites

[0283] The plant metabolomics analysis carried out according to Examples 1.9 to 1.11 showed that in terms of the number of metabolites identified in the six plant exosomes obtained in Example 2.2, layer-by-layer filtration was higher than ultracentrifugation in all cases. Specifically, as shown in Table 3. After Venn diagram analysis, the specific metabolites of layer-by-layer filtration were also higher than those of ultracentrifugation.

[0284] Table 3 Statistical Table of Venn Analysis of Exosomes Extracted from Six Plants Using Layer-by-Layer Filtration and Ultracentrifugation

[0285]

[0286] 2.4.2 Compound Classification Analysis

[0287] Compound classification analysis classifies metabolites into sugars, amino acids, organic acids, lipids, etc. according to their structures and properties. Classified according to the HDMB compound classification hierarchy Superclass, the metabolites in the six plant exosomes were mainly concentrated in six categories, namely lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzenoid compounds. As shown in Figure 2-3.

[0288] · The additional metabolite classifications of GQV01 compared to LyE01 were phenylpropanoids and polyketides (44), organic oxygen compounds (41),

[0289] lipids and lipid-like molecules (34), organic acids and their derivatives (18), lignans, neolignans and related compounds (18), and organic heterocyclic compounds (16);

[0290] · The additional metabolite classifications of RSV01 compared to GsE01 were phenylpropanoids and polyketides (73), lipids and lipid-like molecules (60

[0291] ), organic oxygen compounds (54), organic heterocyclic compounds (22), organic acids and their derivatives (16), and lignans, neolignans and related compounds (16);

[0292] · The additional metabolite classifications of SHV01 compared to DeE01 were phenylpropanoids and polyketides (57), lipids and lipid-like molecules (46

[0293] ), organic oxygen compounds (33), organic heterocyclic compounds (20), and organic acids and their derivatives (10);

[0294] · The metabolites that HJV01 has more than PoE01 are classified into lipids and lipid-like molecules (108), phenylpropanoids and polyketides (76

[0295] ), organic oxygen compounds (72), organic heterocyclic compounds (35), organic acids and their derivatives (25), lignans, neolignans and related compounds (23), benzene compounds (13);

[0296] · The metabolites that DGV01 has more than AnE01 are classified into lipids and lipid-like molecules (129), organic oxygen compounds (94

[0297] ), phenylpropanoids and polyketides (96), organic heterocyclic compounds (30), organic acids and their derivatives (30), lignans, neolignans and related compounds (17), benzene compounds (12);

[0298] · The metabolites that DHV01 has more than ReE01 are classified into lipids and lipid-like molecules (135), phenylpropanoids and polyketides (115

[0299] ), organic oxygen compounds (104), organic heterocyclic compounds (49), organic acids and their derivatives (42), lignans, neolignans and related compounds (16), benzene compounds (13).

[0300] 2.4.3 Lipid classification analysis

[0301] Lipids and lipid-like molecules rank first in the compound classification. The lipid classification mainly includes fatty acyl (FA), glycerolipids (GL), glycerophospholipids (GP), prenol lipids (PR), sphingolipids (SP), steroids (ST) and saccharolipids (SP).

[0302] The lipids and lipid-like molecules of exosomes from six plants were classified and counted. As shown in Table 4, the number of lipid types of plant exosomes extracted by layer-by-layer filtration is more than that by ultracentrifugation. The ones with relatively large content differences between GQV01 and LyE01, HJV01 and PoE01, DGV01 and AnE01, and DHV01 and ReE01 are FA, PR and ST respectively; the ones with relatively large content differences between RSV01 and GsE01 and between SHV01 and DeE01 are FA and PR respectively.

[0303] Table 4 Comparison of lipid classification of exosomes from six plants

[0304]

[0305] 2.5 Analysis of differential metabolites between plant exosomes

[0306] Univariate statistical analysis (t-test) combined with multivariate statistical analysis (OPLS-DA / PLS-DA) and fold change value (FC) were used to jointly screen for differential metabolites. The screening criteria were P < 0.05, VIP > 1, and (FC < 1 or FC > 1, FC was not screened by default) [6].

[0307] As Figure 4 shown, there were a total of 617 significantly differential metabolites in GQV01 compared with LyE01, including 401 up-regulated metabolites and 216 down-regulated metabolites; there were a total of 705 significantly differential metabolites in RSV01 compared with GsE01, including 527 up-regulated metabolites and 178 down-regulated metabolites; there were a total of 770 significantly differential metabolites in SHV01 compared with DeE01, including 537 up-regulated metabolites and 233 down-regulated metabolites; there were a total of 652 significantly differential metabolites in HJV01 compared with PoE01, including 546 up-regulated metabolites and 106 down-regulated metabolites; there were a total of 747 significantly differential metabolites in DGV01 compared with AnE01, including 659 up-regulated metabolites and 88 down-regulated metabolites; there were a total of 777 significantly differential metabolites in DHV01 compared with ReE01, including 657 up-regulated metabolites and 120 down-regulated metabolites.

[0308] 2.6 Analysis of differential metabolic pathways between plant exosomes

[0309] Generally, multiple metabolites with interrelated functions constitute a metabolic pathway, and the cumulative expression differences of multiple metabolites in the metabolic pathway constitute the expression variation of the entire metabolic pathway. If the proportion of a certain pathway involved in differential metabolism is much greater than the proportion of this pathway involved in background metabolites, it is considered that this experimental treatment is related to the change of this metabolic pathway. During enrichment analysis, based on the KEGG database, pathways with significant enrichment were screened out according to the enrichment P value less than 0.05.

[0310] There are 73 metabolic pathways related to differential metabolites between GQV01 and LyE01, and a total of 11 pathways are significantly enriched. There are 78 metabolic pathways related to differential metabolites between RSV01 and GsE01, and a total of 25 pathways are significantly enriched. There are 73 metabolic pathways related to differential metabolites between SHV01 and DeE01, and a total of 20 pathways are significantly enriched. There are 82 metabolic pathways related to differential metabolites between HJV01 and PoE01, and a total of 27 pathways are significantly enriched. There are 81 metabolic pathways related to differential metabolites between DGV01 and AnE01, and a total of 28 pathways are significantly enriched. There are 80 metabolic pathways related to differential metabolites between DHV01 and ReE01, and a total of 21 pathways are significantly enriched.

[0311] As shown in Figure 5, compared with ultracentrifugation, the differential metabolites of plant exosomes extracted by layer-by-layer filtration are mainly upregulated in Metabolism, and the main pathways are mainly tryptophan metabolism, arachidonic acid metabolism, phenylpropanoid biosynthesis, starch and sucrose metabolism; alanine, aspartate and glutamate metabolism; galactose metabolism, ABC transporters, valine, leucine and isoleucine biosynthesis; biosynthesis of cutin, suberin and wax; linoleic acid metabolism, β-alanine metabolism, nucleotide metabolism, histidine metabolism, aminoacyl-tRNA biosynthesis, arginine biosynthesis, biosynthesis of various secondary metabolites.

[0312] 2.7 TEM identification of plant exosomes separated by layer-by-layer filtration

[0313] Through the above analysis of the particle concentration, particle size, yield and plant metabolomics of plant exosomes, it shows that the extraction method of layer-by-layer filtration is superior to ultracentrifugation, and higher-yield plant exosomes can be extracted in less time (equivalent to at least 40 times the yield of exosomes extracted by ultracentrifugation). Therefore, we further perform TEM on the plant exosomes extracted by layer-by-layer filtration.

[0314] The results are as Figure 6 shown. The exosomes of six plants (wolfberry, ginseng, Dendrobium officinale, polygonatum, angelica, rehmannia) all have a typical saucer-like shape of exosomes, and the particle size is in the range of 30 - 200 nm, which meets the definition of extracellular vesicles by the International Society for Extracellular Vesicles (ISEV).

[0315] Specifically for wolfberry exosomes, the results are shown in Figure 7. It has a typical saucer-like shape of exosomes ( Figure 7A ), and the particle size is in the range of 30 - 200 nm (see Figure 7B ), which meets the definition of extracellular vesicles by the International Society for Extracellular Vesicles (ISEV).

[0316] 2.8 PKH67 detection of plant exosomes separated by layer-by-layer filtration

[0317] The plant exosomes prepared in Example 2.2 above were stained in vitro with the lipophilic dye PKH67, and the PKH67-positive particles were detected and analyzed by a nano-flow cytometer.

[0318] As Figure 8A shown, the positive rates of PKH67 for GQV01, RSV01, SHV01, HJV01, DGV01, and DHV01 were 64.5%, 83.3%, 97.4%, 79.9%, 44.0%, and 34.7%, respectively. Specifically for wolfberry exosomes, as Figure 8B the results showed, the positive rate of GQV01 was 68.8%.

[0319] 2.9 Detection of protein content of plant exosomes obtained by sequential filtration

[0320] The protein content of plant exosomes was detected, and it was found that the six plant vesicles were rich in protein. Specifically, as Figure 9 shown in A, the protein content of GQV01 was the highest at 47434.90 μg / mL, and the protein amounts of other plant exosomes were 5862.76 μg / mL for DGV01, 2816.93 μg / mL for DHV01, 2521.15 μg / mL for RSV01, 1940.16 μg / mL for HJV01, and 875.89 μg / mL for SHV01.

[0321] 2.10 Detection of anti-inflammatory activity of plant exosomes obtained by sequential filtration

[0322] The efficacy of the plant exosomes extracted by sequential filtration was detected, mainly by observing the inhibition rate of LPS-induced release of TNF-α from RAW264.7 for evaluation.

[0323] As Figure 9 shown in B, the TNF-α inhibition rate of the positive control group with 1 μg / mL dexamethasone was 91.41%. When adding plant exosomes with a particle concentration of 1×10 9 / mL, the TNF-α inhibition rate of GQV01 was 90.19% ( Figure 9 C), the TNF-α inhibition rate of RSV01 was 86.98%, the TNF-α inhibition rate of SHV01 was 64.40%, the TNF-α inhibition rate of HJV01 was 98.73%, the TNF-α inhibition rate of DGV01 was 87.84%, and the TNF-α inhibition rate of DHV01 was 79.35%.

[0324] The results showed that the plant exosomes extracted by sequential filtration had satisfactory anti-inflammatory and immunomodulatory activities.

[0325] 2.11 Amplification Process

[0326] According to the layer-by-layer filtration method in Examples 1.1 - 1.2, 1000 g of wolfberries, 1000 g of ginseng, and 500 g of Dendrobium officinale were processed to separate and extract exosomes, and verify whether layer-by-layer filtration can process plant raw materials with a mass of about 1000 g or even more. After performing NTA detection according to Example 1.5, the particle concentration, particle size, and final volume of the obtained plant exosomes were counted.

[0327] As shown in Table 5, after amplifying the 100 g of plant raw materials for obtaining exosomes in Example 2.3 by 5 - 10 times (wolfberries and ginseng were amplified 10 times, and Dendrobium was amplified 5 times), about 5000 mL of plant crude extract can be processed by increasing the membrane area in the layer-by-layer filtration method. The particle sizes of the separated and extracted plant exosomes are all within the range of 30 - 200 nm, and the final harvested exosome yield is also considerable.

[0328] Table 5 Particle Sizes and Concentrations of Plant Exosomes after Amplifying the Layer-by-Layer Filtration Process by 5 - 10 Times

[0329]

[0330]

[0331] 2.12 Cosmetic Efficacy Analysis of Representative Metabolites of Wolfberry Exosomes

[0332] Non-targeted metabolomics was used to analyze the metabolites of GQ. A total of 1647 metabolites were identified in GQ. Compound classification analysis classifies metabolites into sugars, amino acids, organic acids, lipids, etc. according to their structures and properties. Comprehensive metabolic databases such as KEGG and HMDB commonly used have metabolite classification information, which can achieve quantitative classification analysis. Most compounds of the same type have similar physicochemical properties or biological functions, facilitating data mining and the elaboration of biological significance.

[0333] After analysis, the top 100 metabolites of GQ total metabolites were screened according to the average content, and the cosmetic efficacy analysis of the metabolites was carried out according to compound classification (https: / / pubmed.ncbi.nlm.nih.gov / ). The results are shown in Table 6. Among them, there are 17 metabolites related to the skin, which are classified as carboxylic acids and their derivatives, flavonoids, organic oxygen compounds, cinnamic acid and its derivatives, coumarin and its derivatives, indole and its derivatives, keto acids and their derivatives, hydroxy acids and their derivatives, and dihydrofuran according to the classification. The possible effects in cosmetics are mainly anti-inflammatory, whitening, promoting collagen synthesis, and repairing the skin barrier

[15] -

[22] .

[0334] Table 6 Efficacy of Representative Metabolites of GQ in Cosmetics

[0335]

[0336] 2.13. Repair and moisturizing effects of GQ on 3D epidermal skin models

[0337] Long-term exposure of the skin to ultraviolet light may lead to skin erythema, swelling, thickening, melanin deposition, cell sunburn, etc. We used UVB irradiation (600 mJ / cm 2 ) to simulate the damage of UV irradiation to the skin, and then performed slicing and H&E staining for histological morphology observation.

[0338] This study used a 3D epidermal skin model Lot number: ES230501, provided by Guangdong Boxi Biotechnology Co., Ltd. The administration concentration of wolfberry exosomes was 1.33×10^8 particles / mL, the administration volume was 0.9 mL, and the administration dose was 1.197×10^8 particles. According to the test grouping, the groups that needed to be irradiated with UVB were irradiated with UVB (irradiation dose: 600 mJ / cm 2 ). After irradiating for 3 min 36 s, the model was placed in a CO2 incubator (37°C, 5% CO2) and incubated for another 24 h. After incubation, the residual test substance on the surface of the model was washed with sterile PBS, and the residual liquid inside and outside the model was wiped off with a sterile cotton swab.

[0339] After UVB irradiation, sunburn cells appeared in the epidermal model( Figure 10 A). By counting the number of sunburn cells, it can be seen that after treatment with GQ, the skin can resist sunburn caused by ultraviolet rays, and GQ has excellent repair effects( Figure 10 A - B). At the same time, we also detected the viability of the skin tissue( Figure 10 C) and the trans-epidermal water loss rate (TEWL; Figure 10 D). The experimental results show that GQ has the ability to relieve the damage of the skin tissue after stimulation and has a repair function on the skin barrier( Figure 10 C), and can prevent the epidermal water loss rate after UVB irradiation( Figure 10 D).

[0340] 2.14 Regulation of GQ on the expression of skin barrier proteins

[0341] To further study the repair effect of GQ on the skin barrier, we sliced the 3D epidermal skin model and performed immunohistochemical detection on skin barrier proteins and integrated optical density (IOD) to count the content of each protein( Figure 11) Among them, filaggrin (FLG), loricrin (LOR), and Claudins are key components in the assembly process of the cornified envelope and the formation of tight junction structures. We specifically performed immunohistochemical detection on the three proteins ( Figure 11 A), and separately counted the expression levels of the three barrier proteins ( Figure 11 B). The results showed that in the epidermal model irradiated with UVB, GQ treatment significantly increased the expression levels of FLG, LOR, and CLDN1, with the increase rates being 77.50%, 130.00%, and 80.00% respectively.

[0342] 2.15 Effect of GQ on skin moisturization

[0343] Regarding the research on moisturization, we detected the expression of aquaporins and moisturizing factors in the epidermal model through experiments. We specifically detected the expression of aquaporin 3 (AQP3) in the epidermal model by immunofluorescence. The results showed that GQ could significantly up-regulate the expression of AQP3 in the skin after the skin was exposed to external stimuli ( Figure 12 A - B). In addition, we also detected the expression of the natural moisturizing factor PCA by HPLC ( Figure 12 C). PCA is formed by the hydrolysis of FLG by Caspase-14, which can effectively adsorb and lock in moisture and strengthen the moisturizing barrier. GQ can significantly increase the content of PCA in the epidermis, and its effect is better than that of the positive control (WY14643) and the unstimulated normal control group (con), indicating its excellent moisturizing effect.

[0344] 2.16 Mitigation effect of GQ on lipid peroxidation

[0345] Lipid peroxide (MDA) is also an important detection index for cell oxidative damage. Through detection by high performance liquid chromatography (HPLC), we found that the content of malondialdehyde in the 3D epidermal model increased significantly after ultraviolet irradiation, while the lipid peroxidation in the GQ treatment group was significantly alleviated ( Figure 13 ).

[0346] 2.17 Effect of plant exosome composition containing GQ on skin barrier

[0347] (1) Subsequently, we pretreated cells for 24 h with four kinds of plant exosomes, namely Lycium barbarum exosomes (GQ), Angelica sinensis exosomes (DG), Panax ginseng exosomes (RS), and Dendrobium officinale exosomes (SH), which were separated by the layer-by-layer filtration method in Examples 1.1 - 1.2, and their mixtures. The ratios of the number of exosome particles of GQ:DG:RS:SH in mix1, mix2, and mix3 were 1:1:1:1, 3:1:1:3, and 1:1:3:3, respectively. And the total concentration of exosomes in the GQ, DG, RS, SH, mix1, mix2, or mix3 groups was 1x10 8 per ml, and the concentration of each component in each mix was converted according to the displayed ratio; the positive drug group pretreated cells with 5 μM ceramide for 24 h; WB was performed to detect the expression of the tight junction protein Claudin 1 (as Figure 14 ).

[0348] The results showed that all four kinds of plant exosomes and exosome mixtures with different ratios could repair the downregulation of the skin tight junction protein Claudin 1 induced by UVB to a certain extent, and the repair ability was RS > mix3 > DG > GQ > mix2 > mix1 > SH( Figure 14 A - B). Among them, the repair abilities of RS, DG, and GQ and the exosome mixture with a ratio of 1:1:3:3 on the skin tight junction protein Claudin 1 were all better than the positive control drug ceramide (5 μM). However, surprisingly, the skin barrier repair effects of the three different ratios of plant exosome mixtures did not exceed those of the single exosomes, indicating that there are interactions between the active components of exosomes, weakening the skin barrier repair effect of the plant exosome mixtures.

[0349] (2) We changed to compound the above three kinds of plant exosomes with a ratio of 1:1:1, and other conditions remained unchanged, where mix1 = GQ:DG:RS, mix2 = GQ:DG:SH, mix3 = GQ:RS:SH, and mix4 = DG:RS:SH.

[0350] The results showed that all experimental groups could repair the downregulation of the skin tight junction protein Claudin 1 induced by UVB to a certain extent, and the repair ability was SH > mix1 > RS > GQ > mix2 > DG > mix3 > mix4. Among them, the repair abilities of GQ, DG, RS, SH, and mix1, mix2, and mix3 on the skin tight junction protein Claudin 1 were all better than the positive control( Figure 15 A - B). In addition, surprisingly, the repair ability of mix1 (GQ:DG:RS = 1:1:1) on the skin tight junction protein Claudin 1 exceeded that of the single GQ exosomes, DG exosomes, and RS exosomes. Therefore, this compounding will be further verified later.

[0351] (3) We further selected two plant exosome mixtures for compounding, with a ratio of 1:1 for both, and other conditions remained unchanged, where mix1 = GQ:DG, mix2 = GQ:RS, and mix3 = DG:RS.

[0352] The results showed that, surprisingly, after two rounds of experiments, we obtained mix1 (GQ:DG = 1:1, Figure 16 A-B) and mix2 (GQ:RS = 1:1, Figure 16 A-B), and the repair ability of these two compounding conditions for the skin barrier protein Claudin 1 was better than that of the corresponding monomeric plant exosomes. These results indicate that the combination of wolfberry exosomes and angelica exosomes has a stronger effect on the skin barrier than the corresponding monomeric plant exosomes.

[0353] Study on the ratio of GQ+DG (Compound 1) and GQ+RS (Compound 2) plant exosome compositions with skin repair effects starting from 2.18

[0354] Combined with Figures 15 - 16 the experimental results, we adjusted the ratios of the two exosome mixtures of GQ+DG and GQ+RS to 1:1, 1:3, and 3:1, and other conditions remained unchanged (the same as in Example 2.17) to determine the barrier repair efficacy of the two exosome mixtures at different ratios.

[0355] The results showed that, surprisingly, we found that when GQ:DG ( Figure 17 A-B) and GQ:RS were 1:1 ( Figure 17 C-D), their repair ability for the skin barrier protein Claudin 1 was the strongest. Therefore, we repeated the experiment on the expression of the barrier protein Claudin 1 induced by UVB with the two plant exosome mixtures with a ratio of 1:1 for many times.

[0356] Summarizing the results of four experiments, when GQ:DG was 1:1 (where the total concentration of the GQ:DG composition was 1x10 8 per ml), the average repair rate could reach 174.3%, higher than the average repair rates of GQ and DG monomers (where the total concentration of GQ was 1x10 8 per ml and the total concentration of DG was 1x10 8 per ml) (Table 7). The compounding groups performed better than the positive control group, and also better than GQ and DG monomers respectively; when GQ:RS was 1:1, the average repair rate could reach 107.6%, higher than the average repair rates of GQ and RS monomers (Table 8). The compounding groups performed better than the positive control group, and also better than GQ and RS monomers respectively.

[0357] Summary of the repair rate results of Lycium barbarum and Angelica sinensis plant exosomes and their mixtures in repairing the down-regulation of the barrier protein Claudin 1 induced by UVB

[0358]

[0359] Table 8 Summary of the repair rate results of Lycium barbarum and Panax ginseng plant exosomes and their mixtures in repairing the down-regulation of the barrier protein Claudin 1 induced by UVB

[0360]

[0361] 2.19 Research on the composition ratio of GQ+DG and GQ+RS plant exosome compositions with skin antioxidant effects (compound, efficacy two)

[0362] During the research process, we found that the mixtures of GQ+DG ( Figure 18 ) and GQ+RS ( Figure 19 ), prepared in the same manner as the previous examples, showed strong antioxidant ability and could significantly reduce the ROS generated in HaCaT cells after UVB irradiation. We detected different compounded exosome particle number ratios, including 1:1, 1:2, 1:3, 1:4 and 2:1, 3:1, 4:1, and the total concentration of exosomes in each composition group of GQ, DG, RS, SH was 1×10 6 per mL, and the concentration of each component in each composition was converted according to the displayed ratio.

[0363] The results showed that all compounded ratios could significantly inhibit the production of ROS induced by UVB. Among them, the antioxidant effects of the treatment groups with GQ:RS = 1:1 ( Figure 18 B) and GQ:DG = 1:1 ( Figure 19 B) were the most significant and were superior to other condition groups. This result was consistent with the experimental group of skin barrier repair efficacy.

[0364] In summary, the above examples illustrate that within the scope of this study, GQ+DG and GQ+RS achieve the best skin care effects under the condition of a 1:1 ratio of mixed exosome particle numbers.

[0365] References

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Claims

1. A method for separating and extracting plant exosomes, specifically a method for separating and extracting plant exosomes by deep filtration, the method comprising the following steps: (a) obtaining plant material; (b) pre-treating the obtained plant material to obtain a crude plant extract; and (c) filtering the obtained crude plant extract layer by layer, Wherein, in step (c), the layer-by-layer filtration process includes a deep filtration step and an optional membrane filtration step.

2. The method according to claim 1, wherein in step (c), at least one membrane filtration step is performed after the deep filtration step; preferably, the membrane filtration includes anti-clogging membrane filtration, or further includes sterilizing membrane filtration; more preferably, the membrane filtration consists of anti-clogging membrane filtration and sterilizing membrane filtration performed sequentially.

3. The method according to claim 2, wherein the pore size of the anti-clogging membrane filtration is about 0.4-0.8 μm, preferably about 0.45-0.8 μm, more preferably about 0.45 μm; the pore size of the sterilization membrane filtration is about 0.1-0.3 μm, preferably about 0.1-0.22 μm, more preferably about 0.22 μm.

4. The method according to any one of claims 1 to 3, wherein in step (c), the deep filtration adopts a deep filtration with a pore size of 1-50 μm, preferably a deep filtration with a pore size of 2-30 μm, and more preferably a deep filtration with a pore size of 6-30 μm.

5. The method according to any one of claims 1 to 4, wherein in step (c), the depth filtration capacity is about 50-200 L / m 2 , the filtration flow rate is about 100-1000LMH; and / or the deep filtration step is performed using a deep capsule filter, preferably a deep capsule filter with a double-layer membrane structure.

6. The method according to any one of claims 1 to 5, wherein the plant material is selected from wolfberry, angelica, ginseng and / or dendrobium.

7. The method of any one of claims 1-6, wherein the method can process plant material weighing at least about 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9000 g, 10000 g, 50000 g, or 100000 g; and / or the method improves yield by at least about 40 times compared to an ultracentrifugation method.

8. Plant exosomes prepared by the method according to any one of claims 1 to 7, or derived from wolfberry, angelica, ginseng and / or dendrobium.

9. The plant exosomes of claim 8, which have more metabolites in one or more of the following aspects compared to the plant exosomes prepared by ultracentrifugation method: lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, organic heterocyclic compounds, organic acids and their derivatives and / or benzene compounds.

10. A composition comprising Lycium barbarum exosomes; optionally, the composition further comprises ginseng exosomes and / or Angelica sinensis exosomes; preferably, the composition does not contain Dendrobium officinale exosomes.

11. A composition comprising any one, any two, any three, or four of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, and angelica exosomes.

12. The composition of claim 10 or 11, wherein the composition comprises wolfberry exosomes and ginseng exosomes.

13. The composition of claim 10 or 11, wherein the composition comprises Lycium barbarum exosomes and Angelica sinensis exosomes. The composition of claim 10 or 11 , wherein the composition comprises wolfberry exosomes, angelica exosomes, and ginseng exosomes.

15. The composition of claim 10 or 11, wherein at least one of the wolfberry exosomes, ginseng exosomes, dendrobium exosomes and angelica exosomes is prepared by the method of any one of claims 1 to 7, or is derived from wolfberry, ginseng, dendrobium, and angelica, respectively.

16. The composition of claim 10 or 11, comprising Lycium barbarum exosomes, ginseng exosomes, dendrobium officinale exosomes and angelica exosomes, wherein the particle number ratio between any two exosomes is in the range of about 1:10 10 -10 10 :1, for example, about 1:10 9 -10 9 :1, 1:10 8 -10 8 :1, 1:10 7 -10 7 :1, 1:10 6 -10 6 :1, 1:10 5 -10 5 :1, 1:10 4 -10 4 :1, 1:10 3 -10 3 :1, 1:10 2 -10 2 :1, 1:10-10:1, 1:9-9:1, 1:8-8:1, 1:7-7:1, 1:6-6:1, 1:5-5:1, 1:4-4:1, 1:3-3:1, 1:2-2:1 or 1:

1.

17. The composition of claim 10 or 11, comprising wolfberry exosomes and angelica exosomes, wherein the ratio of the number of wolfberry exosome particles to the number of angelica exosome particles is about 10 10 :1,10 9 :1,10 8 :1,10 7 :1,10 6 :1,10 5 :1,10 4 :1,10 3 :1,10 2 :1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1:10 8 , 1:10 9 , 1:10 10 .

18. The composition of claim 17, wherein the ratio of the number of wolfberry exosome particles to the number of angelica exosome particles ranges from about at least 1:1, preferably the ratio of the number of wolfberry exosome particles to the number of angelica exosome particles is about 1:

1.

19. The composition of claim 10 or 11, comprising wolfberry exosomes and ginseng exosomes, wherein the ratio of the number of wolfberry exosome particles to the number of ginseng exosome particles is about 10 10 :1,10 9 :1,10 8 :1,10 7 :1,10 6 :1,10 5 :1,10 4 :1,10 3 :1,10 2 :1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1:10 8 , 1:10 9 , 1:10 10 .

20. The composition of claim 19, wherein the ratio of the number of particles of wolfberry exosomes to the number of particles of ginseng exosomes is in the range of at least about 1:

1.

21. The composition of claim 19, wherein The ratio of the number of wolfberry exosome particles to the number of ginseng exosome particles was approximately 1:

1.

22. The composition of claim 10 or 11, comprising wolfberry exosomes and ginseng exosomes, wherein the composition has an improved skin repair effect or antioxidant effect compared to the exosomes of each component, preferably the particle number ratio of wolfberry exosomes to ginseng exosomes is about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, for example 1:

1.

23. The composition of claim 10 or 11, comprising wolfberry exosomes and angelica exosomes, wherein the composition has an improved skin repair effect or antioxidant effect compared to the exosomes of each component, preferably the particle number ratio of wolfberry exosomes to angelica exosomes is about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, for example 1:

1. 24 . The composition of claim 10 or 11 , comprising wolfberry exosomes, angelica exosomes, and ginseng exosomes, and a particle number ratio among the wolfberry exosomes, angelica exosomes, and ginseng exosomes is about 1:1:

1.

25. A plant exosome composition comprising the plant exosomes of claim 8 or 9 or the composition of claims 10-24, and optionally a suitable excipient or carrier.

26. The plant exosome composition of claim 25, which is a pharmaceutical composition, a food composition, a beauty or cosmetic composition, or a nutritional health composition.

27. Use of the plant exosomes of claim 8 or 9, the composition of claims 10-24 or the plant exosome composition of claim 25 or 26 in the preparation of medicines, foods, nutritional supplements or beauty or cosmetics, wherein the product is used to treat or prevent inflammation, preferably skin inflammation, or helps or assists in improving, relieving or controlling inflammation, preferably skin inflammation, or is used or assists in enhancing skin barrier function, repairing, anti-inflammatory, anti-oxidant, anti-aging, whitening or moisturizing in the field of dermatology.

28. The use of claim 27, wherein the medicine, food, nutritional supplement or beauty or cosmetic is used to enhance skin barrier function, such as increasing the synthesis of filaggrin, loricrin and / or CLDN1 protein; for skin repair, such as reducing the production of sunburn cells, improving skin tissue vitality and / or reducing the transepidermal water loss rate of the skin; for skin moisturizing, such as promoting the expression of aquaporin 3 and / or increasing the PCA content; and / or for skin anti-oxidation, such as reducing the MDA content.

29. Use of the plant exosomes of claim 8 or 9, the composition of claims 10-24, or the plant exosome composition of claim 25 or 26 for treating or preventing inflammation, preferably skin inflammation, or for helping or assisting in improving, alleviating or controlling inflammation, preferably skin inflammation, or for or assisting in enhancing skin barrier function, repairing, anti-inflammatory, anti-oxidant, anti-aging, whitening or moisturizing in the field of dermatology.

30. The use of claim 29, wherein the plant exosomes or plant exosome composition is used to enhance skin barrier function, such as increasing the synthesis of filaggrin, loricrin and / or CLDN1 protein; for skin repair, such as reducing the production of sunburn cells, improving skin tissue vitality and / or reducing the transepidermal water loss rate of the skin; for skin moisturizing, such as promoting the expression of aquaporin 3 and / or increasing the PCA content; and / or for skin anti-oxidation, such as reducing the MDA content.

31. A method for treating or preventing inflammation, preferably skin inflammation, comprising administering the plant exosomes of claim 8 or 9, the composition of claims 10-24, or the plant exosome composition of claim 25 or 26 to a subject in need thereof.

32. A method for enhancing skin barrier function, repairing, anti-inflammatory, anti-oxidant, anti-aging, whitening or moisturizing in dermatology, comprising applying the beauty or cosmetic composition and / or nutritional health care composition of claim 11 to a subject in need thereof.

33. A method for improving, alleviating or controlling dermatological symptoms, comprising administering the plant exosomes of claim 8 or 9, the composition of claims 10-24, or the plant exosome composition of claim 25 or 26 to a subject in need thereof.

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