Method for extracting exosomes from plants

Abstract

The present disclosure relates to a method for preparing plant exosomes, such as a method for preparing exosomes from Lycium chinense, Panax ginseng, Dendrobium nobile, Polygonatum sibiricum, Angelica sinensis, and Rehmannia glutinosa, plant exosomes prepared via the method, and use thereof. Specifically, the preparation method for the plant exosomes comprises a layer-by-layer filtration step, the layer-by-layer filtration step sequentially comprising depth filtration and any membrane filtration.

Description

A method for extracting exosomes from plants Technical Field

[0001] The present disclosure relates to methods for extracting exosomes from plants, such as wolfberry, ginseng, dendrobium, polygonatum, angelica, and rehmannia, as well as plant exosomes extracted by the method and their applications. Specifically, the method for preparing plant exosomes includes a layer-by-layer filtration step, which sequentially includes depth filtration and membrane filtration. Background Art

[0002] Exosomes are nanoparticles secreted by cells, typically 30-200 nm in diameter, containing a phospholipid bilayer. They are rich in proteins and nucleic acids. Exosomes can transport active substances such as proteins, mRNA, and microRNA between cells, participating in many important physiological and pathological processes. Their unique role has attracted increasing attention within the industry.

[0003] Currently, exosomes are primarily obtained from milk samples, various cell culture supernatants, and exudates from tumor tissue. However, obtaining exosomes from sources like cell culture supernatants is limited, expensive, and results in low exosome content, resulting in even higher purification costs, which hinders large-scale industrial production of exosomes.

[0004] Various plants are also an important source of exosomes, and the extraction rate of plant-derived exosome-like nanoparticles (PEN) is much higher than that of mammalian cell culture, indicating its economic effectiveness as a nanofactory. [1] Most importantly, recent studies have shown that plant exosomes can be absorbed by intestinal macrophages, playing a role in intercellular communication and immune regulation. [2-3] .

[0005] Correct extraction and purification methods are crucial for the analysis of exosomes because they are often present in overly complex matrices. Research on exosome extraction methods must not only examine the specificity and purity of the extracts, but also consider the simplicity of operation and scale efficiency. Currently, the more commonly used methods for isolating exosomes include ultracentrifugation, polymer precipitation, gel exclusion, antibody affinity precipitation, density gradient centrifugation, membrane filtration, etc. However, each method has its own defects. For example, ultracentrifugation is complicated to operate, technically difficult, and time-consuming. The isolated exosomes may be damaged by high-speed centrifugation and contain a large amount of impurities. The exosomes extracted by polymer precipitation lack specificity and selectivity and have low purity. The gel exclusion method is technically difficult, but it is difficult to ensure the sterility of the isolated exosomes. It is time-consuming and difficult to scale up. Immunoaffinity chromatography usually improves the purity of the sample but reduces efficiency and recovery rate. Membrane filtration improves efficiency and recovery rate but reduces its sample specificity and purity. [4] .

[0006] Typically, mammalian exosomes are isolated from biological fluids, while plant-derived exosomes are isolated from apoplast washes. For mammalian exosomes, current separation and purification technologies primarily include microfluidics, ultracentrifugation, separation techniques based on exosome size, precipitation, and immunoaffinity capture. Available methods for isolating and purifying plant-derived exosomes are primarily based on established techniques for mammalian exosomes, but have varying degrees of disadvantages. For example, with differential centrifugation, due to physiological differences between plants and animals, plant-derived exosomes obtained by this method are often mixed with proteins, nucleic acid aggregates, and other vesicles, requiring further purification using density gradient ultracentrifugation to separate contaminants. For another example, the presence of high molecular weight components in plant juices, such as cellulose and starch, often makes centrifugation difficult. Therefore, a combination of various centrifugation methods (e.g., differential centrifugation combined with sucrose density gradient centrifugation) is required to address the aforementioned shortcomings of traditional centrifugation techniques. Furthermore, while several other separation techniques, such as immunoaffinity capture, ultrafiltration or size exclusion chromatography (SEC), coprecipitation methods, and microfluidics, have been successfully applied to mammalian exosomes, they are less ideal for the separation of plant-derived exosomes. For example, while immunoaffinity capture based on the formation of immune complexes against extracellular vesicle surface antigens is an ideal method for purifying mammalian exosomes, the lack of marker proteins and specific antibodies for plant-derived exosomes limits the application of this technology. Therefore, methods suitable for the extraction of mammalian exosomes are not suitable for the extraction of plant exosomes.

[0007] There is no universal method for the isolation of plant exosomes. Currently, ultracentrifugation is commonly used in research, or a combination of various methods such as extraction, membrane filtration for impurity removal, microfiltration for sterilization, nanofiltration for concentration, and even complexation reaction to obtain plant exosomes. [7] As mentioned above, although ultracentrifugation is generally considered the gold standard for exosome isolation, it can isolate relatively pure exosomes. [5] However, the ultracentrifugation method has many drawbacks and relies on ultra-high centrifugal force and duration to achieve separation by sedimentation of particles to the bottom. Therefore, it is time-consuming and the extraction scale is limited by the processing volume of the ultracentrifuge.

[0008] Therefore, a simple, efficient method suitable for large-scale, industrial-scale isolation and extraction of high-purity plant exosomes is needed. Extracting exosomes from abundant plant materials is expected to reduce production costs and increase yields, which is of great significance for the large-scale application of exosomes. Summary of the Invention

[0009] To overcome the deficiencies and shortcomings of the existing plant exosome extraction methods, the inventors first proposed a method for separating and extracting plant exosomes using layer-by-layer filtration. This method is simple, efficient, and suitable for industrial scale (as shown in Figure 1), and the separated and purified exosomes have a high purity.

[0010] Specifically, this paper uses both layer-by-layer filtration and ultracentrifugation to isolate plant exosomes from wolfberry, ginseng, dendrobium officinale, polygonatum, angelica sinensis, and rehmannia root. By analyzing the physical properties and efficacy of the isolated plant exosomes and combining them with plant metabolomics analysis, the authors compared the differences between the plant exosomes extracted using the two methods, thereby identifying a method suitable for the isolation and extraction of plant exosomes.

[0011] The results showed that compared with ultracentrifugation, layer-by-layer filtration reduced the processing time by three times and increased the yield by at least 40 times for the same mass of plant material. Metabolomics analysis revealed that plant exosomes extracted by layer-by-layer filtration contained more active metabolites. The advantages of layer-by-layer filtration for plant exosome extraction became even more pronounced when the plant material was scaled up 10-fold or even 20-fold. Furthermore, the plant exosomes extracted by layer-by-layer filtration had a typical exosome-like saucer shape, were rich in protein, and exhibited anti-inflammatory and immunomodulatory functional activities in vitro.

[0012] Specifically, the layer-by-layer filtration method disclosed herein is time-efficient compared to ultracentrifugation, achieves higher exosome yields or particle concentrations, prevents membrane clogging due to excessive impurities, and ensures product sterility. Plant exosomes extracted using the layer-by-layer filtration method disclosed herein have a typical exosome-like saucer shape, a particle size range of 30-200 nm, and exhibit excellent anti-inflammatory and immunomodulatory activities.

[0013] The plant exosome extraction method disclosed herein is suitable for large-scale production; preferably, the method can process the amount of plant material corresponding to large-scale production by increasing the membrane area for filtration, for example, 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 of plant material.

[0014] Therefore, in one aspect, the present disclosure provides a method for isolating and extracting plant exosomes, specifically a method for isolating and extracting plant exosomes by deep filtration, the method comprising the following steps:

[0015] (a) obtaining plant material;

[0016] (b) pre-treating the obtained plant material to obtain a crude plant extract; and

[0017] (c) filtering the obtained crude plant extract layer by layer,

[0018] Wherein, in step (c), the layer-by-layer filtration treatment includes a deep filtration step and optionally a membrane filtration step.

[0019] In one embodiment of the method for isolating and extracting plant exosomes disclosed herein, in step (c), the layer-by-layer filtration step comprises using depth filtration, preferably, the depth filtration adopts 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 comprises a depth filtration step, and the depth filtration step adopts a deep capsule filtration (preferably with a pore size of about 1-50 μm), more preferably, the deep capsule filtration is a deep capsule filtration with a pore size of about 2-30 μm.

[0020] 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.

[0021] In one embodiment of the method for isolating and extracting plant exosomes disclosed herein, the layer-by-layer filtration further 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.

[0022] In one embodiment of the method for isolating 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, or about 30-100 mL / min.

[0023] In one embodiment of the method for isolating and extracting plant exosomes disclosed herein, the layer-by-layer filtration in step (c) consists of depth filtration as generally, specifically, or preferably defined herein and membrane filtration as generally, specifically, or preferably defined herein.

[0024] In one embodiment of the method for isolating and extracting plant exosomes disclosed herein, in step (b), the pretreatment comprises one or more of mixing, crushing, grinding, stirring, filtering, and centrifugation to obtain a crude plant extract. In a specific embodiment, the pretreatment comprises mixing, crushing, filtering, and centrifugation.

[0025] 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.

[0026] 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 subrange 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).

[0027] In the method embodiment of the present disclosure, the plant material can be terrestrial or aquatic. Preferably, the plant material comprises wolfberry, ginseng, dendrobium, polygonatum, angelica and / or rehmannia.

[0028] On the other hand, the present disclosure provides a plant exosome prepared by the above-mentioned general or preferred method embodiments; 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 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.

[0029] In another aspect, the present disclosure provides a plant 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 that is acceptable for nutrition, medicine, food, dermatology and / or cosmetics.

[0030] In a specific embodiment, the exosome composition is a pharmaceutical composition. In a specific embodiment, the exosome composition comprises at least one of Lycium barbarum exosomes, Panax ginseng exosomes, Dendrobium candidum exosomes, Polygonatum sibiricum exosomes, Angelica sinensis exosomes, and Rehmannia glutinosa exosomes prepared by the disclosed method; and an optional pharmaceutically acceptable carrier.

[0031] In a specific embodiment, the exosome composition is a food composition comprising plant exosomes prepared by the methods generally or preferably defined in the present disclosure, and optionally other food ingredients and / or edible excipients and / or carriers. In a specific embodiment, the food composition comprises at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes prepared by the methods of the present disclosure, and optionally other food ingredients and / or edible excipients and / or carriers.

[0032] In a specific embodiment, the exosome composition is a cosmetic composition comprising plant exosomes prepared by the methods generally or preferably defined herein, and optionally a cosmetically acceptable excipient or carrier. In a specific embodiment, the cosmetic composition comprises at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes prepared by the methods of the present disclosure, and at least one cosmetically acceptable excipient or carrier.

[0033] In a specific embodiment, the exosome composition is a nutraceutical composition comprising plant exosomes prepared by the methods generally or preferably defined herein, and optionally a nutraceutical-acceptable excipient or carrier. In a specific embodiment, the nutraceutical composition comprises at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes prepared by the methods of the present disclosure, and at least one nutraceutical-acceptable excipient or carrier.

[0034] On the other hand, the present disclosure also provides the use of plant exosomes prepared by the method of the present disclosure or compositions containing the same (including but not limited to pharmaceutical compositions / food compositions / cosmetic or cosmetic compositions / nutritional health care compositions), which are used to treat or prevent - or help (or assist in) improving, alleviating or controlling - inflammation, preferably skin inflammation, or in the field of dermatology for - or assist in - repair, anti-inflammatory, anti-oxidation, anti-aging, whitening or moisturizing.

[0035] On the other hand, the present disclosure also provides the use of plant exosomes prepared by the method of the present disclosure or compositions containing the same (including but not limited to pharmaceutical compositions / food compositions / cosmetic compositions / nutraceutical compositions) in the preparation of products (drugs, foods, nutraceuticals, beauty / cosmetics, etc.), wherein the products are used to treat or prevent - or help (or assist in) improving, alleviating or controlling - inflammation, preferably skin inflammation, or are used or assisted in repair, anti-inflammatory, anti-oxidation, anti-aging, whitening or moisturizing in the field of dermatology.

[0036] 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, comprising administering to a subject in need thereof plant exosomes prepared by the above-mentioned general or preferred method or a composition (pharmaceutical composition / food composition / cosmetic or cosmetic composition / nutraceutical composition) containing the same.

[0037] In another aspect, the present disclosure provides use of the plant exosomes or plant exosome compositions in biomedicine or drug delivery systems.

[0038] In another aspect, the present disclosure further provides a method for preparing a pharmaceutical, food, nutritional supplement, or beauty / cosmetic composition, comprising preparing plant exosomes according to the generally or preferably defined methods of the present disclosure, and incorporating the plant exosomes into the pharmaceutical, food, nutritional supplement, or beauty / cosmetic composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The following drawings illustrate preferred embodiments of the present disclosure. For the purpose of illustrating the present disclosure, the drawings show currently preferred embodiments. However, it should be understood that the present disclosure is not limited to the specific embodiments shown in the drawings.

[0040] FIG1 shows an exemplary process flow of the plant exosome isolation and extraction method disclosed herein.

[0041] Figure 2 shows the compound classification of six plant exosomes extracted by the layer-by-layer filtration method disclosed herein; A: GQV01; B: RSV01; C: SHV01; D: HJV01; E: DGV01; F: DHV01.

[0042] Figure 3 shows the compound classification of six plant exosomes extracted by ultracentrifugation; A: LyE01; B: GsE01; C: DeE01; D: PoE01; E: AnE01; F: ReE01.

[0043] Figure 4 shows a volcano plot of differential metabolite profiles in six plant exosomes extracted using layer-by-layer filtration and ultracentrifugation. A: GQV01 vs. LyE01; B: RSV01 vs. GsE01; C: SHV01 vs. DeE01; D: HJV01 vs. PoE01; E: DGV01 vs. AnE01; F: DHV01 vs. ReE01. The horizontal axis represents the fold change (log2FC) of the metabolite expression difference between the two groups; the vertical axis represents the statistical test value (-log10 p-value) of the difference in metabolite expression. Both horizontal and vertical axes are logarithmically processed.

[0044] Figure 5 shows KEGG pathway enrichment analysis of six plant exosomes extracted using layer-by-layer filtration and ultracentrifugation; A: GQV01 vs. LyE01; B: RSV01 vs. GsE01; C: SHV01 vs. DeE01; D: HJV01 vs. PoE01; E: DGV01 vs. AnE01; F: DHV01 vs. ReE01. The horizontal axis represents the pathway name, and the vertical axis represents the enrichment ratio, 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). A larger ratio indicates a higher degree of enrichment. The color gradient of the column indicates the significance of enrichment. By default, darker colors indicate more significant enrichment of the KEGG term. P values ​​< 0.001 are marked with ***, P values ​​< 0.01 are marked with **, and P values ​​< 0.05 are marked with *.

[0045] Figure 6 shows TEM analysis images of exosomes extracted by layer-by-layer filtration separation; A: GQV01; B: RSV01; C: SHV01; D: HJV01; E: DGV01; F: DHV01.

[0046] Figure 7 shows the in vitro PKH67 staining flow cytometric analysis of exosomes extracted by layer-by-layer filtration separation; A: GQV01; B: RSV01; C: SHV01; D: HJV01; E: DGV01; F: DHV01.

[0047] Figure 8 shows the activity assays of six plant exosomes extracted by layer-by-layer filtration; A: Protein concentration of the six plant exosomes. B: Inhibition of LPS-induced TNF-α release by RAW264.7 cells by the six plant exosomes. The vertical axis represents the TNF-α concentration detected by the ELISA kit. P values ​​< 0.001 are marked with ***, and P values ​​< 0.01 are marked with **.

[0048] Detailed Description of the Invention

[0049] I. Definitions and Terminology

[0050] Unless defined otherwise, 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 belongs.

[0051] As used herein, "vesicles," also called extracellular vesicles, are membrane-enclosed structures that are released by cells in vitro and in vivo. Extracellular vesicles can contain proteins, lipids, and nucleic acids and can mediate intercellular communication between different cells (including different cell types) in vivo. Two types of extracellular vesicles are exosomes and microvesicles.

[0052] As used herein, "exosomes" refer to lipid-bound, cell-secreted vesicles that are released from cells through fusion of multivesicular endosomes (MVEs) with the plasma membrane and mediate intercellular communication through the intercellular transport of proteins and RNA. Typically, exosomes range in size from about 30 nm to about 200 nm, and can also range from 30-150 nm or 30-100 nm, with a double-layer membrane structure and a saucer-shaped morphology.

[0053] As used herein, "plant material" has the meaning generally understood in the art. The plant material can be fresh or dried. Generally, with the exception of certain plants (e.g., wolfberry), it is more preferred to use fresh plant material to produce plant exosomes.

[0054] As used herein, "depth filtration (DF)" refers to the removal of particles (e.g., impurities) from a liquid, such as a solution, using a depth filtration medium to retain particles and impurities throughout the depth of the porous structure of the filter medium through a combination of size (sieving / filtration) and intermolecular interactions (e.g., electrostatic attraction and hydrophobic interactions between oppositely charged surfaces). Depth filtration has been successfully used for the initial clarification of bacterial, yeast, insect, and mammalian cell suspensions, and can be used alone or in combination with centrifugation. The depth filtration medium can comprise cellulose (e.g., cellulose fibers) and / or polypropylene (e.g., polypropylene fibers), and / or filter aids (e.g., activated carbon, diatomaceous earth (DE), and / or perlite), and / or resins (e.g., polymeric resins). In some embodiments, medium-sized particles are less removed by depth filtration; very small particles are effectively removed by Brownian diffusion (plus adsorption) (e.g., particles smaller than the pores of the medium can enter the interior of the medium), while very large particles are captured by physical sieving or interception (e.g., being trapped and adhering to the medium).

[0055] The term "depth filter" or "depth capsule filter" as used herein is to achieve filtration within the depth of the filter material. Such filters are those that contain a matrix of random fibers that are combined to form a complex maze of tortuous flow channels. The separation of particles in these filters is caused by being trapped by or adsorbed onto the fiber matrix. The most frequently used depth filter media for bioprocessing of cell culture broth and other feeds include cellulose fibers, filter aids such as DE and positively charged resin binders. Unlike absolute filters, depth filter media retains particles throughout the porous medium, thereby allowing particles larger than the pore size to be retained. Companies currently in the industry that have deep filter media products include Sartorius, Merck Millipore, Pall, 3M and other companies. Commercially available depth filters include but are not limited to Millistak+Pod depth filter systems, XOHC media (Millipore), Zeta Plus TM Depth filter (3MPurification Company) etc. In the present disclosure, depth filtration can be performed using two or more depth filters arranged in parallel. In this case, commercially available depth filters may include, for example, Millistak+ mini DOHC (Millipore Company) and XOHC filter (Millipore Company) or Pall Corporation filter (Pall Corporation NY), such as Supracap TM Depth Filter Capsules.

[0056] In the context of this document, the terms "cosmetics," "cosmetic compositions," "cosmetic products," or "cosmetic compositions" refer to industrial chemicals or fine chemical products that are applied to any part of the human body, such as the skin, hair, nails, lips, and teeth, by smearing, spraying, or other similar methods for the purpose of cleansing, maintaining, beautifying, modifying, or altering appearance, or correcting body odor or maintaining good condition. In some embodiments, the cosmetics, cosmetic compositions, cosmetic products, or cosmetic compositions of the present disclosure are for non-therapeutic purposes.

[0057] In the present context, the term "nutraceutical composition" refers to a product used to supplement the nutrient requirements of the human body, improve physical health and physical function, and can be administered enterally or parenterally, usually enterally.

[0058] In the present context, "skin" is understood to include the layers thereof, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous layer (including both the uppermost layer or stratum corneum and the lowermost layer or subcutaneous layer). These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, mast cells, neurons and / or adipocytes. The term "skin" also includes the scalp. The term "skin" includes mammalian skin and includes human skin. Similarly, the term "hair, nails and mucous membranes" includes the hair, nails and mucous membranes of mammals (e.g., humans).

[0059] In the context of this article, the term "treatment" encompasses treatment methods, including methods involving the administration of an extract according to the present disclosure to alleviate or eliminate a disease or condition or to reduce or eliminate one or more symptoms associated with the disease or condition. The term "treatment" also encompasses treatment methods that involve alleviating or eliminating the physiological consequences of a disease or condition.

[0060] In the present context, the term "care" refers to maintaining the properties of the skin, hair, nails and / or mucous membranes by improving or maintaining said properties by cosmetic treatment and / or care in healthy subjects as well as in subjects with sensitive skin and subjects showing symptoms of the skin, hair, nails and / or mucous membranes such as, but not limited to, ulcers and skin lesions, psoriasis, dermatitis, acne or rosacea.

[0061] In the context of this document, when the terms "treatment" and "care" are accompanied by the modifiers "cosmetic" and / or "non-therapeutic", it means that the purpose of the treatment or care is to help or assist in improving, alleviating or controlling the condition in question, such as helping or assisting in improving or maintaining the cosmetic properties of the skin, hair, nails and / or mucous membranes, which will affect the aesthetic appearance of the skin, hair, nails and / or mucous membranes, specifically, for example, helping or assisting in repairing, anti-inflammatory, anti-oxidant, anti-aging, whitening or moisturizing, etc., to improve the hydration, elasticity, firmness, radiance, tone or texture of the skin, hair, nails and / or mucous membranes.

[0062] In the context of this article, the term "prevent" refers to the ability of the extracts of the present disclosure to prevent, delay or hinder the occurrence or development of a disease or condition, 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 "prevent" can be used interchangeably with the term "inhibit the occurrence," that is, it refers to the ability of the extracts of the present disclosure to inhibit the occurrence or development of a disease or condition, or to inhibit changes in the cosmetic properties of the skin, hair, nails and / or mucous membranes.

[0063] In the context of this article, when used with respect to a specific numerical value or range of values, the term "about" means that the numerical value associated therewith fluctuates by ±10%, for example, by ±5%, ±2%, or ±1%. For example, the expression "about 100" as used herein includes 90 and 110 and all values ​​therebetween (e.g., 90.5, 95, 101, 105, 109.95, etc.). For a ratio, the term "about" is used to qualify each digit of the given ratio, for example, a ratio of "about 1:1" means a ratio of (0.9-1.1):(0.9-1.1), and for another example, a range of "about nm" or "about n-about m" means 90% n-110% n to 90% m-110% m.

[0064] 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.

[0065] All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0066] II. Preparation of Exosomes

[0067] raw material

[0068] In some embodiments, the raw materials involved in the methods of the present disclosure may include animal materials or plant materials. Preferably, the raw materials are plant or fruit materials. The plant materials can be terrestrial or aquatic. Preferably, the plant materials are succulent plants or fruits.

[0069] The plant material may be part or all of a plant, for example, may be selected from wood, roots, rhizomes, bark, trunks, flowers, petals, sepals, seeds, fruits, stems, leaves and / or embryos, and mixtures of more than one thereof.

[0070] In some embodiments, the plant material is or includes succulent plants or fruits, including but not limited to grapes, cucumbers, watermelons, tomatoes, strawberries, blueberries, cherries, bitter melon, purslane, ginseng, Codonopsis pilosula, Pseudostellaria baicalensis, American ginseng, Salvia miltiorrhiza, Angelica dahurica, Peucedanum chinense, Achyranthes bidentata, Belamcanda chinensis, Polygonum cuspidatum, Phytolacca japonica, Pueraria lobata, Poria cocos, Scrophularia ningpoensis, red dates, Chinese yam, Polygonatum sibiricum, Astragalus membranaceus, garlic, mulberry fruit, wolfberry fruit, ginger, litchi, longan, Epimedium brevicornum, mint, Bupleurum chinense, dandelion, Phragmites australis, honeysuckle, forsythia suspensa, Andrographis paniculata, Houttuynia cordata, rhubarb, aloe vera, senna leaf, Schisandra chinensis, Gastrodia elata, Dendrobium candidum or other succulent plants, or a mixture of more than one thereof.

[0071] In some embodiments, the plant material includes medicinal materials, including but not limited to rehmannia root, polygonatum, morinda officinalis, cooked rehmannia root, ginseng, dendrobium (including dendrobium officinale), deer antler, zezhi, meat date, withered fragrance, chicken blood vine, Sixian, Xuanji, glossy privet fruit, cattle strong, Acanthopanax senticosus, Qianjinba, five-fingered peach, Achyranthes bidentata, Gangqian, mistletoe, dayyun, longan meat, Panlong ginseng, dragon bean, yam, wind tail banana leaf, paper mulberry, white cardamom, angelica, heather leaf, chaos clothing, wolfberry, clove, cinnamon, raspberry, vitex, astragalus, mountain arrowhead, Kochia fruit, inula flower, gastrodia elata, cnidium monnieri, sheep placenta, lemon, atractylodes, mountain lentil, asparagus, white The plant material comprises: Chinese wolfberry, ginseng, dendrobium, angelica sinensis, curculigo, dodder seed, ramie, gypsophila, safflower, safflower, dandelion, cordyceps sinensis, toad oil, polygala, alpinia oxyphylla, mountain conch, green box, tuckahoe, anemarrhena, costus root, aquilaria sinensis, fennel, astragalus, coix seed, codonopsis pilosula, cibotrya japonica, schizonepeta tenuifolia, shizophylla, shizophylla, atractylodes macrocephala, adenophora australis, polygonatum, ox hoof nail, cibotrya japonica, acanthopanax chinensis, drynaria root, eucommia bark, rhizoma dioscoreae, diamond wind, nine-section tea, ant, ginkgo leaf, eucommia leaf, seven-leaf gall, ganoderma lucidum, umbellate, verbena, summer herb, notoginseng, usnea, safflower, bamboo leaf, jujube, wolfberry, ophiopogon japonicus, epimedium, walnut kernel, cistanche deserticola. Preferably, the plant material comprises wolfberry, ginseng, dendrobium, polygonatum, angelica sinensis, rehmannia root or a mixture of more than one of them.

[0072] In some embodiments, the plant material is or includes Dendrobium officinale. The Dendrobium officinale used in the disclosed methods can be dried or fresh, such as fresh or dried stems, or processed traditional Chinese medicine Dendrobium officinale. Examples of Dendrobium officinale that can be used in the disclosed methods include, but are not limited to, Dendrobium candidum, Dendrobium nobile, Dendrobium slender stem, Dendrobium microflorum, Dendrobium chrysotoxum, Dendrobium hornbeam, Dendrobium scutellariae, Dendrobium circumflexum, all or part of Dendrobium iris, or fresh stems thereof, or mixtures of more than one thereof. The disclosed methods can use mixtures of more than one Dendrobium officinale as the plant material for exosome extraction.

[0073] In some embodiments, plant material is or includes ginseng (Panax ginseng CA mate.), is Araliaceae, genus Panax (genus Panax) perennial herb. Ginseng that can be used for the disclosed method can be one or more of the seeds, roots, stems, leaves and fruits of Panax plants, preferably roots, preferably dry ginseng roots, or various processed Chinese medicine slices ginseng. Specifically, the plant in Panax can be Korean ginseng (Panax ginseng), American ginseng (Panax quinquefoli μm), Panax notoginseng (Panax notoginseng), bamboo ginseng (Panax japonic μm), three-leaf ginseng (Panax trifoli μm), Himalayan ginseng (Panax pseudoginseng), Vietnamese ginseng (Panax vietnamensis) etc. Specifically, the ginseng that can be used for the disclosed method can be garden ginseng (cultivated ginseng) or mountain ginseng (wild ginseng), specifically including raw sun-dried ginseng, sugar ginseng, raw sun-dried mountain ginseng, red ginseng, white ginseng etc. The disclosed method can use one or more ginseng species or a mixture of one or more ginseng parts as plant materials to extract exosomes.

[0074] In some embodiments, the plant material is or includes wolfberry (Lycium chinense Miller), a perennial woody plant of the genus Lycium in the Solanaceae family. Forms of wolfberry that can be used in the disclosed methods include dried wolfberry, wolfberry powder, wolfberry extracts, physiologically acceptable salts thereof, and derivatives thereof. Wolfberries that can be used in the disclosed methods include, but are not limited to, Ningxia wolfberry, Chinese wolfberry, black wolfberry, Xinjiang wolfberry, truncate wolfberry, cylindrical wolfberry, Yunnan wolfberry, and Changji wolfberry. Furthermore, wolfberry also includes wolfberry varieties, such as yellow wolfberry, northern wolfberry, or red-branched wolfberry. The disclosed methods can use a mixture of more than one wolfberry as the plant material for exosome extraction.

[0075] In some embodiments, the plant material is or includes Polygonatum sibiricum Delar.ex Redoute, a perennial herb of the genus Polygonatum in the family Asparagaceae, also known as chicken-head ginseng, deer bamboo, pen tube vegetable, and tiger ginger. Polygonatum that can be used in the disclosed methods is particularly rhizome, such as dried rhizome, such as processed Polygonatum such as black bean-processed Polygonatum, honey-processed Polygonatum, wine-processed Polygonatum, or steamed pure Polygonatum. Species of Polygonatum that can be used in the disclosed methods include, but are not limited to, Polygonatum sibiricum Redoute, Polygonatum yunnanensis (Polygonatum coll. et Hemsl.), or Polygonatum cyrtonema Hua, or portions thereof. The disclosed methods can use a mixture of more than one Polygonatum species as the plant material for exosome extraction.

[0076] In some embodiments, the plant material is or includes Angelica sinensis (Oliv.) Diels, a perennial herbaceous plant of the Apiaceae family. Angelica sinensis that can be used in the methods disclosed herein is particularly its root, such as a dried root, in forms including, but not limited to, powder, flakes, or blocks. Types of Angelica sinensis that can be used in the methods disclosed herein include, but are not limited to, Qin Angelica, Sichuan Angelica, Western Angelica, Yunnan Angelica, Eastern Angelica, Taiwan Angelica, Sichuan Angelica, Minxian Angelica, Beihai Angelica, Yamato Angelica, and Korean Angelica. The methods disclosed herein can use a mixture of more than one Angelica species as the plant material for exosome extraction.

[0077] In some embodiments, the plant material is or includes Rehmannia glutinosa (Gaert.) Libosch. ex Fisch. et Mey., a perennial herbaceous plant of the genus Rehmannia in the family Scrophulariaceae. The Rehmannia root used in the disclosed methods can be fresh or dried, the former commonly known as "fresh Rehmannia" and the latter commonly known as "raw Rehmannia," or processed Rehmannia glutinosa, which can be in powdered, flaky, or block form. Types of Rehmannia glutinosa that can be used in the disclosed methods include, but are not limited to, Tianmu Rehmannia, Gaodihuang, Rehmannia glutinosa, Hubei Rehmannia, Split Leaf Rehmannia, and Solanum lycopersicum. The disclosed methods can use a mixture of more than one Rehmannia root as the plant material for exosome extraction.

[0078] Preprocessing

[0079] In some embodiments, the extraction method includes pre-treating the plant material. Pre-treatment techniques are well known in the art and include physical, chemical, and biological pre-treatments, or any combination thereof.

[0080] 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 performed in a sterile environment.

[0081] In some embodiments, the pre-treatment cleaning can be performed with an aqueous carrier or other edible or pharmaceutically acceptable solvent (including organic 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 process further comprises a certain amount of a surfactant in the cleaning solvent. More preferably, the cleaning agent is water or water for injection.

[0082] 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 buffered saline, Ringer's injection, isotonic glucose injection, and Ringer's injection of glucose and lactate. Isotonic agents include isotonic agents selected from sodium chloride, mannitol, lactose, glucose (aqueous or anhydrous), sucrose, glycerol, and sorbitol, or a solution of any 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.

[0083] In some embodiments, the plant material is mixed with the isotonic solution in a certain ratio. For example, the plant material is mixed with the isotonic solution according to the following mass volume ratio (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 volume ratio of the plant material to the isotonic solution is in the range of about 1:2-1:50, about 1:3-1:20 or about 1:5-1:20.

[0084] In some embodiments, the pulverization in the pretreatment includes any suitable method known in the art, including but not limited to dry grinding, wet grinding, and vibration ball milling. For example, the pulverization includes pulverizing the plant or part using a pulverization technique or a wall-breaking technique. The wall-breaking technique uses a wall-breaking machine.

[0085] In some embodiments, the pretreatment can be carried out at any suitable temperature. For example, the pretreatment 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 subrange 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, step (b) of the present disclosure can be carried out at ambient temperature (e.g., 25°C).

[0086] In some embodiments, the stirring, filtering or centrifugation operations in the pretreatment can be selected and adjusted by those skilled in the art according to specific needs.

[0087] Layer-by-layer filtering

[0088] The plant exosome extraction method disclosed herein includes the steps of layer-by-layer filtration.

[0089] The layer-by-layer filtration of the disclosed method 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 performed independently or continuously. In some embodiments, the layer-by-layer filtration step includes a deep 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 deep filtration step, anti-clogging filtration and sterilizing filtration, preferably the anti-clogging filtration may include 0.4-0.8 μm membrane filtration and the sterilizing filtration includes 0.1-0.3 μm membrane filtration.

[0090] The methods of the present disclosure can be used to treat the plants at a suitable rate. For example, for a production batch, the methods of the present disclosure can treat the plants at a rate in the range of 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 subrange between about 10 g / hour and about 1,000 g / hour (e.g., any range between any two of the above production rates).

[0091] The various filtration steps of the layer-by-layer filtration of the method disclosed herein may be the same or different. For example, the filtration pore size is different between different filtration steps. In some embodiments, the layer-by-layer filtration includes one filtration step, wherein the filtration step uses two filtration units with different pore sizes, and preferably the two pore sizes are successively smaller along the direction of the filtrate flow, for example, the first filtration pore size is about 0.45 μm, and the second filtration pore size is about 0.22 μm. In some embodiments, the layer-by-layer filtration includes two 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 are successively smaller along the direction of the filtrate flow, 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; for another example, the pore size in the first filtration step is about 2-30 μm (such as 30 μm), and the pore size in the second filtration step is about 0.45 μm. In some embodiments, the layer-by-layer filtration includes three 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 exactly the same or at least partially different. Preferably, the three pore sizes become smaller in sequence along the flow direction of the filtrate, 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.

[0092] In the method of the present disclosure, the pore size in the filtering 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 size in the filtration step can be a range composed of any two of the above pore sizes, for example, 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. 3-0.6μm, 0.4-1.0μm, 0.4-0.8μm, 0.45-0.8μm, 0.5-100μm, 1-100μm, 1-50μm m, 1-40μm, 2-50μm, 2-40μm, 2-30μm, 2-20μm, 2-10μm, 2-5μm, 2-4μm, 3-50μm, 3-40μm, 3-30μm, 3-20μm, 3-10μm, 3-5μm, 3-4μm, 4-50μm, 4-40μm, 4-30μm, 4 -20μm, 4-10μm, 4-5μm, 5-50μm, 5-40μm, 5-30μm, 5-20μm, 5-10μm, 6-50μm, 6- 40μm, 6-30μm, 6-20μm, 6-10μm, 7-50μm, 7-40μm, 7-30μm, 7-20μm, 7-10μm, 8 -50μm, 8-40μm, 8-30μm, 8-20μm, 8-10μm, 9-50μm, 9-40μm, 9-30μm, 9-20μm, 9 -10 μm, preferably the pore size of the layer-by-layer filtration can be selected from about 1-50 μm, 2-30 μm, 2-20 μm, 6-30 μm, 0.05-1 μm, 0.1-0.8 μm, 0.1-0.22 μm, 0.1-0.3 μm, 0.2-0.8 μm, 0.2-0.6 μm, 0.2-0.4 μm, 0.3-0.8 μm, 0.4-1.0 μm, 0.4-0.8 μm, 0.45-0.8 μm. In some embodiments, more preferably, the pore size in the filtration step can be selected from about 1-50 μm, 0.4-0.8 μm and 0.1-0.3 μm, or a combination thereof, such as about 2-30 μm, about 0.45-0.8 μm or about 0.1-0.22 μm, such as about 6-30 μm, about 0.45 μm or about 0.22 μm.

[0093] In the disclosed method, the filtering step in the layer-by-layer filtering can be performed in one device, wherein the device includes a plurality of separate units with a filtering function. In some embodiments, the layer-by-layer filtering can be performed in two or more devices, wherein each device includes one or more separate units with a filtering function. In some embodiments, the layer-by-layer filtering can be performed in one device, wherein 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 separate units with a filtering function.

[0094] In the disclosed method, the layer-by-layer filtration includes the use of a solvent, such as an organic solvent or an inorganic solvent used in conventional extraction, for example, water, an isotonic aqueous solution of sodium chloride, ethanol, acetone, ether, petroleum ether, ethyl acetate, or a mixture thereof. In some embodiments, the solvent 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 / min, 2 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min, 45 mL / min, 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min, 90 mL / min, 100 mL / min, 200 mL / min, 300 mL / min, 400 mL / min, 500 mL / min, 600 mL / min, 700 mL / min, 800 mL / min, 900 mL / min, or 1,000 mL / min, or from about 1 mL / min to about The flow rate of the solvent may be as high as about 1 L / min or even higher. In some embodiments, the flow rate of the solvent in the layer-by-layer filtration of the disclosed method is about 20 mL / min to about 1000 mL / min, for example, about 20 mL / min to about 500 mL / min, about 20 to about 200 mL / min, about 30 to about 100 mL / min.

[0095] 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, for example, 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), for example, about 0°C to about 35°C, about 0°C to about 30°C, 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 (for example, 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 deep 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.

[0096] In general, any suitable filter 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 acid-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylysine or polyglutamic acid, polyethersulfone (PES) (unmodified), polyethersulfone (mPES), polyvinylidene fluoride (PVDF), cellulose acetate, nitrocellulose, MCE (mixed cellulose esters), ultra-high molecular weight polyethylene (UPE), polyfluorotetraethylene (PTFE), nylon, polysulfone, polyacrylonitrile, polypropylene, polyvinyl chloride, polycarbonate, ceramics, diatomaceous earth, glass fiber, resin-bonded glass fiber and combinations thereof. The available forms of filter material include but are not limited to microporous membrane, homogeneous membrane, asymmetric membrane.

[0097] In some embodiments, layer-by-layer filtration comprises filtering using a filter membrane comprising the following polymers: nylon, copolymers of acrylic acid, polysulfone, polyvinylidene fluoride, cellulose esters and cellulose esters. The filter membrane is typically made of a polymeric support material, such as PTFE (polytetrafluoroethylene), PES (polyethersulfone), PVP (polyvinyl pyrrolidine), PVDF (polyvinylidene fluoride), nylon (polyamide), PP (polypropylene), cellulose (including cellulose esters), PEEK (polyethyl ether ether ketone), nitrocellulose, glass fiber, resin-bonded glass fiber, etc. In some embodiments, the polymer can be a hydrophilic polymer. Hydrophilic membranes include, but are not limited to, Bioassure (from Cuno); EverLUX TM Polyethersulfone; STyLUX TMPolyethersulfone (all from Meissner); Millex GV, Millex HP, Millipak 60, Millipak 200 and Durapore CVGL01TP3 membranes (from Millipore); Fluorodyne TM EX EDF membrane, Supor TM EAV、Supor TM EBV, Supor TM EKV (both from Pall); Sartopore TM (from Sartorius); hydrophilic PES membranes from Sterlitech; and WFPES PES membranes from Wolftechnik.

[0098] In other embodiments, the layer-by-layer filtration of the present disclosure, such as depth filtration, may further include adsorbents such as silica particles, diatomaceous earth, or carbon particles.

[0099] In some embodiments, the intrinsic properties of membrane can be changed by processing membrane surface.For example, it is known to prepare hydrophilic or hydrophobic membrane by processing with coating membrane surface with other materials (for example other polymers, graphite, silicon etc.).For example, the membrane used in layer by layer filtration can be positively charged modified, for example, the filter membrane in layer by layer filtration can be a kind of charge-modified polyvinylidene fluoride (PVDF) membrane produced by Micropore company, or the film of employing nylon 66 or positively charged polyethersulfone sulfate obtained from Pall company.Filter membrane can be sterilized (for example autoclaving) before use, to ensure its aseptic.

[0100] Deep filtration

[0101] The present disclosure provides a method for preparing plant exosomes, comprising a layer-by-layer filtration step, wherein the layer-by-layer filtration step has the function or effect of preventing or avoiding clogging. The method of the present disclosure can use any filtration method that has 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.

[0102] In some embodiments, the present disclosure provides a method for preparing plant exosomes, comprising a layer-by-layer filtration step, wherein the layer-by-layer filtration step comprises depth filtration. Preferably, in some embodiments, the depth filtration is achieved by a depth filter, such as a depth capsule filter.

[0103] In some embodiments, the depth filter is a commercially available depth filter, such as a depth filter manufactured by Pall Corporation (NY), such as Supracap TM Depth filters; also available from Millipore (Billerica, MA) such as MilliStak TM series of activated carbon filters; other depth filters may also be used: 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 may be a filter product of Pall Corporation (NY), such as Supracap TM Depth Filter Capsules, such as Supracap TM 50depth filter capsules, or Supracap TM 100Depth Filter Capsules.

[0104] The depth filtration capacity range that can be used for the disclosed method is about 50-200 L / m 2 , for example 50-150L / m 2 40-100L / m 2 .

[0105] For purposes of the present disclosure, a depth filter can be any filter having a depth filtration function. In some embodiments, a 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 a pore size range of about 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, 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 size of the depth filtration can be selected from about 1-50 μm, about 2-30 μm, about 2-20 μm, about 6-30 μm, more preferably a depth filter having a pore size of about 2-30 μm, such as a depth capsule filter having a pore size of about 2-30 μm, such as a Supracap. TM Depth Filter Capsules filter.

[0106] For purposes of the present disclosure, a depth filter can be any type of depth filter, such as a filter plate filter, a filter disc filter, a filter cartridge filter, a filter stack filter, a depth capsule filter, or a cartridge filter. In some embodiments, the depth filtration of the present disclosure is performed using a depth capsule filter. Depth capsule filters are commonly used for clarifying and filtering fermentation broths and cell culture fluids, filtering serum and blood products, filtering enzyme preparations, removing impurities from chemicals, decarbonization filtration, or filtering colloidal or viscous materials. The present inventors have unexpectedly discovered that depth capsule filters are also suitable for extracting plant materials, and are particularly suitable for the method of extracting plant exosomes disclosed herein.

[0107] The deep capsule filter may generally comprise a single-layer membrane structure or a double-layer membrane structure. The deep capsule filter used in the method of the present disclosure may be a single-layer membrane structure, which may, for example, comprise cellulose fibers, and / or filter aids (such as diatomaceous earth and perlite) and / or resins. In some embodiments, the deep capsule filter comprises two layers of membranes, wherein the upper filter plate has a large pore size to intercept large-sized particles and protect the lower filter plate; the lower filter plate has a small pore size to further intercept small particles and ensure the clarity of the filtered liquid. For example, the pore size of the first membrane layer may be about 11-30 μm, and the pore size of the second membrane layer may be about 6-15 μm; or the pore size of the first membrane layer may be about 8-20 μm, and the pore size of the second membrane layer may be about 2-4 μm.

[0108] In some embodiments, the material of the depth 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).

[0109] For the present disclosure, any suitable flow rate may be used for the deep filtration. For example, the filtration flow rate for the deep filtration of the disclosed method may range from 100 to 1000 LMH (1 m3 / h). 2 The liquid volume of the membrane package), for example, about 100LMH, about 200LMH, about 500LMH, about 800LMH, about 120-500LMH, about 500-1000LMH, or a range consisting of any two points between 100-1000LMH. Alternatively, the flow rate of the depth filtration of the disclosed method can be expressed in mL / min, for example, the depth filtration is performed using a flow rate in the range of about 1 mL / min to about 1,000 mL / min, such as, but not limited to, 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. Preferably, the feed rate of the present disclosure is about 20 mL / min to about 1000 mL / min, such as about 20 mL / min to about 500 mL / min, about 20-200 mL / min.

[0110] For purposes of the present disclosure, any suitable pressure can be used for the depth filtration. For example, the depth filtration can be performed 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 subrange within about 0.1 psi to about 100 psi (e.g., any range between any two of the foregoing pressures).

[0111] For the present disclosure, the layer-by-layer filtration (e.g., deep 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 (e.g., any range between any two of the above-mentioned time points) within about 10 minutes to about 10 hours. Preferably, for example, for the 500ml plant crude extract obtained by pretreatment, when using the layer-by-layer filtration comprising deep filtration and membrane filtration (e.g., 0.45 μm membrane filtration and 0.22 μm membrane filtration) defined in the present disclosure, the processing time is about 0.5 hour to about 2 hours, preferably about 1 hour, while the same plant crude extract is centrifuged twice at 12000g for 70min, consuming about 3 hours.

[0112] The disclosed method can be used to process the plants at a suitable rate. For example, for a production batch, the disclosed method processes plants, such as crude plant extracts, at a rate ranging from 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 subrange (e.g., any range between any two of the above-mentioned production rates) from about 10 g / hour to about 1,000 g / hour.

[0113] The method for preparing plant exosomes by layer-by-layer filtration (e.g., deep filtration and optional other filtration steps) disclosed herein can obtain the following amount of plant exosomes per 100 g of plant: 1×10 10 5×10 10 10×10 10 11×10 10 12×10 10 13×10 10 14×10 10 15×10 10 16×10 10 17×10 10 18×10 10 19×10 10 20×10 10 22×10 10 24×10 10 pcs, 26, ×10 1028×10 10 30×10 10 32×10 10 34×10 10 36×10 10 38×10 10 40×10 10 42×10 10 44×10 10 46×10 10 48×10 10 50×10 10 52×10 10 54×10 10 56×10 10 58×10 10 60×10 10 66×10 10 In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 20×10 10 -50×10 10 , preferably about 30×10 10 -40×10 10 For example, about 33×10 10 In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 20×10 10 -60×10 10 , preferably about 30×10 10 -50×10 10 For example, about 39×10 10 In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 10×10 10 -30×10 10 , preferably about 10×10 10 -20×10 10 For example, about 12×10 10 In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 10×10 10 -40×10 10 , preferably about 10×10 10 -30×10 10 For example, about 22×10 10 In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 30×10 plant exosomes per 100 g of Angelica sinensis. 10 -70×10 10 pcs, preferably about 40×1010 -60×10 10 For example, about 50×10 10 In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 35×10 10 -80×10 10 pcs, preferably about 40×10 10 -60×10 10 For example, about 56×10 10 Plant exosomes.

[0114] The deep filter described in the present disclosure comprises a deep filter medium. In some embodiments, the deep filter 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 filter medium can be in the form of a filter plate. In some embodiments, the deep filter medium layer is optionally selected from one or more of Supradur P filter plate, K300 filter plate, and BECOPAD P 270 filter plate. 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 plate used in the present disclosure can be produced by American companies such as 3M, PALL, and EATON. Its commonly used models include 3M's SP series filter plates, PALL's P series filter plates, and EATON's BECO series filter plates.

[0115] In some embodiments, the depth filtration medium can be distributed in a gradient or mixed distribution. In some embodiments, the depth filtration medium can be distributed in a gradient. In some embodiments, the deep capsule filtration device has a double-layer membrane structure with a gradient distribution, and the pore size of the two membrane layers decreases along the direction of the filtrate flow. For example, the pore size of the first membrane layer can be about 11-30 μm, and the pore size of the second membrane layer can be about 6-15 μm; or the pore size of the first membrane layer can be about 8-20 μm, and the pore size of the second membrane layer can be about 2-4 μm.

[0116] In some embodiments, the deep filtration medium may further include a membrane filtration layer, for example, the filtration pore size of the membrane filtration layer is preferably about 0.1 to 10 μm. In some embodiments, the deep filtration medium of the present disclosure may be used in conjunction with a membrane filtration medium. In some embodiments, the membrane filtration layer is disposed at the liquid outlet end of the deep filtration medium. In some embodiments, the membrane filtration layer is disposed at the liquid inlet end of the deep filtration medium. In some embodiments, the membrane filtration material after deep capsule filtration is generally selected from hydrophilic materials with low protein adsorption, such as polyethersulfone (PES), polyvinylidene fluoride (PVDF), polypropylene (PP), etc.

[0117] In some embodiments, the depth filtration medium may further include a filter aid. In some embodiments, the filter aid may include diatomaceous earth, perlite, talc, silica gel, activated carbon, asbestos, molecular sieves, clay, etc. In some embodiments, the filter aid may be a mineral-based filter aid such as perlite, diatomaceous earth, or sand, or an activated carbon filter aid derived from natural materials such as wood or coconut shells.

[0118] In some embodiments, the deep filtration described in the present disclosure can be performed using 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 deep filtration can be performed using a peristaltic pump.

[0119] In some embodiments, the flow rate of the pump used for deep filtration can be in the range of about 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 deep filtration of the present disclosure is a peristaltic pump with a flow rate of, for example, about 20-1000 mL / min, such as about 20-500 mL / min, about 20-200 mL / min, or about 30-100 mL / min.

[0120] In some embodiments, the depth filter is rinsed with an aqueous solution during the depth filtration described herein, preferably with an isotonic solution, more preferably with an isotonic sodium chloride solution, and most preferably with sodium chloride injection. For example, in the depth filtration described herein, the depth filter capsule is preferably rinsed with sodium chloride injection at a flow rate of, for example, about 20-1000 mL / min, such as about 20-500 mL / min, about 20-200 mL / min, or about 30-100 mL / min, before the filtration begins.

[0121] In some embodiments, during the 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. For example, during the depth filtration described in the present disclosure, preferably at the end of the depth filtration, the depth filter capsule is cleaned with sodium chloride injection at a flow rate of, for example, about 20-1000 mL / min, such as about 20-500 mL / min, about 20-200 mL / min, or about 30-100 mL / min.

[0122] Membrane filtration

[0123] In some embodiments, layer-by-layer filtration further comprises membrane filtration. For example, the layer-by-layer filtration of the present disclosure comprises depth filtration and membrane filtration. For example, in the layer-by-layer filtration, the membrane filtration may comprise anti-clogging membrane filtration and sterilizing membrane filtration. In some embodiments, anti-clogging membrane filtration may be performed first, and then sterilizing membrane filtration may be performed optionally. In some embodiments, anti-clogging membrane filtration may be performed before sterilizing membrane filtration. In some embodiments, one or more anti-clogging membrane filtrations may be performed, followed by one or more sterilizing membrane filtrations.

[0124] 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 a range composed of any two of the above pore sizes, for example, but not limited to the following range: 0.0 5-2μm, 0.05-1μm, 0.05-0.8μm, 0.05-0.6μm, 0.05-0.4μm, 0.1-2μm, 0.1-1μm, 0.1-0.8μm, 0.1-0.6μm, 0.1-0.4μm, 0.1-0.3μ m, 0.1-0.22μm, 0.2-1μm, 0.2-0.8μm, 0.2-0.6μm, 0.2-0.4μm, 0.3-0.8μm, 0.3-0.6μm, 0.4-1.0μm, 0.4-0.8μm, 0.45-0.8μm. In some preferred embodiments, the pore size range of the membrane filtration 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.

[0125] In some embodiments, the layer-by-layer filtration of the present disclosure includes membrane filtration after deep filtration. For example, membrane filtration can include membrane filtration of about 0.4-0.8 μm (e.g., about 0.45 μm) and membrane filtration of about 0.1-0.3 μm (e.g., about 0.22 μm). In some embodiments, membrane filtration of about 0.4-0.8 μm (e.g., about 0.45 μm) can be performed first, and then membrane filtration of about 0.1-0.3 μm (e.g., about 0.22 μm) can be optionally performed. In some embodiments, membrane filtration of about 0.4-0.8 μm (e.g., about 0.45 μm) can be performed before membrane filtration of about 0.1-0.3 μm (e.g., about 0.22 μm). In some embodiments, one or more membrane filtrations at about 0.4-0.8 μm (e.g., about 0.45 μm) may be performed, followed by one or more membrane filtrations at about 0.1-0.3 μm (e.g., about 0.22 μm). Preferably, one or more membrane filtrations, preferably one membrane filtration at about 0.4-0.8 μm (e.g., about 0.45 μm), may be performed before the membrane filtration at about 0.1-0.3 μm (e.g., about 0.22 μm) to reduce or prevent clogging of the 0.22 μm membrane filter.

[0126] In some embodiments, the membrane filtration includes passing through one or more series membrane filters. In some embodiments, the number range of series membrane filters is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 series filters, preferably 1-4, more preferably 2-3. The cut-off sizes of all series membrane filters can be the same or different. In some embodiments, at least 2 of the series membrane filters have different cut-off sizes. In some embodiments, size exclusion decreases from large to small along the series membrane filter. For example, in 3 membrane filters in series, the first filter can be an approximately 0.45 μm filter, the second filter can be a 0.3 μm filter, and the last filter can be an approximately 0.22 filter.

[0127] 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, nitrocellulose membrane filters, glass fibers, resin-bonded glass fibers, or bead filters.

[0128] For the methods disclosed herein, the membrane used for membrane filtration can be any filter material commonly used in the art, such as the various filter materials and membrane materials described above in the layer-by-layer filtration section. In some embodiments, the anti-clogging membrane filtration is a resin-bonded glass fiber membrane, preferably with a pore size of approximately 0.45-0.8 μm. In some embodiments, the sterilizing membrane filtration is a polyethersulfone (PES) membrane, preferably with a pore size of approximately 0.1-0.22 μm.

[0129] In some embodiments, any suitable pressure can be used to carry out the membrane filtration.For example, the feed pressure of about 0.1psi to about 100psi can be used to carry out the membrane filtration, for example, about 0.1psi, 0.5psi, 1psi, 2psi, 3psi, 4psi, 5psi, 6psi, 7psi, 8psi, 9psi, 10psi, 20psi, 30psi, 40psi, 50psi, 60psi, 70psi, 80psi, 90psi, 100psi, or any sub-range (for example, any range between any two of the above-mentioned feed pressures) in about 0.1psi to about 100psi. Any suitable permeate end pressure can be used to carry out the membrane filtration. For example, the membrane filtration can be performed using a permeation pressure in the range of about 0.1 psi to about 100 psi, for example, 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 subrange within about 0.1 psi to about 100 psi (e.g., any range between any two of the above pressures).

[0130] In some embodiments, 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 peristaltic pump to carry out. In some embodiments, membrane filtration can use a peristaltic pump. In some embodiments, the flow rate of the pump used for membrane filtration can be following range: 1-1000mL / min, 100-1000mL / min, 200-1000mL / min, 500-1000mL / min, 10-800mL / min, 10-500mL / min, 20-1000mL / min, 20-500mL / min, 20-300mL / min, 20-200mL / min, 30-100mL / min, 40-100mL / min or 50-100mL / min. Preferably, the flow rate of the membrane filtration pump 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.

[0131] In some embodiments, the membrane filter is rinsed and / or cleaned using an aqueous solution, preferably an aqueous isotonic solution, more preferably an isotonic sodium chloride solution, during membrane filtration as described herein. Preferably, the membrane filter is cleaned using sodium chloride injection at a flow rate of about 20-1000 mL / min, for example, about 20-500 mL / min, about 20-200 mL / min, or about 30-100 mL / min.

[0132] In some embodiments, the membrane filtration can be carried out using any suitable flow rate. For example, the membrane filtration can be carried out using a flow rate ranging from about 1 mL / min to about 1,000 mL / min, for example, from about 1 mL / min to about 800 mL / min, from about 1 mL / min to about 500 mL / min, from about 1 mL / min to about 200 mL / min, from about 10 mL / min to about 1,000 mL / min, from about 10 mL / min to about 800 mL / min, from about 10 mL / min to about 500 mL / min, from about 10 mL / min to about 200 mL / min, from about 20 mL / min to about 1000 mL / min, from about 20 mL / min to about 800 mL / min, from about 20 mL / min to about 500 mL / min, from about 20 mL / min to about 200 mL / min, from about 50 mL / min to about 1000 mL / min, from about 50 mL / min to about 500 mL / min, from about 50 mL / min to about 200 mL / min, In large-scale production embodiments, the flow rate of membrane filtration can be as high as about 1 L / min or even higher. Preferably, the filtration flow rate of the present disclosure is about 20-1000 mL / min, such as about 20-200 mL / min, about 30-100 mL / min.

[0133] In some embodiments, any suitable time limit can be used to carry out described membrane filtration.For example, for a production batch, described membrane filtration can be carried out in the time range of about 10 minutes to about 10 hours, for example, 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 about 10 minutes to any sub-range (for example, any range between any two above-mentioned time points) in about 10 hours.Preferably, for the 500mL plant crude extract obtained by pre-treatment, carry out about 1 hour membrane filtration, preferably, when using to comprise that depth filtration, anti-clogging membrane filtration such as about 0.45 μm filters and sterilizing membrane filtration such as about 0.22 μm filters layer by layer filtration, the processing time is about 0.5 hour-about 2 hours, preferably about 1 hour.

[0134] In some embodiments, the method of the present disclosure comprising depth filtration and membrane filtration can obtain plant exosomes at a yield of about 1×10 10 5×10 10 10×10 10 11×10 10 12×10 10 13×10 10 14×10 10 15×1010 16×10 10 17×10 10 18×10 10 19×10 10 20×10 10 22×10 10 24×10 10 pcs, 26, ×10 10 28×10 10 30×10 10 32×10 10 34×10 10 36×10 10 38×10 10 40×10 10 42×10 10 44×10 10 46×10 10 48×10 10 50×10 10 52×10 10 54×10 10 56×10 10 58×10 10 66×10 10 In some embodiments, the membrane filtration of the present disclosure can obtain about 20×10 10 -50×10 10 , preferably about 33×10 10 In some embodiments, the membrane filtration of the present disclosure is sufficient to obtain about 30×10 10 -50×10 10 , preferably about 39×10 10 In some embodiments, the membrane filtration of the present disclosure can obtain about 10×10 10 -20×10 10 , preferably about 12×10 10 In some embodiments, the membrane filtration of the present disclosure is sufficient to obtain about 20×10 10 -30×10 10 , preferably about 22×10 10 In some embodiments, the membrane filtration of the present disclosure can obtain about 45×10 10 -60×10 10 , preferably about 50×10 10In some embodiments, the membrane filtration of the present disclosure is sufficient to obtain about 50×10 10 -65×10 10 , preferably about 56×10 10 Plant exosomes.

[0135] Preferred Implementation

[0136] In one embodiment, the method for preparing plant exosomes disclosed herein comprises the following steps:

[0137] (a) obtaining plant materials;

[0138] (b) pre-treating the obtained plant raw material to obtain a crude plant extract;

[0139] (c) filtering the crude plant extract layer by layer;

[0140] Wherein, the layer-by-layer filtration process includes a deep filtration step and an optional membrane filtration step.

[0141] In one embodiment, the method of preparing plant exosomes disclosed herein comprises the following steps:

[0142] (a) obtaining plant materials;

[0143] (b) pre-treating the obtained plant material, preferably mixing, crushing, filtering and centrifuging to obtain a crude plant extract; (c) filtering the crude plant extract layer by layer to obtain a filtrate;

[0144] wherein the layer-by-layer filtration process comprises a deep filtration step, preferably the deep 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

[0145] (d) The filtrate in step (c) is subjected to membrane filtration, preferably anti-clogging membrane filtration and / or 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, 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 0.45 μm anti-clogging membrane filtration and 0.22 μm sterilizing membrane filtration performed sequentially to obtain plant exosomes.

[0146] In the above embodiment, the obtained plant material is pretreated in step (b), wherein the pretreatment includes mixing the plant material with an isotonic solution (preferably sodium chloride injection), crushing the plant material using a wall breaking machine to obtain a plant residue liquid, filtering the plant residue liquid with a filter to obtain a plant juice, and centrifuging the plant juice to collect the supernatant to obtain a crude plant extract.

[0147] In the above embodiments, the plant material can be mixed and pulverized on a large scale in step (b). In some specific embodiments, the plant material is mixed and pulverized in step (b) on a mass of about 1000 g or even more, for example, about 500-1000 g, about 50-200 g, or about 100 g. In some embodiments, step (b) is performed at about 4°C.

[0148] In the above embodiment, the pulverized slag liquid is stirred in step (b), preferably at a stirring speed of about 50-500 rpm, more preferably about 100-250 rpm, and more preferably about 200±20 rpm. In some embodiments, the stirring time is about 10-200 min, preferably about 2-100 min, and more preferably about 30 min.

[0149] In some preferred embodiments, the centrifugation in step (b) is performed at about 1000-10000 g. In some preferred embodiments, the centrifugation in step (b) is performed 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 performed at about 4° C.

[0150] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes wolfberry.

[0151] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes ginseng.

[0152] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes Dendrobium, preferably Dendrobium officinale.

[0153] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes Polygonatum sibiricum.

[0154] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes Angelica sinensis.

[0155] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes Rehmannia glutinosa.

[0156] In a specific embodiment of the above method for preparing plant exosomes, the layer-by-layer filtration process includes a deep filtration step, wherein the deep filter is a deep capsule filter with a pore size of about 1-50 μm, preferably about 2-30 μm, and a loading range of about 50-200 L / m 2 , for example 50-150L / m 2 40-100L / m 2 ; Preferably the depth filter is a depth filter produced by Pall Corporation (Pall Corporation, NY), which is Supracap TM Depth Filter Capsules.

[0157] In a specific embodiment of the above-mentioned method for preparing plant exosomes, in step (4), the filtrate in step (3) is filtered at about 0.45 μm and then at about 0.22 μm, and the final filtrate is collected to obtain plant exosomes.

[0158] Technical Effects

[0159] The layer-by-layer filtration method disclosed herein has the following advantages over the ultracentrifugation method:

[0160] (1) Lower time consumption;

[0161] (2) higher exosome production or particle concentration;

[0162] (3) It can prevent the filter membrane from being blocked due to excessive impurities and ensure the sterility of the product;

[0163] (4) The extracted plant exosomes have excellent metabolomic performance;

[0164] (5) The extracted plant exosomes have a typical exosome-like saucer shape and a particle size range of 30–200 nm;

[0165] (6) The extracted plant exosomes have anti-inflammatory and immunomodulatory activities.

[0166] The plant exosome preparation method disclosed herein is suitable for small-scale, pilot-scale and large-scale production. In some embodiments, the exosome preparation method disclosed herein is particularly suitable for large-scale production, and the amount of plant material that can be processed is, for example, at least about 100g, 200g, 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1000g, 2000g, 3000g, 4000g, 5000g, 6000g, 7000g, 8000g, 9000g, 10000g, 50000g, or even up to 100000g. Preferably, the exosome preparation method disclosed herein can process plant material within about 1000g, more preferably plant material within about 10000g, and most preferably plant material within about 100000g.

[0167] In particular, the layer-by-layer filtration process of the present disclosure is particularly suitable for large-scale production, for example, on the basis of the preferred deep filtration and membrane filtration of the present disclosure (as shown in the embodiments), large-scale production can be achieved by increasing the membrane area of ​​the filter. The layer-by-layer filtration of the present disclosure can filter at least about 100g, 200g, 300g, 400g, 500g, 600g, 700g, 800g, 900g, 1000g, 2000g, 3000g, 4000g, 5000g, 6000g, 7000g, 8000g, 9000g, 10000g, 50000g, or even 100000g of plant material or its corresponding pre-treated liquid by appropriately increasing the membrane area in the layer-by-layer filtration.

[0168] III.Exosomes

[0169] The plant 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 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 subrange within 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 method of the present disclosure have a diameter of about 30-450 nm, preferably a diameter of about 30-250 nm, and more preferably a diameter of about 30-200 nm.

[0170] The plant exosomes may have any suitable shape, including but not limited to a saucer shape, a sphere shape, and a disc shape. Preferably, the plant exosomes prepared according to the method of the present disclosure have a saucer shape.

[0171] Plant exosomes prepared according to the disclosed methods have more metabolites than plant exosomes prepared by ultracentrifugation in one or more of the following aspects: lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzene compounds. In some embodiments, the number of lipids in plant exosomes prepared according to the disclosed methods is greater than that in plant exosomes prepared by ultracentrifugation. As shown in Table 4 of the Examples below, the lipid classes include fatty acyl (FA), glycerolipids (GL), glycerophospholipids (GP), prenol lipids (PR), sphingolipids (SP), steroids (ST), and glycolipids (SL). In some embodiments, the number of fatty acyl compounds in plant exosomes prepared according to the disclosed methods is 190-300, preferably 200-250. In some embodiments, the number of glycerolipid compounds in the plant exosomes prepared according to the methods of the present disclosure is 2-10, preferably 3-9, and more preferably 4-8. In some embodiments, the number of glycerophospholipid compounds in the plant exosomes prepared according to the methods of the present disclosure is 2-10, preferably 3-8, and more preferably 4-7. In some embodiments, the number of isoprene glycolipid compounds in the plant exosomes prepared according to the methods of the present disclosure is 190-400, preferably 200-350. In some embodiments, the number of sphingolipid compounds in the plant exosomes prepared according to the methods of the present disclosure is 2-9, preferably 2-5, and more preferably 3. In some embodiments, the number of steroid compounds in the plant exosomes prepared according to the methods of the present disclosure is 10-100, preferably 20-50, and more preferably 20-40. In some embodiments, the number of glycolipid compounds in the plant exosomes prepared according to the methods of the present disclosure is 0-5, preferably 2-5, and most preferably 2-3.

[0172] The plant exosomes prepared according to the method of the present disclosure have upregulated metabolism compared to the plant exosomes prepared by the ultracentrifugation method ( 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, cutin, suberin and wax biosynthesis, linoleic acid metabolism, β-alanine metabolism, nucleotide metabolism, histidine metabolism, aminoacyl-tRNA biosynthesis, arginine biosynthesis, and biosynthesis of various secondary metabolites.

[0173] Specifically, compared with Lycium barbarum exosomes prepared by ultracentrifugation, the Lycium barbarum exosomes prepared according to the method disclosed herein had 73 differential metabolite-related pathways, and 11 pathways were significantly enriched. Compared with ginseng exosomes prepared by ultracentrifugation, the ginseng exosomes prepared according to the method disclosed herein had 78 differential metabolite-related pathways, and 25 pathways were significantly enriched. Compared with Dendrobium officinale exosomes prepared by ultracentrifugation, the Dendrobium officinale exosomes prepared according to the method disclosed herein had 73 differential metabolite-related pathways, and 20 pathways were significantly enriched. Compared with Polygonatum sibiricum exosomes prepared by ultracentrifugation, the Polygonatum sibiricum exosomes prepared according to the method disclosed herein had 82 differential metabolite-related pathways, and 27 pathways were significantly enriched. Compared with Angelica sinensis exosomes prepared by ultracentrifugation, the Angelica sinensis exosomes prepared according to the method disclosed herein had 81 differential metabolite-related pathways, and 28 pathways were significantly enriched. Compared with the Rehmannia glutinosa exosomes prepared by the method of the present invention and the Rehmannia glutinosa exosomes prepared by the ultracentrifugation method, there are 80 differential metabolite-related pathways, and 21 significantly enriched pathways.

[0174] In some embodiments, the wolfberry exosomes prepared according to the method of the present invention have a total of 617 significantly different metabolites compared with the wolfberry exosomes prepared by the ultracentrifugation method, 401 upregulated metabolites, and 216 downregulated metabolites; the ginseng exosomes prepared according to the method of the present invention have a total of 705 significantly different metabolites compared with the ginseng exosomes prepared by the ultracentrifugation method, 527 upregulated metabolites, and 178 downregulated metabolites; the dendrobium exosomes prepared according to the method of the present invention have a total of 770 significantly different metabolites compared with the dendrobium exosomes prepared by the ultracentrifugation method, 537 upregulated metabolites, and 178 downregulated metabolites. 233; there are 652 significantly different metabolites in the polygonatum exosomes prepared according to the method of the present invention compared with the polygonatum exosomes prepared by the ultracentrifugation method, 546 up-regulated metabolites, and 106 down-regulated metabolites; there are 747 significantly different metabolites in the angelica exosomes prepared according to the method of the present invention compared with the angelica exosomes prepared by the ultracentrifugation method, 659 up-regulated metabolites, and 88 down-regulated metabolites; there are 777 significantly different metabolites in the rehmannia exosomes prepared according to the method of the present invention compared with the rehmannia exosomes prepared by the ultracentrifugation method, 657 up-regulated metabolites, and 120 down-regulated metabolites.

[0175] In some embodiments, in an in vitro staining analysis of plant exosomes using the lipophilic dye PKH67, the positive rates of Lycium barbarum exosomes, ginseng exosomes, Dendrobium officinale exosomes, Polygonatum sibiricum exosomes, Angelica sinensis exosomes, and Rehmannia glutinosa exosomes prepared according to the disclosed methods were 64.5%, 83.3%, 97.4%, 79.9%, 44.0%, and 34.7%, respectively. In a protein content assay of plant exosomes, the protein contents of Lycium barbarum exosomes, Angelica sinensis exosomes, Rehmannia glutinosa exosomes, ginseng exosomes, Polygonatum sibiricum exosomes, and Dendrobium officinale exosomes prepared according to the disclosed methods were 47434.90 μg / mL, 5862.76 μg / mL, 2816.93 μg / mL, 2521.15 μg / mL, 1940.16 μg / mL, and 875.89 μg / mL, respectively.

[0176] In some embodiments, the plant exosomes prepared by the method of the present disclosure (wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes) show good anti-inflammatory effects, and the TNF-α inhibition rates shown in the examples are all above about 60%, or even close to 100%.

[0177] IV. Composition

[0178] The present disclosure provides a plant exosome composition, comprising at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes, preferably at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes prepared by the method for preparing plant exosomes generally, preferably, or specifically defined in the present disclosure.

[0179] Specifically, the plant exosome composition provided by the present disclosure is a pharmaceutical composition, which, in addition to comprising at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes (preferably at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes prepared by the method for preparing plant exosomes generally, preferably, or specifically defined in the present disclosure), optionally further comprises a pharmaceutically acceptable carrier or other active ingredients. The carrier is commonly 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, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0180] Specifically, the plant exosome composition provided by the present disclosure is a food composition or a nutraceutical composition, which comprises at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes, preferably at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes prepared by the method for preparing plant exosomes generally, preferably, or specifically defined in the present disclosure. When the food composition or nutraceutical composition of the present disclosure is used as a food additive, the composition can be directly added together with other foods or food ingredients and used according to conventional methods. Typically, when preparing food or beverages, the food composition or nutraceutical composition of the present disclosure can be added 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.

[0181] The nutritional health-care composition of the present disclosure can itself be in the form of a nutritional supplement or health-care product. In addition to the plant exosomes of the present disclosure, it can also contain other nutrients and / or nutritional health-acceptable excipients or carriers, such as various proteins, fats, vitamins, prebiotics, probiotics, electrolytes, flavorings, colorants, pectin and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonates used in carbonated beverages, etc.

[0182] Foods to which the above-mentioned food composition or nutritional health composition can be added include, but are not limited to, meat, sausage, bread, chocolate, sugar, fast food, biscuits, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea beverages, alcoholic beverages, and vitamin complexes. In some embodiments, the food composition of the present disclosure can be prepared into a functional food.

[0183] 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 gum), 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 topically or transdermally, or by any other suitable route (such as a parenteral route).

[0184] Specifically, the exosome composition provided herein is a cosmetic / cosmetic composition comprising at least one of wolfberry exosomes, ginseng exosomes, dendrobium exosomes, polygonatum exosomes, angelica exosomes, and rehmannia exosomes, preferably prepared using the methods for preparing plant exosomes generally, preferably, or specifically defined herein. The cosmetic / cosmetic composition of the present disclosure typically comprises at least one cosmetic / cosmetic-acceptable excipient or auxiliary material. The cosmetic / cosmetic-acceptable excipient or auxiliary material can be selected from the group consisting of solvents, solubilizers, preservatives, antioxidants, pH adjusters, penetration enhancers, liposomes, humectants, thickeners, chelating agents, skin feel modifiers, surfactants, emulsifiers, propellants / propellants, fragrances, pigments, and other functional additives. The cosmetic composition may be in the form of, but not limited to, soap, face cleanser, cleansing foam, cleansing lotion, cleansing cream, shower gel, skin softener, skin gel, skin lotion, skin cream, essence, eye cream, facial mask, aerosol or spray, lotion, skin softener, toner, astringent, lotion, milk-based lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, foundation, essence, nourishing essence, pressed powder, body lotion, and cleanser. These cosmetic compositions may be prepared by methods well known to those skilled in the art.

[0185] When the cosmetic composition of the present disclosure is a paste, cream, or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silica gel, bentonite, silicon dioxide, talc, zinc oxide, and the like can be used as carrier components. When the cosmetic composition of the present disclosure is a powder or spray, lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate, polyamide powder, and the like can be used as carrier components. In particular, when the formulation is a spray, a propellant such as a 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, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol oil, glycerol fatty 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, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth gum, etc. can be used as a carrier component.

[0186] V. Application

[0187] Many diseases are accompanied by inflammatory responses, including skin-related inflammation (such as dermatitis, acne, pimples, chloasma, etc.), so reducing the level of inflammation will be beneficial for disease treatment and care. Therefore, in some embodiments, the present disclosure provides a use of plant exosomes or a composition comprising the same, which is used to treat or prevent, or helps / assists in improving, alleviating or controlling inflammation, such as skin inflammation. In other embodiments, the present disclosure provides a use of plant exosomes or a composition comprising the same, which is used for or helps / assists in cosmetic, non-therapeutic treatment and / or care of skin, hair, nails and / or mucous membranes, preferably, for or helps / assists in cosmetic, non-therapeutic treatment and / or care of inflammation of the skin, hair, nails and / or mucous membranes.

[0188] In some embodiments, the plant exosomes or compositions comprising the same disclosed herein can be used for applications such as repair, anti-inflammatory, anti-oxidation, anti-aging, whitening, and moisturizing in the field of skin. For example, plant exosomes or compositions comprising the same can be used for anti-inflammatory applications, preferably for anti-inflammatory applications in the field of skin. For another example, plant exosomes or compositions comprising the same can be used for antioxidant applications. For another example, plant exosomes or compositions comprising the same can be used for applications in the field of skin repair. For another example, plant exosomes or compositions comprising the same can be used for anti-aging applications. For another example, plant exosomes or compositions comprising the same can be used for whitening applications. For another example, plant exosomes or compositions comprising the same can be used for moisturizing applications.

[0189] Thus, the present disclosure also provides the use of plant exosomes or compositions comprising the same in the preparation of products (pharmaceuticals, foods, nutritional supplements, beauty / cosmetics). In some embodiments, the pharmaceuticals are used to treat or prevent inflammation, such as dermatitis, acne, pimples, melasma, etc. In some embodiments, the nutritional supplements contribute to or assist in antioxidant, repair, anti-aging, whitening, and / or moisturizing effects.

[0190] 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-inflammatory, anti-oxidation, anti-aging, whitening or moisturizing), comprising administering to a subject in need thereof plant exosomes prepared by the aforementioned general or preferred method or a composition (pharmaceutical composition / food composition / cosmetic or cosmetic composition / nutritional health composition) containing the same.

[0191] For the above-mentioned methods and uses of the present disclosure, plant exosomes or compositions comprising the same can be administered orally, topically or parenterally. In particular, topical or transdermal application can be implemented by iontophoresis, ultrasound phagophoresis, electroporation, mechanical pressure, osmotic pressure gradient, occlusive therapy, microinjection, microneedles (or microneedle arrays), needle-free injection by pressure, microelectric patches, facial masks or any combination thereof. Among them, some technologies that create channels in the skin (such as microneedles or lasers) themselves as a certain treatment method can be used in combination with plant exosomes or compositions comprising the same to help plant exosomes or compositions comprising the same be absorbed and reach deeper skin layers, thereby enhancing their activity. The local area of ​​such combined treatment will be determined by the nature of the condition, disorder and / or disease to be treated and / or processed.

[0192] The daily dosage and frequency of administration of the plant exosomes or compositions comprising the same may vary depending on various factors, such as the stage of the disease to be treated, age, health status, the presence of complications, etc. In one aspect, the composition can be administered 1-3 times daily at a daily dose 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.

[0193] The plant exosomes disclosed herein 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 respiratory system related diseases includes treating COVID-19, suppressing lung inflammation, treating pulmonary fibrosis or preventing pulmonary fibrosis and lung inflammation. In some embodiments, the treatment or prevention of digestive system related diseases includes promoting colitis recovery, treating colon cancer, treating acute and chronic colitis, treating ulcerative colitis, treating chronic periodontitis, preventing alcohol damage to the liver, treating IBD and inhibiting CAC progression, preventing colitis, preventing colitis caused by dextran sodium sulfate, treating DSS-induced colitis, inhibiting colon cancer liver metastasis, inhibiting Clostridium difficile infection, treating liver inflammation, preventing acute liver damage 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 circulatory system related diseases includes improving myocardial damage caused by doxorubicin, preventing damage to the vascular system by various stressors, or preventing oxidative stress in human mesenchymal stromal cells. In some embodiments, the treatment or prevention of nervous system related diseases includes treating gliomas, treating brain gliomas, inhibiting the development of gliomas, or stimulating neural differentiation of stem cells. In some embodiments, the treatment or prevention of endocrine system related diseases includes preventing insulin resistance and obesity, or preventing or inhibiting obesity-related gastrointestinal inflammation caused by a high-fat, high-sugar diet. In some embodiments, the treatment or prevention of urogenital system related diseases includes inhibiting the occurrence and development of mammalian breast cancer, or treating cervical cancer. In some embodiments, the treatment or prevention of musculoskeletal system related diseases includes promoting wound healing.

[0194] In addition, the plant exosomes disclosed herein can also be used as drug delivery vehicles. In some embodiments, the plant exosomes can be used as drug delivery vehicles to transport various drug molecules used to treat the aforementioned 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 a delivery vehicle for treating plant diseases caused by fungal infections. In some embodiments, different plant exosomes have different properties, such as variations in size, charge, and stability. These properties inevitably affect the drug loading in the plant exosomes. For example, in certain embodiments, the plant exosomes used as drug delivery vehicles are smaller in size, such as 30-80 μm, 30-60 μm, or 30-50 μm. Due to their higher surface area to volume ratio, these smaller plant exosomes have a greater drug loading capacity, faster release rate, and higher stability. In other embodiments, plant exosomes can be used as drug delivery vehicles to transport positively charged drug molecules. Plant exosomes are usually negatively charged due to the presence of phosphate, so under the influence of electrostatic attraction, positively charged molecules are more effectively absorbed and encapsulated by these negatively or neutral plant exosomes. Example

[0195] The following is a further description of the technical solution of the present disclosure in conjunction with specific embodiments, but the scope of protection of the present invention is not limited to these embodiments. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0196] The experimental methods and means for which specific conditions are not specified in the following examples are generally carried out under the conventional conditions for such experimental operations in the art. The experimental equipment for which specific sources and models are not marked in the following examples 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 unless otherwise specified and can be used directly without further purification. Unless otherwise stated, percentages and parts are percentages by weight and parts by weight, respectively. Unless otherwise stated, the ratios of liquids are volume ratios, and the temperatures used in the present invention are all degrees Celsius.

[0197] 1. Experimental Methods

[0198] 1.1 Preparation of crude plant extract

[0199] Wash the plants (wolfberry, ginseng, dendrobium officinale, polygonatum, angelica, and rehmannia roots or stems) separately and wipe dry with a clean cloth. Weigh the plants to be treated and mix the weighed plants with sodium chloride injection according to the mass-to-volume ratio (m / v, g / mL) of plant mass: sodium chloride injection volume = 1:3-1:20. Use a wall breaker to crush the plants and collect the crushed plant residue into a measuring bucket. Place the measuring bucket on ice media and stir at a speed of 200±20rpm for 30 minutes. Use a filter to pour in the plant residue to separate the plant residue from the juice and collect the plant juice. Centrifuge the collected plant juice at 4,000g for 15 minutes at 4°C. In a biosafety cabinet, discard the precipitate and collect the supernatant P1 as the crude plant extract.

[0200] 1.2 Isolation and extraction of plant exosomes using layer-by-layer filtration method

[0201] S1 deep filtration: In a biosafety cabinet, place a peristaltic pump, pump tubing, and a 2-30 μm deep capsule filter (Supracap TM Depth Filter Capsules, Pall, filtration capacity approximately 50-200 L / m 2 ) assembly. Rinse the deep filter capsule with sodium chloride injection at a flow rate of 20-200 mL / min and drain the liquid. Filter P1 at a flow rate of 20-200 mL / min and collect the filtrate P2. Remove the filter and rinse the tubing with sodium chloride injection at a flow rate of 20-200 mL / min. Drain the liquid and remove the pump tubing.

[0202] S2 0.45 μm filtration: In a biosafety cabinet, connect a peristaltic pump, pump tubing, and a 0.45 μm membrane filter (PreFlow membrane filter capsules, Pall) to the peristaltic pump. Rinse the 0.45 μm membrane filter with sodium chloride injection at a flow rate of 20-200 mL / min and drain. Filter P2 at a flow rate of 20-200 mL / min and collect the filtrate, P3. Remove the filter and rinse the tubing with sodium chloride injection at a flow rate of 20-200 mL / min. Drain and remove the pump tubing.

[0203] S3 0.22 μm filtration: In a biosafety cabinet, place the peristaltic pump, pump tubing, and 0.22 μm membrane filter (Supor TM EX Grade ECV in Mini Kleenpak TMConnect a peristaltic pump to a 0.22 μm membrane filter. Rinse the filter with sodium chloride injection at a flow rate of 20-200 mL / min and drain the solution. Filter P3 at a flow rate of 20-200 mL / min and collect the filtrate to obtain plant exosomes. Remove the filter and rinse the tubing with sodium chloride injection at a flow rate of 20-200 mL / min. Drain the solution and remove the pump tubing.

[0204] 1.3 Isolation and extraction of plant exosomes using ultracentrifugation

[0205] The collected crude plant extract P1 was centrifuged at 120,000 g at 4°C for 70 minutes using an ultracentrifuge. After centrifugation, the supernatant was removed and the pellet was resuspended in sodium chloride injection. After a further centrifugation at 120,000 g at 4°C for 70 minutes, the pellet was resuspended in sodium chloride injection to obtain plant exosomes.

[0206] 1.4 Transmission electron microscopy

[0207] 5 μL of each prepared plant exosome sample was dripped onto a copper grid and incubated at room temperature for 5 minutes. After incubation, excess liquid was blotted off with absorbent paper. A drop of 2% uranyl acetate was added to the grid and incubated at room temperature for 1 minute. After incubation, excess liquid was blotted off with absorbent paper. The grid was then dried at room temperature for approximately 20 minutes. Transmission electron microscopy (TEM) was performed using a nano-transmission electron microscope (FEI, Tecnai G2 Spirit BioTwin).

[0208] 1.5 nanometer particle size tracking analysis

[0209] Nanoparticle Tracking Analysis (NTA) was used to measure the particle size and concentration of each plant exosome prepared above: the standard stock solution was diluted to 1000 times the calibration stock solution (1 μL of the standard stock solution can be used to prepare 1 mL of the calibration stock solution according to the ratio). 100 μL of the prepared standard stock solution was added to 25 mL of pure water and diluted to a 250,000-fold calibration solution. When testing the sample, the sample was generally diluted 1000 times first. After calibrating the instrument (Particle Metrix, PMX120) with the calibration stock solution, the test sample was injected into the sample cell with a syringe, the sample concentration was measured, and the test was repeated three times.

[0210] 1.6 In vitro and in vitro labeling of plant exosomes by PKH67

[0211] Dilute 1 mM PKH67 (a green fluorescent tracer dye) 50-fold to 20 μM with Diluent C (a universal membrane marker diluent). Then, mix 5 μL of 20 μM PKH67 with 5 μL of the prepared plant exosomes and incubate at room temperature for 15 minutes. Detect the positive rate of PKH67 in the plant exosomes using a flow cytometer.

[0212] 1.7 Trace protein detection

[0213] Prepare 40% SDS with pure water, filter it, and then dilute it with pure water to 2% SDS as a standard curve diluent. TM According to the instructions for the Protein Assay Kit (Thermo Fisher Scientific), prepare the Working Reagent (WR) by mixing the reagents MA:MB:MC in a ratio of 25:24:1. Prepare a standard curve of 200, 40, 20, 10, 5, 2.5, 1, 0.5, and 0 μg / mL using a 2.0 mg / mL BSA stock solution in diluent. Dilute the plant exosomes in the diluent and add an equal volume of WR. Incubate at 100 rpm at 37°C for 2 hours. Measure the absorbance of the standards and samples at 570 nm using a microplate reader to calculate the protein concentration of the samples.

[0214] 1.8 TNF-α inhibition rate detection

[0215] RAW264.7 cells (mouse mononuclear macrophage leukemia cells) were cultured in DMEM basal medium supplemented with 10% FBS for 48 h and the concentration was 1.875 × 10 4 / cm 2 Cells were seeded in 96-well plates at a density of 100 μg / mL. The cells were cultured in a 37.0°C, 5% CO2 incubator for 24 h. Plant exosomes (1×10 9 Cells were pretreated with LPS (final concentration 5 ng / mL) and dexamethasone (final concentration 1 μg / mL) for 24 hours, followed by LPS (final concentration 5 ng / mL) for 4 hours. The cell supernatant was collected and centrifuged at 500g for 5 minutes at 4°C. TNF-α concentrations were measured using a Mouse TNF-α ELISA kit (R&D Systems), and the inhibition rate was calculated as follows:

[0216] Inhibition rate (%) = 1-(average concentration of experimental group / average concentration of LPS-treated group).

[0217] 1.9 Sample preparation for non-targeted metabolomics analysis of plant exosomes

[0218] 100 μL of plant exosome liquid sample was pipetted into a 1.5 mL centrifuge tube, and 400 μL of extraction solution (acetonitrile:methanol = 1:1, containing 0.02 mg / mL of internal standard L-2-chlorophenylalanine) was added. After vortex mixing for 30 seconds, the sample was extracted with low-temperature ultrasonic technology for 30 minutes (5°C, 40 kHz). The sample was placed at -20°C for 30 minutes. After centrifugation at 13,000 g for 15 minutes at 4°C, the supernatant was removed, dried with nitrogen, and reconstituted with 100 μL of reconstitution solution (acetonitrile:water = 1:1). The sample was extracted with low-temperature ultrasonic technology for 5 minutes (5°C, 40 kHz). After centrifugation at 13,000 g for 10 minutes at 4°C, the supernatant was transferred to a vial with an internal cannula for analysis. Equal volumes of all sample metabolites were mixed to prepare quality control samples (QC). During the instrument analysis process, a QC sample was inserted into every 5-15 samples to examine the repeatability of the entire analysis process.

[0219] 1.10 LC-MS / MS analysis

[0220] Equal volumes of all sample metabolites were mixed to prepare quality control samples. During the instrument analysis, a QC sample was inserted into every 5-15 samples to examine the repeatability of the entire analysis process. Sample mass spectrometry signals were acquired in positive and negative ion scanning modes with a mass scan range of 70-1050 m / z. The sheath gas flow rate was 50 psi, the auxiliary gas flow rate was 13 psi, the auxiliary gas heating temperature was 425°C, the positive mode ion spray voltage was set to 3500 V, the negative mode ion spray voltage was set to -3500 V, the ion transfer tube temperature was 325°C, and the normalized collision energy was 20-40-60 V cyclic collision energy. The primary mass spectrometry resolution was 60,000, the secondary mass spectrometry resolution was 7500, and data were acquired in DDA mode.

[0221] 1.11 Substance Identification and Analysis

[0222] After the LC-MS data were loaded onto the computer, they were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. This ultimately resulted in a data matrix of retention time, mass-to-charge ratio, and peak intensity. The MS and MSMS mass spectrometry information was then matched with the public metabolic databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ), as well as the Metlin-built library, to obtain metabolite information.

[0223] The searched data matrix was uploaded to the Majorbio Cloud platform (cloud.majorbio.com) for analysis. The data matrix was first preprocessed as follows: The 80% rule was used to remove missing values, retaining variables with at least 80% non-zero values ​​in at least one sample group. Missing values ​​were then filled (using the minimum value in the original matrix). To minimize errors caused by sample preparation and instrument instability, the response intensities of the sample mass spectrometry peaks were normalized using sum normalization to obtain a normalized data matrix. Variables with a relative standard deviation (RSD) greater than 30% in QC samples were also removed, and log10 logarithmization was performed to obtain the final data matrix for subsequent analysis.

[0224] The preprocessed data matrix was then subjected to principal component analysis (PCA) and orthogonal least squares discriminant analysis (OPLS-DA) using the ropls package (Version 1.6.2) in R. Seven rounds of cross-validation were used to assess model stability. Significantly differentially expressed metabolites were selected based on the variable weights (VIPs) obtained from the OPLS-DA model and the student's t-test p-values. Metabolites with a VIP > 1 and a p < 0.05 were considered significantly differentially expressed.

[0225] The differential metabolites were annotated with metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https: / / www.kegg.jp / kegg / pathway.html) to identify the pathways in which the differential metabolites participated. Pathway enrichment analysis was performed using the Python package scipy.stats, and the most relevant biological pathways to the experimental treatments were identified using Fisher's exact test.

[0226] 2. Experimental Results and Analysis

[0227] 2.1 Screening of extraction methods

[0228] For ginseng, wolfberry and dendrobium, based on the above Example 1.1, the following different filtration treatment combinations were performed and the final results were evaluated. The specific extraction methods and corresponding results are shown in the following table:

[0229] Among them, “+” represents that it contains relevant processing, and “-” represents that it does not contain relevant processing.

[0230] 2.2 Extraction of plant exosomes (PEN) using layer-by-layer filtration and ultracentrifugation methods

[0231] Exosomes were isolated and extracted from plants (wolfberry, ginseng, dendrobium officinale, polygonatum, angelica sinensis, and rehmannia root) using the layer-by-layer filtration method described in Examples 1.1-1.2. Specifically, the plants (up to 1000 g) were mixed (adding a sodium chloride injection solvent according to the mass-to-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 was removed by centrifugation at 4000 g for 15 minutes to obtain a crude plant extract, P1. Large impurities in P1 were removed by S1 (depth filtration) to obtain P2. Larger particles in P2 were pre-filtered by S2 (0.45 μm membrane filtration) to obtain P3. Finally, sterile filtration was performed by S3 (0.22 μm membrane filtration) to harvest sterile exosomes. On average, 500 mL of crude plant extract P1 took approximately 1 hour to process.

[0232] Exosomes were isolated and extracted from the six plants using the ultracentrifugation method described in Example 1.3. The crude plant extract P1 was centrifuged twice at 12,000 g for 70 min at 4°C. The average extraction time for 500 mL was approximately 3 h.

[0233] Table 1 Names and labels of PEN isolated from six plants using ultracentrifugation and layer filtration

[0234] The following tests were performed on the exosome samples obtained from the plants described above. The results demonstrated the properties and effects of the exosomes described above. As a representative example, the test results for 100 g of exosomes obtained from each of the plants described above (group names are shown in Table 1) are provided below.

[0235] 2.3 Physical properties of plant exosomes

[0236] After 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 were counted.

[0237] As shown in Table 2, the particle size of plant exosomes isolated and extracted by both layer-by-layer filtration and ultracentrifugation methods was within the range of 30-200 nm, but the yield of plant exosomes extracted by layer-by-layer filtration was significantly higher than that of exosomes extracted by ultracentrifugation. The results indicate that the layer-by-layer filtration method significantly outperforms ultracentrifugation in terms of both the efficiency of the exosome isolation method and the final yield of the harvest.

[0238] Table 2 Particle size and concentration of plant exosomes

[0239] 2.4 Qualitative metabolomics analysis of plant exosomes

[0240] 2.4.1 Analysis of unique metabolites

[0241] Plant metabolomics analysis conducted according to Examples 1.9 to 1.11 showed that the number of metabolites identified in the six plant exosomes obtained in Example 2.2 was higher with layer-by-layer filtration than with ultracentrifugation, as shown in Table 3. Venn diagram analysis also revealed that the number of metabolites unique to layer-by-layer filtration was higher than that with ultracentrifugation.

[0242] Table 3 Venn analysis statistics of exosomes extracted from six plants using layer-by-layer filtration and ultracentrifugation

[0243] 2.4.2 Compound classification analysis

[0244] Compound classification analysis categorizes metabolites into sugars, amino acids, organic acids, lipids, and other groups based on their structure and properties. Using the HDMB compound classification hierarchy (Superclass), metabolites in the six plant exosomes are primarily concentrated into six categories: lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzene compounds. This is shown in Figures 2-3.

[0245] The additional metabolites of GQV01 compared with LyE01 were categorized into phenylpropanoids and polyketides (44), organic oxygen compounds (41), lipids and lipid-like molecules (34), organic acids and their derivatives (18), lignans, neolignans and related compounds (18), and organic heterocyclic compounds (16).

[0246] The additional metabolites of RSV01 compared with GsE01 were categorized into phenylpropanoids and polyketides (73), lipids and lipid-like molecules (60), organic oxygen compounds (54), organic heterocyclic compounds (22), organic acids and their derivatives (16), and lignans, neolignans, and related compounds (16).

[0247] The additional metabolites of SHV01 compared with DeE01 were classified into phenylpropanoids and polyketides (57), lipids and lipid-like molecules (46), organic oxygen compounds (33), organic heterocyclic compounds (20), and organic acids and their derivatives (10).

[0248] The additional metabolites in HJV01 compared with PoE01 were classified into lipids and lipid-like molecules (108), phenylpropanoids and polyketides (76), organic oxygen compounds (72), organic heterocyclic compounds (35), organic acids and their derivatives (25), lignans, neolignans and related compounds (23), and benzene compounds (13).

[0249] The additional metabolites of DGV01 compared with AnE01 were categorized into lipids and lipid-like molecules (129), organic oxygen compounds (94), phenylpropanoids and polyketides (96), organic heterocyclic compounds (30), organic acids and their derivatives (30), lignans, neolignans and related compounds (17), and benzene compounds (12).

[0250] The additional metabolites of DHV01 compared with ReE01 were classified into lipids and lipid-like molecules (135), phenylpropanoids and polyketides (115), organic oxygen compounds (104), organic heterocyclic compounds (49), organic acids and their derivatives (42), lignans, neolignans and related compounds (16), and benzene compounds (13).

[0251] 2.4.3 Lipid classification analysis

[0252] Lipids and lipid-like molecules rank first in the compound classification. The lipid classifications mainly include fatty acyl (FA), glycerolipids (GL), glycerophospholipids (GP), prenol lipids (PR), sphingolipids (SP), steroids (ST) and glycolipids (SP).

[0253] Lipids and lipid-like molecules were classified and counted from the six plant exosomes. As shown in Table 4, the amount of plant exosome lipids extracted by layer-by-layer filtration was greater than that extracted by ultracentrifugation. The most significant differences in the content of FA, PR, and ST between GQV01 and LyE01, HJV01 and PoE01, DGV01 and AnE01, and DHV01 and ReE01 were respectively. The most significant differences in the content of FA and PR between RSV01 and GsE01, and between SHV01 and DeE01 were respectively.

[0254] Table 4 Comparison of lipid classification of exosomes from six plants

[0255] 2.5 Analysis of differential metabolites between plant exosomes

[0256] Univariate statistical analysis (t-test) combined with multivariate statistical analysis (OPLS-DA / PLS-DA) and fold change (FC) were used to screen differential metabolites. The screening conditions were P < 0.05 and VIP > 1 and (FC < 1 or FC > 1, FC was not screened by default) [6].

[0257] As shown in Figure 4, there are 617 significantly different metabolites between GQV01 and LyE01, of which 401 are upregulated and 216 are downregulated; there are 705 significantly different metabolites between RSV01 and GsE01, of which 527 are upregulated and 178 are downregulated; there are 770 significantly different metabolites between SHV01 and DeE01, of which 537 are upregulated and 23 are downregulated. 3; there were 652 significantly different metabolites between HJV01 and PoE01, of which 546 were upregulated and 106 were downregulated; there were 747 significantly different metabolites between DGV01 and AnE01, of which 659 were upregulated and 88 were downregulated; there were 777 significantly different metabolites between DHV01 and ReE01, of which 657 were upregulated and 120 were downregulated.

[0258] 2.6 Analysis of Different Metabolic Pathways among Plant Exosomes

[0259] Typically, multiple functionally interconnected metabolites form a metabolic pathway, and the cumulative expression differences of multiple metabolites within a metabolic pathway constitute the expression variation of the entire metabolic pathway. If the proportion of differentially expressed metabolites participating in a particular pathway is significantly greater than the proportion of background metabolites participating in this pathway, the experimental treatment is considered to be associated with changes in this metabolic pathway. In enrichment analysis, based on the KEGG database, pathways with significantly enriched expression were screened based on an enrichment P value less than 0.05.

[0260] Compared with LyE01, GQV01 had 73 differential metabolite-related pathways, of which 11 were significantly enriched. Compared with GsE01, RSV01 had 78 differential metabolite-related pathways, of which 25 were significantly enriched. Compared with DeE01, SHV01 had 73 differential metabolite-related pathways, of which 20 were significantly enriched. Compared with PoE01, HJV01 had 82 differential metabolite-related pathways, of which 27 were significantly enriched. Compared with AnE01, DGV01 had 81 differential metabolite-related pathways, of which 28 were significantly enriched. Compared with ReE01, DHV01 had 80 differential metabolite-related pathways, of which 21 were significantly enriched.

[0261] As shown in Figure 5, the differential metabolites of plant exosomes extracted by layer-by-layer filtration were mainly upregulated in Metabolism compared with those extracted by ultracentrifugation. The main pathways were tryptophan metabolism, arachidonic acid metabolism, phenylpropanoid biosynthesis, starch and sucrose metabolism; alanine, aspartic acid and glutamate metabolism; galactose metabolism, ABC transporters, valine, leucine and isoleucine biosynthesis; cutin, suberin and wax biosynthesis; linoleic acid metabolism, β-alanine metabolism, nucleotide metabolism, histidine metabolism, aminoacyl-tRNA biosynthesis, arginine biosynthesis, and biosynthesis of various secondary metabolites.

[0262] 2.7 TEM identification of plant exosomes obtained by layer-by-layer filtration separation

[0263] The above analysis of plant exosome particle concentration, particle size, yield, and plant metabolomics showed that the layer-by-layer filtration extraction method is superior to ultracentrifugation and can extract a higher yield of plant exosomes in less time (equivalent to at least 40 times the yield of ultracentrifugation extraction). Therefore, we further performed TEM on the plant exosomes extracted by layer-by-layer filtration.

[0264] The results are shown in Figure 6. The exosomes of six plants (wolfberry, ginseng, dendrobium officinale, polygonatum, angelica, and rehmannia) all have a typical exosome-like saucer shape, with a particle size ranging from 30 to 200 nm, which is consistent with the definition of extracellular vesicles by the International Society for Extracellular Vesicles (ISEV).

[0265] 2.8 Detection of PKH67 in plant exosomes isolated by layer-by-layer filtration

[0266] The plant exosomes prepared in Example 2.2 above were stained in vitro using the lipophilic dye PKH67, and the PKH67-positive particles were detected and analyzed using a nanoflow cytometer.

[0267] As shown in FIG7 , the PKH67 positive rates of GQV01, RSV01, SHV01, HJV01, DGV01, and DHV01 were 64.5%, 83.3%, 97.4%, 79.9%, 44.0%, and 34.7%, respectively.

[0268] 2.9 Protein content detection of plant exosomes obtained by layer-by-layer filtration separation

[0269] Protein content analysis of plant exosomes revealed that the six plant vesicles were rich in protein. Specifically, as shown in Figure 8A, GQV01 had the highest protein content, at 47,434.90 μg / mL. Other plant exosomes contained 5,862.76 μg / mL of protein, followed by DGV01, 2,816.93 μg / mL, DHV01, RSV01, 2,521.15 μg / mL, HJV01, and 875.89 μg / mL.

[0270] 2.10 Detection of anti-inflammatory activity of plant exosomes separated by layer-by-layer filtration

[0271] The efficacy of plant exosomes extracted by layer-by-layer filtration was tested, mainly by observing the inhibition rate of LPS-induced TNF-α release from RAW264.7.

[0272] As shown in Figure 8B, the TNF-α inhibition rate of 1 μg / mL dexamethasone in the positive control group was 91.41%. 9 / mL of plant exosomes, the TNF-α inhibition rate of GQV01 was 90.19%, the TNF-α inhibition rate of RSV01 was 86.98%, the TNF-α inhibition rate of SHV01 was 64.40%, the TNF-α inhibition rate of HJV01 was 98.73%, the TNF-α inhibition rate of DGV01 was 87.84%, and the TNF-α inhibition rate of DHV01 was 79.35%.

[0273] The results showed that the plant exosomes extracted using layer-by-layer filtration had satisfactory anti-inflammatory and immunomodulatory activities.

[0274] 2.11 Scale-up process

[0275] Following the layer-by-layer filtration method described in Examples 1.1-1.2, 1000 g of wolfberry, 1000 g of ginseng, and 500 g of Dendrobium officinale were processed to isolate and extract exosomes. This was to verify whether layer-by-layer filtration could process approximately 1000 g or more of plant material. After NTA analysis according to Example 1.5, the obtained plant exosomes were analyzed for particle concentration, particle size, and final volume.

[0276] As shown in Table 5, after scaling up the 100g plant material used to obtain exosomes by 5-10 times compared to Example 2.3 (10 times for wolfberry and ginseng, and 5 times for dendrobium), approximately 5000mL of crude plant extract could be processed by increasing the membrane area in the layer-by-layer filtration method. The isolated exosomes all had particle sizes within the 30-200nm range, and the final exosome yield was also quite high.

[0277] Table 5 Particle size and concentration of plant exosomes after 5-10 times scaling up of the layer-by-layer filtration process

[0278] References

[0279] [1] Zhang M, Viennois E, Prasad M, Zhang Y, Wang L, Zhang Z, Han MK, Xiao B, Xu C, Srinivasan S, Merlin D. Edible ginger-derived nanoparticles: A novel therapeutic approach for the prevention and treatment of inflammatory bowel disease and colitis-associated cancer. Biomaterials. 2016 Sep; 101:321-40.

[0280] [2]Mu J, Zhuang

[0281] [3]Baldini N,Torreggiani E,Roncuzzi L,Perut F,Zini N,Avnet S.Exosome-like Nanovesicles Isolated from Citrus limon L.Exert Antioxidative Effect.Curr Pharm Biotechnol.2018;19(11):877-885.

[0282] [4]Mu N,Li J,Zeng L,et al.Plant-Derived Exosome-Like Nanovesicles:Current Progress and Prospects.Int J Nanomedicine.2023;18:4987-5009.Published 2023 Sep 5.

[0283] [5]Théry C,Witwer KW,Aikawa E,et al.Minimal information for studies of extracellular vesicles 2018(MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.J Extracell Vesicles.2018;7(1):1535750.

[0284] [6]Wang X,Li J,Zhang A H.Urine metabolic phenotypes analysis of extrahepatic cholangiocarcinoma disease using ultra-high performance liquid chromatography-mass spectrometry[J].RSC Advances,2016,6(67):63049-63057.

[0285] [7]CN108384743A.

Claims

1. 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: (a) Obtaining plant materials; (b) Pretreating the obtained plant materials to obtain a crude plant extract; and (c) Subjecting the obtained crude plant extract to layer-by-layer filtration, wherein, in step (c), the layer-by-layer filtration comprises a depth 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 carried out after the depth filtration step; preferably, the membrane filtration comprises anti-clogging membrane filtration, or further comprises sterilizing membrane filtration; more preferably, the membrane filtration consists of anti-clogging membrane filtration and sterilizing membrane filtration carried out in sequence.

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 sterilizing 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 - 3, wherein in step (c), the depth filtration uses a depth filtration with a pore size of 1 - 50 μm, preferably a depth filtration with a pore size of 2 - 30 μm, more preferably a depth filtration with a pore size of 6 - 30 μm.

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

6. The method according to any one of claims 1 - 5, wherein the plant materials are selected from Dendrobium officinale, Panax ginseng, Lycium barbarum, Polygonatum sibiricum, Angelica sinensis, Rehmannia glutinosa or a mixture of any one or more of them.

7. The method according to any one of claims 1 - 6, wherein the weight of the plant raw materials that the method can process is 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 yield of the method is increased by at least about 40 times compared with the ultracentrifugation method.

8. Plant exosomes prepared by the method according to any one of claims 1 - 7.

9. The plant exosomes of claim 8, which have more metabolites in one or more of the following aspects compared with 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 / or benzenoid compounds.

10. A plant exosome composition comprising the plant exosomes of 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 nutraceutical 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 a drug, food, nutritional health product or beauty or cosmetic product, wherein the product is used for treating or preventing inflammation, preferably skin inflammation, or helps or aids in improving, alleviating or controlling inflammation, preferably skin inflammation, or is used or aids in repair, anti - inflammation, antioxidant, anti - aging, whitening or moisturizing in the field of dermatology.

13. 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 helps or aids in improving, alleviating or controlling inflammation, preferably skin inflammation, or is used or aids in repair, anti - inflammation, antioxidant, anti - aging, whitening or moisturizing in the field of dermatology.

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

15. A method for repair, anti - inflammation, antioxidant, anti - aging, whitening or moisturizing in dermatology, which comprises administering the beauty or cosmetic composition and / or nutritional health composition of claim 11 to a subject in need thereof.

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

Images