Lycium barbarum exosome as well as preparation method and application thereof

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, and to the plant exosomes extracted by this method and their applications. Specifically, the method for preparing plant exosomes of the present disclosure includes a stepwise filtration step, and the stepwise filtration step sequentially includes depth filtration and membrane filtration. Background Art

[0002] Exosomes are a type of nanoparticle secreted by cells with a phospholipid bilayer, usually 30 - 200 nm in diameter, and contain abundant proteins, nucleic acid substances, etc. Exosomes can transmit active substances such as proteins, mRNAs, and microRNAs between cells and participate in many important physiological and pathological processes, and their unique roles 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, the material sources for obtaining exosomes from sources such as cell culture supernatants are limited, the cost is high, the content of exosomes obtained is low, and the cost after purification is even higher, 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 the analysis of exosomes 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 the operation and the 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 is complicated in operation, high in technical difficulty, time-consuming, the separated exosomes may be damaged by high-speed centrifugation, and contain a large amount of contaminating proteins; the exosomes extracted by polymer precipitation lack specificity and selectivity and have low purity; the technical difficulty of gel exclusion is relatively high, but it is difficult to ensure that the separated exosomes are sterile, time-consuming and 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 derived from mammals are isolated from biological fluids, while exosomes derived from plants are isolated from apoplast washings. For mammalian-derived exosomes, current isolation and purification techniques mainly include microfluidic techniques, ultracentrifugation methods, size-based separation techniques, 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 the physiological differences between plants and animals, exosomes derived from plants obtained by this method are often contaminated with proteins, nucleic acid aggregates, and other vesicles, thus requiring further purification using density gradient ultracentrifugation 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, the isolation of plant-derived exosomes is not ideal. 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 mammalian exosome extraction are not applicable to plant exosome extraction.

[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 defects and depends 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 the internal tissues and fluids from the external environment. [8] Exposure to ultraviolet rays can damage the stratum corneum of the skin, resulting in a decrease in the expression of intracellular barrier proteins, thereby impairing the skin barrier function. [9]Meanwhile, the skin is also a target organ for ultraviolet-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 resisting skin photoaging and skin diseases.

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

[11] It contains rich nutritional components, including carotenoids, vitamin A, calcium, and iron. Generally, 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, especially goji berry exosomes, at the industrial level is needed to reduce the production cost and increase the yield of exosomes, while 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 characteristics 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 separating and extracting 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 exosome yield or particle concentration, can prevent filter membrane blockage caused by 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 material 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 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 one 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.

[0030] On the other hand, the present disclosure provides a plant exosome (preferably wolfberry exosome), which is prepared by the method embodiment generally or preferably defined above; preferably, the exosome has a diameter of about 30-450 nm, preferably a diameter of about 30-250 nm, more preferably a diameter of about 30-200 nm; 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 wolfberry exosomes prepared by the method of the present disclosure; and an optional pharmaceutically acceptable carrier.

[0033] In a specific embodiment, the exosome composition is a food composition comprising plant exosomes prepared by the method 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 wolfberry exosomes prepared by the method of the present disclosure, and optionally other food ingredients and / or edible excipients and / or carriers.

[0034] 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 a beauty / cosmetic acceptable excipient or carrier. In a specific embodiment, the beauty composition comprises wolfberry exosomes prepared by the methods of the present disclosure, and at least one beauty / cosmetic acceptable excipient or carrier.

[0035] 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 a nutraceutical acceptable excipient or carrier. In a specific embodiment, the nutraceutical composition comprises wolfberry exosomes prepared by the methods of the present disclosure, and at least one nutraceutical acceptable excipient or carrier.

[0036] 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 a composition comprising 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.

[0037] 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 a composition comprising the same (including but not limited to pharmaceutical compositions / food compositions / beauty compositions / nutraceutical compositions) in the preparation of a product (drug, food, nutraceutical, beauty / cosmetic, 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.

[0038] 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 comprising the same (pharmaceutical composition / food composition / beauty or cosmetic composition / nutraceutical composition).

[0039] On the other hand, the present disclosure provides the use of the above-mentioned plant exosomes (preferably wolfberry exosomes) or plant exosome compositions in a biomedical or drug delivery system.

[0040] On the other hand, the present disclosure also provides methods for preparing pharmaceutical, food, nutritional health product, beauty / cosmetic compositions, including 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, food, nutritional health product, beauty / cosmetic compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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 figures. However, it should be understood that the present disclosure is not limited to the specific embodiments shown in the figures.

[0042] Figure 1 An exemplary step flow showing the method for isolating and extracting plant exosomes of the present disclosure.

[0043] Figure 2 Compound classification of wolfberry exosomes extracted by the layer-by-layer filtration method of the present disclosure: GQV01.

[0044] Figure 3 Compound classification of wolfberry exosomes extracted by the ultracentrifugation method: LyE01.

[0045] Figure 4 Volcano plot of differential metabolites of wolfberry exosomes extracted by layer-by-layer filtration and ultracentrifugation: GQV01 vs LyE01. The abscissa is the fold change value of the metabolite expression difference between the two groups, i.e., log2FC; the ordinate is the statistical test value of the metabolite expression change difference, i.e., -log10(p value), and the values of both the abscissa and ordinate are logarithmically processed.

[0046] Figure 5 KEGG pathway enrichment analysis diagram of wolfberry exosomes extracted by layer-by-layer filtration and ultracentrifugation: GQV01 vs LyE01. The abscissa represents the pathway name, and the ordinate represents the enrichment rate, which is the ratio of the number of metabolites enriched in the pathway (Metabolite nμmber) to the number of metabolites annotated to the pathway (Background nμmber). 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 *.

[0047] Figure 6(A - B) shows the TEM analysis diagram of wolfberry exosomes isolated and extracted by layer-by-layer filtration: GQV01.

[0048] Figure 7 Showing the particle size and concentration of GQV01 analyzed by nanoparticle tracking analysis.

[0049] Figure 8 (A - B) shows the in vitro PKH67 staining flow cytometry detection of different batches of wolfberry exosomes extracted by layer-by-layer filtration: GQV01.

[0050] Figure 9 Shows the activity detection of wolfberry exosomes extracted by layer-by-layer filtration; A: Protein concentration of wolfberry exosomes. B: Wolfberry exosomes inhibit the release of TNF-α induced by LPS in RAW264.7. The ordinate is the concentration of TNF-α detected by the Elisa kit. Those with P value < 0.001 are marked as ***; C: Inhibitory rate of wolfberry exosomes on TNF-α release.

[0051] Figure 10 Shows the repair effect of wolfberry on the 3D skin model irradiated with UV. A. Representative tissue sections of the 3D skin model tissue morphology (H&E staining), arrows point to sunburn cells after UV irradiation; B. Quantitative analysis of sunburn cells in the tissue morphology sections of the 3D skin model (H&E staining); C. Quantitative analysis of the tissue viability detection results; D. TEWL detection results. When using the t-test method for statistical analysis, 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).

[0052] Figure 11 Shows the effect of wolfberry on the expression of skin barrier-related proteins in the 3D skin model irradiated with UV. A. Representative immunohistochemical pictures 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 using the t-test method for statistical analysis, 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, the normal group (con), the UV modeling group (UV), the positive control group (WY14643), and the drug treatment group (GQ).

[0053] Figure 12Show the effect of wolfberry on the moisturizing effect of 3D skin models irradiated by UV. A. Representative immunofluorescence 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).

[0054] Figure 13 Show the detection results of lipid peroxidation (malondialdehyde) in 3D skin models irradiated by 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). Detailed description of the invention

[0055] I. Definitions and Terms

[0056] 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.

[0057] As used in the context of the present disclosure, "vesicles", also known as extracellular vesicles, are membrane-enclosed structures that are released by cells into the extracellular and intracellular spaces. 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.

[0058] As used in the context herein, "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 transfer 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.

[0059] "Plant material" as used in the context of this text has the meaning commonly understood in the art. The plant raw material 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 material.

[0060] "Depth filtration (DF)" as used in the context of this text means using a depth filtration medium to remove particles (such as impurities) from a liquid such as a solution, by a combination of size (screening / filtration) and intermolecular interactions (such as electrostatic attraction and hydrophobic interactions between oppositely charged surfaces), intercepting particles and impurities in the entire deep part of the porous structure of the filtration medium. Depth filtration has been successfully used for the preliminary clarification of bacterial, yeast, insect, and mammalian cell suspensions, and can be used alone or in combination with centrifugation. The depth filtration medium can include cellulose (such as cellulose fibers) and / or polypropylene (such as polypropylene fibers), and / or filter aids (such as activated carbon, diatomaceous earth (DE), and / or perlite), and / or resins (such as 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) (for example, particles smaller than the voids of the medium can enter the interior of the medium), while very large particles are captured by physical screening or interception (for example, intercepted and attached to the medium).

[0061] The term "depth filter" or "depth capsule filter" as used herein achieves filtration within the depth of the filter material. Such filters are those that contain 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. Different from absolute filters, depth filter media retain particles throughout the porous medium, thus allowing retention of particles larger than the pore size. Companies in the current industry that have depth filter medium products include Sartorius, Merck Millipore, PALL, 3M, etc. Commercially available depth filters include, but are not limited to, the Millistak+Pod depth filter system, XOHC medium (Millipore Corporation), Zeta Plus TMDepth filters (such as those from 3M Purification). 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 Pall corporation NY filters, such as Supracap TM Depth Filter Capsules.

[0062] In the context of this disclosure, the terms "cosmetic", "cosmetic composition", "beauty product" or "beauty composition" refer to any chemical industrial 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, lips, teeth, etc., for the purpose of cleansing, maintaining, beautifying, modifying and changing 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.

[0063] 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, which can be administered through the gastrointestinal tract or parenterally, usually through the gastrointestinal tract.

[0064] 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" includes the skin of mammals and includes human skin. Similarly, the terms "hair, nails and mucosa" include the hair, nails and mucosa of mammals (such as humans).

[0065] In the context of this disclosure, the term "treatment" encompasses treatment methods, including methods involving administering 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.

[0066] In the context of the present text, 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 on healthy subjects, subjects with sensitive skin, and subjects presenting 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.).

[0067] In the context of the present text, when the terms "treatment" and "care" are modified by "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 conditions involved, such as contributing to or assisting 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, contributing to or assisting in repair, anti-inflammatory, 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.

[0068] In the context of the present text, the term "prevention" refers to the ability of the extracts of the present disclosure to prevent, delay, or hinder the appearance 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 "inhibit occurrence", that is, it refers to the ability of the extracts of the present disclosure to inhibit the appearance or development of a disease or disorder, or to inhibit changes in the cosmetic properties of the skin, hair, nails, and / or mucous membranes.

[0069] In the context of the present text, 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, fluctuates by ±5%, ±2%, or ±1%. For example, as used herein, the expression "about 100" includes 90 and 110 and all values therebetween (such as 90.5, 95, 101, 105, 109.95... etc.). For ratios, the term "about" is used to limit 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). Again, for example, the range of "about n - m" or "about n - about m" means 90%n - 110%n to 90%m - 110%m.

[0070] 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 merely exemplary.

[0071] All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0072] II. Preparation of Exosomes

[0073] Raw materials

[0074] The raw materials involved in the method of the present disclosure are plant materials. Preferably, the plant materials can be terrestrial. More preferably, the plant materials are wolfberries.

[0075] The plant materials can be part or all of the plant, for example, can be selected from wood, roots, rhizomes, barks, trunks, flowers, petals, sepals, seeds, fruits, stems, leaves and / or embryos and mixtures of more than one of them.

[0076] In some embodiments, the plant materials include wolfberries (Lycium chinense Miller), which are perennial woody plants of the genus Lycium in the Solanaceae family. The forms of wolfberries that can be used in the method of the present disclosure include dried wolfberries, wolfberry powder, wolfberry extracts, their physiologically acceptable salts and their derivatives. The wolfberries that can be used in the method of the present disclosure include, but are not limited to, Lycium barbarum, Lycium chinense, Lycium ruthenicum, Lycium dasystemum, Lycium truncatum, Lycium cylindricum, Lycium yunnanense, Lycium changjiense. In addition, wolfberries also include wolfberry varieties, such as Lycium barbarum var. auranticarpum, Lycium chinense var. potaninii or Lycium chinense var. rubricaulium. The method of the present disclosure can use a mixture of more than one kind of wolfberry as plant materials for exosome extraction.

[0077] Pretreatment

[0078] In some embodiments, the extraction method includes pretreatment of the plant materials (preferably wolfberries). Pretreatment techniques are well known in the art and include physical, chemical, and biological pretreatments, or any combination thereof.

[0079] 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.

[0080] 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.

[0081] In some embodiments, the isotonic solution used in the pretreatment includes 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.

[0082] 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.

[0083] 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, comminution includes comminuting a plant or a part thereof using powdering technology or cell wall breaking technology. The cell wall breaking technology uses a cell wall breaker.

[0084] 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, still 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 (for example, about 25°C).

[0085] 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.

[0086] Layer-by-layer filtration

[0087] The method for extracting plant exosomes of the present disclosure includes a step of filtering layer by layer.

[0088] 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.

[0089] 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 the method of the present disclosure to process the plant 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).

[0090] The individual filtration steps 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 comprises 1 filtration step, wherein the filtration step uses two filtration units with different pore sizes, preferably the two pore sizes decrease sequentially 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 comprises 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 sequentially 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 comprises 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 sequentially 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.

[0091] 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 a 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 combinations 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.

[0092] 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.

[0093] 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 can have a suitable flow rate. For example, the solvent can have a flow rate in the range of about 1 mL / minute, 2 mL / minute, 3 mL / minute, 4 mL / minute, 5 mL / minute, 6 mL / minute, 7 mL / minute, 8 mL / minute, 9 mL / minute, 10 mL / minute, 15 mL / minute, 20 mL / minute, 25 mL / minute, 30 mL / minute, 35 mL / minute, 40 mL / minute, 45 mL / minute, 50 mL / minute, 60 mL / minute, 70 mL / minute, 80 mL / minute, 90 mL / minute, 100 mL / minute, 200 mL / minute, 300 mL / minute, 400 mL / minute, 500 mL / minute, 600 mL / minute, 700 mL / minute, 800 mL / minute, 900 mL / minute, or 1,000 mL / minute, or any sub-range within about 1 mL / minute to about 1,000 mL / minute, such as 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 1,000 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 1,000 mL / minute, about 50 mL / minute to about 500 mL / minute, about 50 mL / minute to about 200 mL / minute. In embodiments for large-scale production, the flow rate of the solvent can be as high as about 1 L / minute 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 / minute to about 1,000 mL / minute, such as about 20 mL / minute to about 500 mL / minute, about 20 to about 200 mL / minute, about 30 to about 100 mL / minute.

[0094] 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 (such as 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 is carried out at about 4°C, and the membrane filtration of the layer-by-layer filtration of the present disclosure is carried out at about 4°C.

[0095] Generally speaking, 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.

[0096] In some embodiments, the layer-by-layer filtration includes using a filter membrane for filtration, and the filter membrane contains 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 (both from Meissner); Millex GV, Millex HP, Millipak 60, Millipak 200, and Durapore CVGL01TP3 membranes (from Millipore); Fluorodyne TM EX EDF membrane, Supor TM EAV, Supor TM EBV, Supor TM EKV (all from Pall); Sartopore TM (from Sartorius); Hydrophilic PES membrane from Sterlitech; and WFPES PES membrane from Wolftechnik.

[0097] 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.

[0098] In some embodiments, the inherent properties of the membrane can be changed 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 positive charges. 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 (such as autoclaved) before use to ensure its sterility.

[0099] Depth filtration

[0100] The present disclosure provides a method for preparing plant exosomes (preferably wolfberry exosomes), which contains 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.

[0101] In some embodiments, the present disclosure provides a method for preparing plant exosomes (preferably wolfberry exosomes), which contains 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.

[0102] 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 example, Supracap TM 50 depth filter capsules, or Supracap TM 100 Depth Filter Capsules.

[0103] 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 .

[0104] For the present disclosure, a depth filter can be any filter having a depth filtration function. In some embodiments, the depth filter can be a filter having any pore size. In some embodiments, the depth filters used in the present disclosure include depth filters having an aperture range of approximately: 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.

[0105] 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 carried out by a depth capsule filter. Depth capsule filters are generally applied in the clarification filtration of fermentation broth and cell culture broth, 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 colloids or viscous materials. The inventors 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.

[0106] Depth capsule filters 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 is approximately 8 - 20 μm, and the pore size of the second layer of membrane is approximately 2 - 4 μm.

[0107] 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).

[0108] For the present disclosure, any suitable flow rate can be used for the deep filtration. For example, the filtration flow rate range for the deep filtration applicable to the method 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 of the method 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.

[0109] 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 (for example, any range between any two of the above pressures).

[0110] For the present disclosure, the layer-by-layer filtration (such as depth filtration and optional other filtration steps) can be carried out for any suitable time period. For example, for a 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 (such as 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 (such as 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.

[0111] The method of the present disclosure can be used to process the plant (preferably wolfberry) at a suitable rate. For example, for a production batch, the rate range for the method of the present disclosure to process the plant, such as crude plant extract, 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 (such as any range between any two of the above production rates).

[0112] The method for preparing plant exosomes by layer-by-layer filtration (such as 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 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, 15×10 10 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, 26×1010 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 goji berries, preferably about 30×10 10 -40×10 10 pieces, such as about 33×10 10 pieces of plant exosomes.

[0113] The deep filter described in the present disclosure includes a deep filtration medium. In some embodiments, the deep filtration medium can 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 can 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 can 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 Company, the P series filter plates of PALL Company, and the BECO series filter plates of EATON Company.

[0114] In some embodiments, the depth filtration medium may be in a gradient distribution or a mixed distribution. In some embodiments, the depth filtration medium may be in a gradient distribution. In some embodiments, the depth 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 membrane can be selected to be about 11 - 30 μm, and the pore size of the second layer of membrane can be about 6 - 15 μm; or the pore size of the first layer of membrane can be about 8 - 20 μm, and the pore size of the second layer of membrane can be about 2 - 4 μm.

[0115] In some embodiments, the depth 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 depth filtration medium of the present disclosure can be used in combination with a membrane filtration medium. In some embodiments, the membrane filtration layer is disposed at the liquid outlet end of the depth filtration medium. In some embodiments, the membrane filtration layer is disposed at the liquid inlet end of the depth filtration medium. In some embodiments, the membrane filtration material after depth capsule filtration generally selects a low-protein adsorption hydrophilic material, such as polyethersulfone (PES), polyvinylidene fluoride (PVDF), polypropylene (PP), etc.

[0116] In some embodiments, the depth 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.

[0117] In some embodiments, a pump can be used for the depth filtration described in the present disclosure. In some embodiments, the pump may 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.

[0118] In some embodiments, the flow rate of the pump used for 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 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.

[0119] In some embodiments, in the depth filtration of the present disclosure, an aqueous solution is used to rinse the depth filter, preferably an isotonic solution, 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 of 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.

[0120] In some embodiments, in depth filtration, an aqueous solution is used to clean the depth filter, preferably an isotonic solution, 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 of the present disclosure, preferably at the end of 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.

[0121] Membrane filtration

[0122] In some embodiments, 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 said layer-by-layer filtration, membrane filtration can include anti-clogging membrane filtration and sterilizing membrane filtration. In some embodiments, anti-clogging membrane filtration can be carried out first, and then optionally sterilizing membrane filtration. In some embodiments, anti-clogging membrane filtration can be carried out before sterilizing membrane filtration. In some embodiments, anti-clogging membrane filtration can be carried out one or more times, and then sterilizing membrane filtration can be carried out one or more times.

[0123] The pore size of the membrane filtration of the present disclosure can be 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 pore size range can be the range composed of any two of the above pore sizes, such as but not limited to about the following ranges: 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. In some preferred embodiments, the pore size range of the membrane filtration can be selected from about 0.4 - 0.8 μm and about 0.1 - 0.3 μm or a combination thereof, such as about 0.45 - 0.8 μm and about 0.1 - 0.22 μm.

[0124] In some embodiments, the layer-by-layer filtration of the present disclosure includes membrane filtration after deep 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) can be carried out. 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, the membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm) can be carried out one or more times, and then the membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm) can be carried out one or more times. 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 time of the membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm), to reduce or prevent the blockage of the 0.22 μm membrane filtration.

[0125] 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 to 4, more preferably 2 to 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 a series of 3 membrane filters, the first filter can be a ~0.45 μm filter, the second filter can be a 0.3 μm filter, and the last filter can be a ~0.22 filter.

[0126] Exemplary membrane filters for use in 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.

[0127] For the methods of the present disclosure, the material of the membrane used for membrane filtration can be any filtration material conventionally used in the art, such as the various filtration materials and membrane materials described in the layer-by-layer filtration section above. In some embodiments, the anti-clogging membrane filtration is a resin-bonded glass fiber 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.

[0128] In some embodiments, the membrane filtration can be carried out using any suitable pressure. For example, the membrane filtration can be carried out using a feed 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 (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, the membrane filtration can be carried out using a permeate 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 (e.g., any range between any two of the above pressures).

[0129] 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 the 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.

[0130] 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. Preferably, an injection solution of sodium chloride is used 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.

[0131] In some embodiments, any suitable flow rate can be used for the membrane filtration. For example, a flow rate in the range of about 1 mL / min to about 1,000 mL / min can be used for the membrane filtration, 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 of large-scale production, the flow rate of the 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.

[0132] In some embodiments, any suitable time limit can be used for the membrane filtration. 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 (for example, any range between any two of the above time points). Preferably, for 500 mL of the crude plant extract obtained by pretreatment, the 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.

[0133] In some embodiments, the method of the present disclosure that includes depth filtration and membrane filtration can obtain the following yields of plant exosomes per 100 g of plants: 1×10 10 particles, 5×10 10 particles, 10×10 10 particles, 11×10 10 particles, 12×10 10 particles, 13×10 10 particles, 14×10 10 particles, 15×10 10 particles, 16×10 10 particles, 17×10 10 particles, 18×10 10 particles, 19×10 10 particles, 20×10 10 particles, 22×10 10 particles, 24×10 10 particles, 26×10 10 particles, 28×10 10 particles, 30×10 10 particles, 32×10 10 particles, 34×10 10 particles, 36×10 10 particles, 38×10 10 particles, 40×10 10 particles, 42×10 10 particles, 44×10 10 particles, 46×10 10 particles, 48×10 10 particles, 50×10 10 particles, 52×10 10 particles, 54×10 10 particles, 56×10 10 particles, 58×10 10 particles, 66×10 10 particles or more. In some embodiments, the membrane filtration of the present disclosure can obtain about 20×10 10 -50×10 10 particles, preferably about 33×10 10 plant exosomes per 100 g of goji berries.

[0134] Preferred embodiments

[0135] In one embodiment, the method for preparing plant exosomes of the present disclosure includes the following steps:

[0136] (a) Obtain plant raw materials;

[0137] (b) Pretreat the obtained plant raw materials to obtain a crude plant extract;

[0138] (c) Perform layer-by-layer filtration on the crude plant extract;

[0139] Among them, the layer-by-layer filtration includes a depth filtration step and an optional membrane filtration step.

[0140] In one embodiment, the method for preparing plant exosomes of the present disclosure includes the following steps:

[0141] (a) Obtain plant raw materials;

[0142] (b) Pretreat the obtained plant raw materials, preferably by mixing, crushing, filtering, and centrifuging to obtain a crude plant extract; (c) Perform layer-by-layer filtration on the crude plant extract to obtain a filtrate;

[0143] Among them, the layer-by-layer filtration includes a depth filtration step. Preferably, the depth filtration step uses a depth filtration with a pore size of about 1 - 50 μm, more preferably a depth filtration with a pore size of about 2 - 30 μm; and

[0144] (d) Perform membrane filtration on the filtrate in step (c), preferably anti-clogging membrane filtration and / or sterilizing membrane filtration, such as 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, 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-clogging membrane filtration with a pore size of 0.45 μm and sterilizing membrane filtration with a pore size of 0.22 μm performed sequentially to obtain plant exosomes.

[0145] In the above embodiment, the obtained plant materials are pretreated in step (b), where the pretreatment includes mixing the plant materials with an isotonic solution (preferably sodium chloride injection solution), crushing the plant raw materials using a blender to obtain a plant residue solution, filtering the plant residue solution through a filter screen to obtain a plant juice, and centrifuging the plant juice and collecting the supernatant to obtain a crude plant extract.

[0146] In the above embodiment, large-scale mixing and crushing of the plant materials 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, for example about 100 g, are mixed and crushed. In some embodiments, step (b) is performed at about 4°C.

[0147] 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.

[0148] 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 at least 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.

[0149] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes wolfberry.

[0150] In a specific embodiment of the above method for preparing plant exosomes, the step-by-step filtration treatment includes a depth filtration step, wherein the depth filter is a depth 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 depth filter is a depth filter produced by Pall Corporation (NY), which is Supracap TM Depth Filter Capsules.

[0151] In a specific embodiment of the above method for preparing plant exosomes, in step (d), the filtrate in step (c) is sequentially 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.

[0152] Technical effects

[0153] The step-by-step filtration method of the present disclosure has the following advantages compared with the ultracentrifugation method:

[0154] (1) Lower time consumption;

[0155] (2) Higher exosome yield or particle concentration;

[0156] (3) It can prevent the filter membrane from being blocked due to excessive impurities and can ensure the sterility of the product;

[0157] (4) The extracted plant exosomes have excellent metabolomic performance;

[0158] (5) The extracted plant exosomes have a typical exosome-like saucer shape and a particle size in the range of 30 - 200 nm;

[0159] (6) The extracted plant exosomes have anti-inflammatory and immunomodulatory activities.

[0160] The method for preparing the plant exosomes (preferably wolfberry exosomes) of the present disclosure is suitable for small-scale, pilot-scale, and large-scale production. In some embodiments, the method for preparing exosomes 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, and even up to 100000 g. Preferably, the method for preparing exosomes 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 materials.

[0161] In particular, the layer-by-layer filtration treatment of the present disclosure is particularly suitable for large-scale production. For example, on the basis of the deep filtration and membrane filtration preferably used in the present disclosure (as shown in the examples), large-scale production is achieved by increasing the membrane area of the filter. Through the layer-by-layer filtration of the present disclosure, by appropriately increasing the membrane area in the layer-by-layer filtration, 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, and even 100000 g of plant material or its corresponding pretreatment solution can be filtered.

[0162] III. Exosomes

[0163] The plant exosomes (preferably wolfberry exosomes) prepared according to the methods 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 from 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 from about 10 nm to about 10 μm (e.g., any range between any two of the above particle sizes). Preferably, the plant exosomes prepared according to the methods of the present disclosure have a diameter of from about 30 - 450 nm, preferably from about 30 - 250 nm, more preferably from about 30 - 200 nm.

[0164] 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 methods of the present disclosure have a saucer shape.

[0165] In some embodiments, the wolfberry exosomes provided by the present disclosure contain one or more of the following metabolites: lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzenoid compounds.

[0166] In some embodiments, the wolfberry exosomes provided by the present disclosure contain at least lipids and lipid-like molecules, and the lipids and lipid-like molecules account for at least about 20% of the total number of metabolites, preferably about 20% - 50%, 30% - 40%, 30% - 35%, or 30% - 34%, more preferably about 33% of the total number of metabolites.

[0167] In some embodiments, the wolfberry exosomes provided by the present disclosure contain at least lipids and lipid-like molecules and organic oxygen compounds, and the lipids and lipid-like molecules and organic oxygen compounds account for at least about 30%, at least about 40%, at least about 50%, or at least about 60% of the total number of metabolites, preferably about 30% - 60%, 40% - 55%, 45% - 52%, or 47% - 51%, more preferably about 50% of the total number of metabolites; preferably wherein the lipids and lipid-like molecules account for at least about 20% of the total number of metabolites, preferably about 20% - 50%, 30% - 40%, 30% - 35%, or 30% - 34%, more preferably about 33% of the total number of metabolites; or preferably the organic oxygen compounds account for 10% - 20% of the total number of metabolites, such as about 16%.

[0168] In some embodiments, the wolfberry exosomes provided by the present disclosure comprise lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, wherein the lipids and lipid-like molecules account for at least about 20% of the total number of metabolites, preferably about 20%-50%, 30%-40%, 30%-35%, or 30%-34%, and more preferably about 33% of the total number of metabolites; wherein the phenylpropanoids and polyketides account for 10%-20% of the total number of metabolites, such as about 15%; or the organic oxygen compounds account for 10%-20% of the total number of metabolites, such as about 16%.

[0169] In some embodiments, the wolfberry exosomes provided by the present disclosure comprise lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, preferably wherein the lipids and lipid-like molecules account for at least about 20% of the total number of metabolites, preferably about 20%-50%, 30%-40%, 30%-35%, or 30%-34%, and more preferably about 33% of the total number of metabolites; the organic oxygen compounds account for 10%-20% of the total number of metabolites, such as about 16%; or the phenylpropanoids and polyketides account for 10%-20% of the total number of metabolites, such as about 15%.

[0170] In some embodiments, the wolfberry exosomes prepared according to the method of the present disclosure comprise lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzene compounds, preferably wherein the lipids and lipid-like molecules account for at least about 20% of the total number of metabolites, preferably about 20%-50%, 30%-40%, 30%-35%, or 30%-34%, and more preferably about 33% of the total number of metabolites; the organic oxygen compounds account for 10%-20% of the total number of metabolites, such as about 16%; the phenylpropanoids and polyketides account for 10%-20% of the total number of metabolites, such as about 15%; the organic heterocyclic compounds account for 7%-17% of the total number of metabolites, such as about 12%; the organic acids and their derivatives account for 5%-15% of the total number of metabolites, such as about 10%; or the benzene compounds account for 5%-15% of the total number of metabolites, such as about 11%.

[0171] The plant exosomes (preferably wolfberry exosomes) prepared according to the method of the present disclosure have more metabolites in one or more of the following aspects compared to the plant exosomes prepared by the ultracentrifugation method: 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.

[0172] In some embodiments, the wolfberry exosomes provided by the present disclosure contain about 1×10 5 to 1×10 10 particles / mL, preferably 1×10 6 to 1×10 9 particles / mL, more preferably 1×10 7 to 1×10 9 particles / mL, for example, about 1.33×10 8 particles / mL.

[0173] Compared with plant exosomes prepared by the ultracentrifugation method, the plant exosomes (preferably wolfberry exosomes) prepared according to the method of the present disclosure have up-regulated metabolism ( Figure 5 ), 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.

[0174] Specifically, there are 73 metabolic pathways related to differential metabolites between wolfberry exosomes prepared according to the method of the present disclosure and wolfberry exosomes prepared by the ultracentrifugation method, and a total of 11 pathways are significantly enriched.

[0175] In some embodiments, there are 617 significantly differential metabolites between wolfberry exosomes prepared according to the method of the present disclosure and wolfberry exosomes prepared by the ultracentrifugation method, 401 up-regulated metabolites, and 216 down-regulated metabolites.

[0176] In some embodiments, in the analysis of in vitro staining of plant exosomes with the lipophilic dye PKH67, the positive rates of wolfberry exosomes prepared according to the method of the present disclosure are 64.5% or 68.8% respectively. In the detection of the protein content of plant exosomes, the protein content of wolfberry exosomes prepared according to the method of the present disclosure is 47434.90 μg / mL.

[0177] 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%, even close to 100%.

[0178] IV. Compositions

[0179] The present disclosure provides a plant exosome composition comprising wolfberry exosomes, preferably wolfberry exosomes prepared by the method for preparing plant exosomes as generally or preferably or specifically defined in the present disclosure.

[0180] Specifically, the plant exosome composition provided by the present disclosure is a pharmaceutical composition, which, in addition to containing wolfberry exosomes (preferably wolfberry exosomes prepared by the method for preparing plant exosomes defined generally or preferably or specifically in the present disclosure), optionally further contains 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, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0181] Specifically, the plant exosome composition provided by the present disclosure is a food composition or a nutraceutical composition, which contains wolfberry exosomes, preferably wolfberry exosomes prepared by the method for preparing plant exosomes defined generally or preferably or specifically in the present disclosure. In the case of using the food composition or nutraceutical composition of the present disclosure 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.

[0182] The nutraceutical composition of the present disclosure itself can be in the form of a nutritional supplement or a health 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.

[0183] The 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.

[0184] The plant exosome composition (pharmaceutical composition / nutraceutical composition / food composition) of the present disclosure can be in different types of 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 through topical or transdermal routes, through any other suitable routes (such as parenteral routes).

[0185] Specifically, the exosome composition provided by the present disclosure is a cosmetic / cosmeceutical composition, which contains wolfberry exosomes, preferably wolfberry exosomes prepared by the method for preparing plant exosomes generally or preferably or specifically defined by the present disclosure. The cosmetic / cosmeceutical composition of the present disclosure usually contains at least one excipient or adjuvant acceptable in cosmetics / cosmeceuticals. The "excipient or adjuvant acceptable in cosmetics / cosmeceuticals" can be selected from: solvents, solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, humectants, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, propellants / aerosols, fragrances, pigments, and other efficacy additives. The forms of the cosmetic composition are, for example but not limited to, soaps, facial soaps, facial cleansers, cleansing foams, cleansing lotions, cleansing creams, body washes, skin softeners, skin care gels, skin care lotions, skin care creams, essence, eye creams, facial masks, aerosols or sprays, lotions, 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 body cleansing lotions, etc. These forms of the cosmetic composition can be prepared by methods well-known to those skilled in the art.

[0186] When the cosmetic composition of the present disclosure is a paste, cream or gel, animal fibers, plant fibers, waxes, paraffin wax, starch, tragacanth, cellulose derivatives, polyethylene glycol, silica gel, bentonite, 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 ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan trioleate, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc. can be used as carrier components.

[0187] V. Uses

[0188] The exosomes or compositions for any use of the present disclosure may include the exosomes or compositions generally or specifically of 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 the 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 defined particle densities, different metabolite species and their different percentages of metabolite species.

[0189] Many diseases are accompanied by an inflammatory response, including skin-related inflammations (such as dermatitis, acne, pimples, melasma, etc.). Therefore, reducing the inflammation level will be beneficial to the treatment and care of diseases. Accordingly, in some embodiments, the present disclosure provides the use of a plant exosome (preferably a wolfberry exosome) or a composition containing the same for treating or preventing, or assisting in improving, alleviating or controlling inflammation, such as skin inflammation. In other embodiments, the present disclosure provides the use of a plant exosome (preferably a wolfberry exosome) or a composition containing the same for, or assisting in, a cosmetic, non-therapeutic treatment and / or care of the skin, hair, nails and / or mucous membranes, preferably for, or assisting in, a cosmetic, non-therapeutic treatment and / or care of the inflammation of the skin, hair, nails and / or mucous membranes.

[0190] In some embodiments, the plant exosomes of the present disclosure (preferably wolfberry exosomes) or a composition containing the same can be used in the field of skin for repair, anti-inflammation, antioxidant, anti-aging, whitening, moisturizing and other purposes. For example, the plant exosomes or a composition containing the same can be used for anti-inflammatory purposes, preferably for anti-inflammatory purposes in the field of skin. For another example, the plant exosomes or a composition containing the same can be used for antioxidant purposes. For another example, the plant exosomes or a composition containing the same can be used for repair purposes in the field of skin. For another example, the plant exosomes or a composition containing the same can be used for anti-aging purposes. For another example, the plant exosomes or a composition containing the same can be used for whitening purposes. For another example, the plant exosomes or a composition containing the same can be used for moisturizing purposes.

[0191] 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 helps or aids in enhancing skin barrier function, antioxidant, repair, anti-aging, whitening and / or moisturizing.

[0192] 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 (drug composition / food composition / beauty or cosmetic composition / nutritional health composition) prepared by the methods defined generally or preferably as described above.

[0193] Skin diseases in most cases begin with the deterioration or breakdown of the skin barrier function, and filaggrin, loricrin (LOR), and Claudins proteins play important roles in the skin barrier function. A decrease in filaggrin, loricrin (LOR), and Claudins proteins leads to poor skin barrier and moisturizing ability, 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 Lycium barbarum exosomes of the present disclosure or a composition containing the same have enhanced skin barrier function; or treat, prevent, or contribute to (or be used as an adjunct to) improving, alleviating, or controlling skin barrier damage. In some embodiments, the Lycium barbarum exosomes of the present disclosure or a composition containing the same can promote the synthesis of filaggrin. In some embodiments, the Lycium barbarum exosomes of the present disclosure or a composition containing the same can promote the synthesis of filaggrin, loricrin (LOR), and / or Claudins proteins. The Lycium barbarum exosomes of the present disclosure or a composition containing the same can promote the synthesis of filaggrin, for example, with a promotion rate of 30.00% - 150.00% for profilaggrin synthesis, preferably 50.00% - 100.00%, and most preferably 75.00% - 80.00%. The Lycium barbarum exosomes of the present disclosure or a composition containing the same can promote the synthesis of loricrin, for example, with a promotion rate of 50.00% - 200.00% for loricrin synthesis, preferably 80.00% - 150.00%, and most preferably 125.00% - 135.00%. In some embodiments, the Lycium barbarum exosomes of the present disclosure or a composition containing the same can promote the synthesis of Claudins proteins (such as CLDN1), for example, with a promotion rate of 50.00% - 140.00% for Claudins protein synthesis, preferably 60.00% - 100.00%, and most preferably 75.00% - 85.00%.

[0194] 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 adjuvant to) improving, alleviating, 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 generation 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 aggressions, 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 the presence 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 adjuvant to) improving, alleviating, 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.

[0195] In some embodiments, the wolfberry 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, glucosaminoglycans, mucopolysaccharides, and sodium PCA (pyrrolidone carboxylic acid). Pyrrolidone carboxylic acid (PCA) or sodium PCA (pyrrolidone carboxylic acid) is a natural moisturizer with excellent hygroscopic properties and is present in large amounts in the stratum corneum of the skin as the most important component of the natural moisturizing factor. PCA is formed by the hydrolysis of FLG by Caspase-14, which can effectively adsorb and lock in moisture and strengthen the moisturizing barrier. In some embodiments, the wolfberry exosomes of the present disclosure or a composition comprising the same can treat, prevent, or contribute to (or be used adjunctively for) improving, alleviating, or controlling skin dryness symptoms or related diseases thereof, wherein the skin dryness symptoms are, for example, selected from 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, for example, selected from 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 wolfberry exosomes of the present disclosure or a composition comprising the same promote skin humidification or hydration. In some embodiments, the wolfberry exosomes of the present disclosure or a composition comprising the same are used for skin moisturization. In some embodiments, the wolfberry exosomes of the present disclosure or a composition comprising the same can increase the content of PCA.

[0196] Malondialdehyde (MDA) is produced due to the peroxidation reaction of tissue or organ membrane lipids, 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, ROS attacks the polyunsaturated fatty acids in phospholipids on the biological membrane, triggering a lipid peroxidation chain reaction in the membrane, ultimately leading to an increase in MDA content. Ultraviolet irradiation can induce lipid peroxidation reactions in the membrane, 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 of the present disclosure or a composition containing the same can treat, prevent, or contribute to (or be adjuvantly used for) improving, alleviating, and controlling oxidative damage to skin cells. In some embodiments, the Lycium barbarum exosomes of the present disclosure or a composition containing the same are used for skin antioxidant purposes. In some embodiments, the Lycium barbarum exosomes of the present disclosure or a composition containing the same can reduce or alleviate the ROS generated by ultraviolet irradiation. In some embodiments, the Lycium barbarum exosomes of the present disclosure or a composition containing the same can alleviate the content of lipid peroxides in the skin, for example, reduce the content of MDA.

[0197] In some embodiments, the present disclosure provides the use of the plant exosomes (preferably Lycium barbarum exosomes) of the present disclosure or a composition containing the same in the preparation of products / cosmetics / compositions / beauty products / drugs having functions of enhancing skin barrier function, antioxidant, repair, anti-aging, whitening, firming and anti-wrinkle, and / or moisturizing. Optionally, the plant exosomes (preferably Lycium barbarum exosomes) or a composition containing the same are the sole active ingredient or the main active ingredient for enhancing skin barrier function, antioxidant, repair, anti-aging, whitening, firming and anti-wrinkle, and / or moisturizing.

[0198] In some embodiments, the Lycium barbarum exosomes provided by the present disclosure for the above functions contain about 1×10 5 to 1×10 10 particles / mL, preferably 1×10 6 to 1×10 9 particles / mL, more preferably 1×10 7 to 1×10 9 particles / mL, for example, about 1.33×10 8 particles / mL.

[0199] For the above-described 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 applications can be carried out by iontophoresis, sonophoresis, electroporation, mechanical pressure, osmotic gradients, occlusion therapy, microinjection, microneedles (or microneedle arrays), needle-free injection by pressure, by microelectrical patches, masks, or any combination thereof. Among them, some techniques that create channels in the skin (such as microneedles or lasers) can themselves be used as a certain treatment means and can be combined with the therapy of plant exosomes or compositions containing the same, helping 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 therapy will be determined by the nature of the condition, disorder, and / or disease to be treated and / or addressed.

[0200] The daily dosage and dosing frequency of the plant exosomes (preferably wolfberry 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 1 - 3 times a day at a daily dosage of, for example, 1 μg / kg to 200 mg / kg, more specifically 50 μg / kg to 50 mg / kg. However, the dosage does not limit the scope of the present disclosure in any way.

[0201] 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 acute liver injury caused by GalN / LPS, 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 brain glioma, 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-mentioned 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

[0202] The technical solutions of the present disclosure will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited to these examples. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0203] 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 directly used 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 invention are in degrees Celsius (°C).

[0204] 1. Experimental methods

[0205] 1.1 Preparation of crude plant extract

[0206] Wash the goji berries clean and dry the moisture 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 blender to crush the plant, and collect the crushed plant residue liquid into a measuring cylinder. Place the measuring cylinder on an ice medium, and use a stirrer at a rotation speed of 200 ± 20 rpm and stir for 30 min. Use a filter screen to pour in 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.

[0207] 1.2 Separation and extraction of plant exosomes using the method of layer-by-layer filtration

[0208] S1 Depth filtration: In a biosafety cabinet, connect a peristaltic pump, pump tubing, and a depth capsule filter (Supracap) with a pore size of about 2 - 30 μm TMDepth Filter Capsules, Pall, filtration capacity about 50 - 200L / m 2 ) Connect them together in combination. Rinse the depth filter capsule with sodium chloride injection solution at a flow rate of 20 - 200 mL / min, and finally empty 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 solution at a flow rate of 20 - 200 mL / min, and finally empty the liquid and remove the k pump tube.

[0209] 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 solution at a flow rate of 20 - 200 mL / min, and finally empty 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 solution at a flow rate of 20 - 200 mL / min, and finally empty the liquid and remove the pump tube.

[0210] 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 solution at a flow rate of 20 - 200 mL / min, and finally empty the liquid. Filter P3 at a flow rate of 20 - 200 mL / min and collect the filtrate to obtain plant exosomes. Remove the filter, clean the pipeline with sodium chloride injection solution at a flow rate of 20 - 200 mL / min, and finally empty the liquid and remove the pump tube.

[0211] 1.3 Separate and extract plant exosomes by ultracentrifugation

[0212] Centrifuge the collected crude plant extract P1 at 120,000g, 4°C for 70 min using an ultracentrifuge. After centrifugation, discard the supernatant and resuspend the precipitate with sodium chloride injection solution. After centrifuging again at 120,000g, 4°C for 70 min, resuspend the precipitate with sodium chloride injection solution to obtain plant exosomes.

[0213] 1.4 Transmission electron microscopy detection

[0214] Take 5 μL of each of the above-prepared plant exosome samples and drop them on a copper grid, and incubate at room temperature for 5 min; after incubation, blot the excess liquid on one side with absorbent paper; add a drop of 2% uranyl acetate to the copper grid and incubate at room temperature for 1 min; after incubation, blot 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.

[0215] 1.5 Nanoparticle size tracking analysis

[0216] Use nanoparticle tracking analysis technology (Nanoparticle Tracking Analysis, NTA) to measure the particle size and concentration of each of the above-prepared plant exosomes: Dilute the standard stock solution to a calibration mother liquor 1000 times (1 μL of the standard stock solution can be formulated into 1 mL of the calibration mother liquor according to the ratio). Take 100 μL of the above-prepared standard mother liquor 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 liquor, inject the test sample into the sample pool with a syringe, measure the sample concentration, and repeat three times. 1.6 In vitro labeling of plant exosomes with PKH67

[0217] 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.

[0218] 1.7 Trace protein detection

[0219] 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 the reagents MA:MB:MC = 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 the diluent. After diluting the plant exosomes with the diluent, add an equal volume of WR, and shake at 100 rpm and 37 °C for 2 hours in a constant temperature shaker. Detect the absorbance of the standards and samples at 570 nm using a microplate reader, and calculate the protein concentration of the samples.

[0220] 1.8 Detection of TNF-α Inhibition Rate

[0221] 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 96-well plates 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:

[0222] Inhibition rate (%) = 1 - (average concentration of the experimental group / average concentration of the LPS treatment group).

[0223] 1.9 Sample Preparation for Untargeted Metabolomics Analysis of Plant Exosomes

[0224] 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 (QC). During the instrument analysis process, insert one QC sample into every 5 - 15 samples to examine the repeatability of the entire analysis process.

[0225] 1.10 LC-MS / MS Analysis

[0226] 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 the positive and negative ion scanning mode, 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 at 3500 V, the negative mode ion spray voltage is set at -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.

[0227] 1.11 Substance Identification and Analysis

[0228] 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.

[0229] 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 more than 80% non-zero values 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 total 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 QC samples > 30%, and perform log10 logarithmic transformation to obtain the final data matrix for subsequent analysis.

[0230] 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) value obtained from the OPLS-DA model and the p-value of the student's t-test. Metabolites with VIP > 1 and p < 0.05 are significantly different metabolites.

[0231] Annotate the metabolic pathways of the differential metabolites through the KEGG database (Kyoto Encyclopedia of Genes and Genomes, https: / / www.kegg.jp / kegg / pathway.html) to obtain the pathways participated by the differential metabolites. Use the Python software package scipy.stats for pathway enrichment analysis and obtain the biological pathways most relevant to the experimental treatment through Fisher's exact test.

[0232] 2. Experimental Results and Analysis

[0233] 2.1 Screening of Extraction Methods

[0234] For wolfberries, based on Example 1.1 above, 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:

[0235]

[0236] Among them, "+" represents the presence of relevant treatments, and "-" represents the absence of relevant treatments.

[0237] 2.2 Extract plant exosomes (PEN) using the methods of layer-by-layer filtration and ultracentrifugation respectively

[0238] Use the layer-by-layer filtration method in Examples 1.1 - 1.2 to isolate and extract exosomes from wolfberries. Specifically, first, wolfberries (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 residue is 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.

[0239] Use the ultra - centrifugation method in Example 1.3 to isolate and extract exosomes from wolfberries. 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.

[0240] Table 1 Name labels of PEN isolated from wolfberries using ultra - centrifugation and layer - by - layer filtration

[0241] Plant Category Ratio Layer-by-Layer Filtration Ultracentrifugation Wolfberry 1:5 GQV01 LyE01

[0242] The following various tests are carried out on each plant exosome sample obtained from the above operations. The results show that they have the various characteristics and effects of exosomes described in the detailed part of the above invention. As a representative example, the following gives the test results of exosomes (name labels as shown in Table 1) obtained from 100 g of wolfberries after the above treatment.

[0243] 2.3 Physical properties of plant exosomes

[0244] 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 statistically analyzed.

[0245] 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 superior to ultra - centrifugation.

[0246] Table 2 Particle size and concentration of plant exosomes

[0247]

[0248] 2.4 Qualitative metabolomics analysis of plant exosomes

[0249] 2.4.1 Analysis of Special Metabolites

[0250] 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 wolfberry exosomes obtained in Example 2.2, filtration layer by layer was higher than ultracentrifugation. Specifically, as shown in Table 3. After using Venn diagram analysis, the metabolites unique to filtration layer by layer were also higher than those of ultracentrifugation.

[0251] Table 3 Statistical Table of Venn Analysis of Exosomes Extracted from Wolfberry by Filtration Layer by Layer and Ultracentrifugation

[0252]

[0253] 2.4.2 Compound Classification Analysis

[0254] Compound classification analysis is to classify 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 wolfberry exosomes are mainly concentrated in six categories, namely lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzene compounds. As Figures 2 - 3 shown.

[0255] · The metabolite classifications that GQV01 has more than LyE01 are phenylpropanes and polyketides (44), organic oxygen compounds (41),

[0256] lipids and lipid-like molecules (34), organic acids and their derivatives (18), lignans, neolignans and related compounds (18), and organic heterocyclic compounds (16)

[0257] 2.4.3 Lipid Classification Analysis

[0258] Lipids and lipid-like molecules rank first in compound classification. Lipid classification mainly includes fatty acyls (FA), glycerolipids (GL), glycerophospholipids (GP), prenol lipids (PR), sphingolipids (SP), steroids (ST), and saccharolipids (SP).

[0259] The lipid and lipid-like molecules of wolfberry exosomes were classified and counted. As shown in Table 4, the number of lipid types in plant exosomes extracted by filtration layer by layer is more than that of ultracentrifugation. The lipid types with relatively large content differences between GQV01 and LyE01 are FA, PR, and ST.

[0260] Table 4 Comparison of lipid classification of wolfberry exosomes

[0261]

[0262] 2.5 Analysis of differential metabolites among plant exosomes

[0263] Univariate statistical analysis (t-test) combined with multivariate statistical analysis (OPLS-DA / PLS-DA) and fold change value (FC) were used to jointly screen differential metabolites. The screening conditions were P < 0.05, VIP > 1 and (FC < 1 or FC > 1, FC was not screened by default) [6].

[0264] 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.

[0265] 2.6 Analysis of differential metabolic pathways among plant exosomes

[0266] 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 the 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 according to the enrichment P value less than 0.05.

[0267] There were 73 pathways related to differential metabolites in GQV01 compared with LyE01, and a total of 11 pathways were significantly enriched.

[0268] As Figure 5 shown, the differential metabolites of plant exosomes extracted by layer-by-layer filtration were mainly up-regulated in Metabolism compared with ultracentrifugation. The main pathways were 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.

[0269] 2.7 TEM identification of plant exosomes separated by layer-by-layer filtration

[0270] Through the above analysis of the particle concentration, particle size, yield of plant exosomes, and plant metabolomics, it is shown that the extraction method of layer-by-layer filtration is superior to ultracentrifugation, and higher yields of plant exosomes can be extracted in less time (equivalent to at least 40 times the yield of exosomes extracted by ultracentrifugation). Therefore, we further performed TEM on the plant exosomes extracted by layer-by-layer filtration.

[0271] The results are as Figure 6A and 6B shown. Lycium barbarum exosomes have a typical exosome-like saucer shape, with particle sizes in the range of 30 - 200 nm (see Figure 7 ), which conforms to the definition of extracellular vesicles by the International Society for Extracellular Vesicles (ISEV).

[0272] 2.8 Detection of PKH67 of plant exosomes separated by layer-by-layer filtration

[0273] 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.

[0274] As Figure 8A and 8B shown, the positive rates of PKH67 of GQV01 were 64.5% or 68.8%.

[0275] 2.9 Detection of protein content of plant exosomes separated by layer-by-layer filtration

[0276] The protein content of plant exosomes was detected, and it was found that the protein content of Lycium barbarum exosomes was rich. Specifically, as Figure 9 shown in A, the protein content of GQV01 was as high as 47434.90 μg / mL.

[0277] 2.10 Detection of anti-inflammatory activity of plant exosomes separated by layer-by-layer filtration

[0278] The efficacy of the plant exosomes extracted by layer-by-layer filtration was detected, mainly by observing the inhibition rate of LPS-induced TNF-α release from RAW264.7.

[0279] 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 Lycium barbarum exosomes with a particle concentration of 1×10 9 / mL, the TNF-α inhibition rate of GQV01 was 90.19%.

[0280] The results show that the Lycium barbarum exosomes extracted by layer-by-layer filtration have satisfactory anti-inflammatory and immunomodulatory activities.

[0281] 2.11 Amplification Process

[0282] According to the layer-by-layer filtration method in Examples 1.1 - 1.2, 1000 g of wolfberries 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.

[0283] As shown in Table 5, after magnifying the 100 g of plant raw materials for obtaining exosomes in Example 2.3 by 5 - 10 times (wolfberries magnified 10 times), it is possible to process approximately 5000 mL of plant crude extract 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.

[0284] Table 5 Particle Sizes and Concentrations of Plant Exosomes after Magnifying the Layer-by-Layer Filtration Process by 5 - 10 Times

[0285]

[0286] 2.12 Cosmetic Efficacy Analysis of Representative Metabolites of Wolfberry Exosomes

[0287] 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 metabolite databases such as KEGG and HMDB commonly used have metabolite classification information, enabling 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.

[0288] After analysis, the top 100 metabolites of GQ total metabolites were screened according to the average content, and the cosmetic efficacy analysis of 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] .

[0289] Table 6 Efficacy of Representative Metabolites of GQ in Cosmetics

[0290]

[0291] 2.13. Repair and moisturizing effects of GQ on 3D epidermal skin models

[0292] 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 to the skin caused by UV irradiation, and then performed slicing and H&E staining for histological morphology observation.

[0293] This study used a 3D epidermal skin model Batch 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 (the irradiation dose was 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 the incubation was completed, 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.

[0294] 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 being stimulated, 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).

[0295] 2.14 Regulation of GQ on the expression of skin barrier proteins

[0296] To further study the repair effect of GQ on the skin barrier, we sliced the 3D epidermal skin model and performed immunohistochemical detection and integrated optical density (IOD) statistics on the skin barrier proteins to measure 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.

[0297] 2.15 Effect of GQ on skin moisturization

[0298] 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. From the results, it can be seen that GQ can significantly up-regulate the expression of AQP3 in the skin after the skin undergoes 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 efficacy.

[0299] 2.16 Mitigation effect of GQ on lipid peroxidation

[0300] 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 ).

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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. A method according to any one of claims 1 to 5, wherein the plant material is selected from wolfberries.

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.

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 plant exosome composition comprising the plant exosomes according to claim 8 or 9, and optionally a suitable excipient or carrier.

11. The plant exosome composition of claim 10, which is a pharmaceutical composition, a food composition, a beauty or cosmetic composition, or a nutritional health composition.

12. Use of the plant exosomes of claim 8 or 9 or the plant exosome composition of claim 10 or 11 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.

13. The use of claim 12, 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.

14. Use of the plant exosomes of claim 8 or 9 or the plant exosome composition of claim 10 or 11 for treating or preventing inflammation, preferably skin inflammation, or for helping or assisting in improving, alleviating or controlling inflammation, preferably skin inflammation, or for enhancing skin barrier function, repairing, anti-inflammatory, anti-oxidant, anti-aging, whitening or moisturizing in the field of dermatology.

15. The use of claim 14, 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.

16. A method for treating or preventing inflammation, preferably skin inflammation, comprising administering the plant exosome composition of claim 10 or 11 to a subject in need thereof.

17. 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.

18. A method for improving, alleviating or controlling dermatological symptoms, comprising administering the plant exosome composition of claim 10 or 11 to a subject in need thereof.

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