Dendrobium nobile exosome as well as preparation method and application thereof

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to a method for extracting exosomes from plants such as Dendrobium, and the plant exosomes extracted by this method and their applications. Specifically, the method for preparing plant exosomes of the present disclosure includes a step-by-step filtration step, and the step-by-step filtration step sequentially includes depth filtration and membrane filtration. Background Art

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

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

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

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

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

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

[0008] The skin is an important protective barrier against external stimuli such as bacteria and ultraviolet rays. The skin barrier is mainly composed of the stratum corneum and keratinocytes, and its main function is to protect the internal tissues and fluids from the external environment. Ultraviolet irradiation can cause damage to the stratum corneum of the skin, resulting in a decrease in the expression of intracellular barrier proteins, thereby impairing the skin barrier function. At the same time, the skin is also a target organ for ultraviolet-induced oxidative damage. The energy carried by UVB can activate endogenous photosensitive substances in the skin, generating various reactive oxygen species such as singlet oxygen, oxygen free radicals, and hydrogen peroxide. Therefore, inhibiting the intracellular reactive oxygen species reaction induced by ultraviolet rays and the disruption of the permeable skin barrier is one of the key factors in combating skin photoaging and skin diseases.

[0009] Therefore, there is a need for a simple, efficient method for large-scale separation and extraction of high-purity plant exosomes suitable for industrialization levels to reduce the production cost and increase the yield of exosomes, while the exosomes have beneficial effects on the skin. Summary of the Invention

[0010] To overcome the deficiencies and shortcomings of the existing methods for extracting plant exosomes, the inventors first proposed a method for separating and extracting plant exosomes using layer-by-layer filtration. This method is simple, efficient, suitable for industrial scale (as Figure 1 shown), and the separated and purified exosomes have a high purity.

[0011] In this study, plant extracellular vesicles were extracted by the layer-by-layer filtration method. After comparison, it was found that the anti-aging effect of the extracellular vesicles of Dendrobium officinale (SH) was better than that of other plants, and the anti-aging effect of the exosomes of Dendrobium officinale obtained by this extraction method was better than that of the exosomes of Dendrobium officinale obtained by ultracentrifugation (UC) and the polysaccharides of Dendrobium officinale obtained by ethanol extraction (EE). We further elucidated the efficacy and mechanism of SH in anti-aging and firming through experiments.

[0012] Specifically, the present disclosure uses two methods, layer-by-layer filtration and ultracentrifugation, to separate plant exosomes from Dendrobium (preferably Dendrobium officinale). By analyzing the physical properties and efficacy of the separated plant exosomes, and combining plant metabolomics analysis, the differences between the plant exosomes extracted by the two methods are compared, and a method suitable for the separation and extraction of plant exosomes is thus screened out.

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

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

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

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

[0017] (a) Obtaining plant material;

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

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

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

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

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

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

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

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

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

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

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

[0029] In the method embodiments of the present disclosure, the plant material may be terrestrial or aquatic. Preferably, the plant material is selected from Dendrobium (preferably Dendrobium officinale).

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

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

[0032] In a specific embodiment, the exosome composition is a pharmaceutical composition. In a specific embodiment, the exosome composition comprises exosomes of Dendrobium officinale (preferably Dendrobium officinale) prepared by the method of the present disclosure; and an optional pharmaceutically acceptable carrier.

[0033] In a specific embodiment, the exosome composition is a food composition, which comprises plant exosomes prepared by the methods generally or preferably defined in the present disclosure, and optionally other food ingredients and / or edible excipients and / or carriers. In a specific embodiment, the food composition comprises Dendrobium (preferably Dendrobium officinale) exosomes prepared by the methods of the present disclosure, and optionally other food ingredients and / or edible excipients and / or carriers.

[0034] In a specific embodiment, the exosome composition is a beauty (or cosmetic) composition, which comprises plant exosomes prepared by the methods generally or preferably defined in the present disclosure, and optionally beauty / cosmetic acceptable excipients or carriers. In a specific embodiment, the beauty composition comprises Dendrobium (preferably Dendrobium officinale) exosomes prepared by the methods of the present disclosure, and at least one beauty / cosmetic acceptable excipient or carrier.

[0035] In a specific embodiment, the exosome composition is a nutraceutical composition, which comprises plant exosomes prepared by the methods generally or preferably defined in the present disclosure, and optionally nutraceutical acceptable excipients or carriers. In a specific embodiment, the nutraceutical composition comprises Dendrobium (preferably Dendrobium officinale) exosomes prepared by the methods of the present disclosure, and at least one nutraceutical acceptable excipient or carrier.

[0036] On the other hand, the present disclosure also provides the use of plant exosomes (preferably Dendrobium, more preferably Dendrobium officinale exosomes) prepared by the methods of the present disclosure or compositions containing the same (including but not limited to pharmaceutical compositions / food compositions / beauty or cosmetic compositions / nutraceutical compositions) for treating or preventing—or contributing to (or assisting in) improving, alleviating or controlling—inflammation, preferably skin inflammation, or for—or assisting in—repairing, anti-inflammation, antioxidant, anti-aging, whitening or moisturizing in the field of dermatology.

[0037] On the other hand, the present disclosure also provides the use of plant exosomes (preferably Dendrobium, more preferably Dendrobium officinale exosomes) prepared by the methods of the present disclosure or compositions containing the same (including but not limited to pharmaceutical compositions / food compositions / beauty compositions / nutraceutical compositions) in the preparation of products (drugs, foods, nutraceuticals, beauty / cosmetics, etc.) for treating or preventing—or contributing to (or assisting in) improving, alleviating or controlling—inflammation, preferably skin inflammation, or for or assisting in repairing, anti-inflammation, antioxidant, anti-aging, whitening or moisturizing in the field of dermatology.

[0038] On the other hand, the present disclosure provides a method for treating or preventing inflammation, preferably skin inflammation, or treating or preventing dermatological symptoms, or assisting in improving, alleviating, or controlling dermatological symptoms, which includes administering to a subject in need thereof plant exosomes (preferably Dendrobium, more preferably Dendrobium officinale exosomes) prepared by the aforementioned general or preferred defined methods or a composition (pharmaceutical composition / food composition / beauty or cosmetic composition / nutritional and health composition) containing the same.

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

[0040] On the other hand, the present disclosure also provides a method for preparing drugs, foods, nutritional health products, beauty / cosmetic compositions, including preparing plant exosomes (preferably Dendrobium, more preferably Dendrobium officinale exosomes) according to the general or preferred defined methods of the present disclosure, and incorporating the plant exosomes into the drugs, foods, nutritional health products, beauty / cosmetic compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following drawings illustrate the preferred embodiments of the present disclosure. For the purpose of illustrating the present disclosure, the currently preferred embodiments are shown in the figures. However, it should be understood that the present disclosure is not limited to the specific embodiments shown in the figures.

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

[0043] Figure 2 Compound classification SHV01 of Dendrobium officinale exosomes extracted by the layer-by-layer filtration method of the present disclosure.

[0044] Figure 3 Compound classification DeE01 of Dendrobium officinale exosomes extracted by the ultracentrifugation method.

[0045] Figure 4 Volcano plot of differential metabolites of Dendrobium officinale exosomes extracted by layer-by-layer filtration and ultracentrifugation: SHV01 vs DeE01. The abscissa is the fold change value of the metabolite expression difference between the two groups, i.e., log2FC; the ordinate is the statistical test value of the metabolite expression amount change difference, i.e., -log10(p value), and the numerical values of the abscissa and ordinate are logarithmically processed.

[0046] Figure 5Show the KEGG pathway enrichment analysis chart of Dendrobium officinale exosomes extracted by layer-by-layer filtration and ultracentrifugation: SHV01 vs DeE01. The abscissa represents the pathway name, and the ordinate represents the enrichment rate, which is the ratio of the number of metabolites enriched in this pathway (Metabolite nμmber) to the number of metabolites annotated to this pathway (Background nμmber). The larger the ratio, the higher the degree of enrichment. The color gradient of the columns represents the significance of enrichment. By default, the darker the color, the more significantly enriched the KEGG term. Among them, those with P value < 0.001 are marked as ***, those with P value < 0.01 are marked as **, and those with P value < 0.05 are marked as *.

[0047] Figure 6 Show the TEM analysis chart of Dendrobium officinale exosomes SHV01 extracted by layer-by-layer filtration separation.

[0048] Figure 7 Show the particle size and concentration of SHV01 by nanoparticle tracking analysis.

[0049] Figure 8 Show the in vitro PKH67 staining flow cytometry detection of Dendrobium officinale exosomes extracted by layer-by-layer filtration separation SHV01.

[0050] Figure 9 Show the activity detection of Dendrobium officinale exosomes extracted by layer-by-layer filtration; A: Protein concentration of Dendrobium officinale exosomes. B: Dendrobium officinale exosomes inhibit the release of TNF-α induced by LPS. The ordinate is the concentration of TNF-α detected by the Elisa kit. Among them, those with P value < 0.001 are marked as ***; C: Inhibitory rate of TNF-α release by Dendrobium officinale exosomes.

[0051] Figure 10 Show the physical characterization of extracellular vesicles from different plants. A. TEM analysis of the shape of extracellular vesicles from Dendrobium officinale (SH); B. Nanoparticle tracking analysis of the particle size and concentration of extracellular vesicles from Dendrobium officinale; C. Nanoflow cytometry analysis of the PKH67 positive rate of extracellular vesicles from Dendrobium officinale; D. TEM analysis of the shape of extracellular vesicles from Rehmannia glutinosa (DH); E. Nanoparticle tracking analysis of the particle size and concentration of extracellular vesicles from Rehmannia glutinosa; F. Nanoflow cytometry analysis of the PKH67 positive rate of extracellular vesicles from Rehmannia glutinosa; G. TEM analysis of the shape of extracellular vesicles from Angelica sinensis (DG); H. Nanoparticle tracking analysis of the particle size and concentration of extracellular vesicles from Angelica sinensis; I. Nanoflow cytometry analysis of the PKH67 positive rate of extracellular vesicles from Angelica sinensis.

[0052] Figure 11Show the promoting effect of extracellular vesicles from different plant cells on the basal collagen level of fibroblasts. A. WB was used to detect the difference in the level of type I collagen expression in 3T3 cells. The concentration of the extracellular vesicle treatment group was 1×10 8 particles / mL, and the specific treatment groups and corresponding mixed plants are marked in the figure (con: blank control group; TGF-β: positive control group; SH: Dendrobium officinale; DH: Rehmannia glutinosa; DG: Angelica sinensis); B. Semi-quantitative analysis of the WB results in Figure A; C. Statistical difference analysis of the results of three repeated experiments was performed using two-tailed Student's T test, and the mean and SD values are marked in red. *: P-value < 0.05.

[0053] Figure 12 Show the comparison of the improvement of UVA-induced cell swelling by the active ingredients of Dendrobium officinale obtained by different extraction processes. A. Normal cells were imaged after being fluorescently labeled with phalloidin, and representative cell morphologies are listed on the right; B. After 5 J / cm 2 UVA stimulation to establish an HSF model, representative images of swollen cells are listed on the right; C. HSF cells fluorescently labeled with phalloidin under a fluorescence microscope. The concentrations of the SH and UC treatment groups were both 1×10 8 particles / mL, and the concentration of the EE treatment group was 0.025% (v / v). The specific treatment groups are marked in the figure (con: blank control group; UVA: ultraviolet irradiation group; SH: layer-by-layer filtration; UC: ultracentrifugation; EE: ethanol extraction); D. Quantitative analysis of the fluorescence imaging results in Figure A. The difference analysis was performed using two-tailed Student's T test. # indicates the difference between the data group and the con group, : P-value < 0.001, * indicates the difference from the UVA group, **: P-value < 0.01, ***: P-value < 0.001, ****: P-value < 0.0001.

[0054] Figure 13 Show the promoting effect of the active ingredients of Dendrobium officinale obtained by different extraction processes on the basal collagen level of fibroblasts. A. WB was used to detect the difference in the level of type I collagen expression in 3T3 cells. The concentrations of the SH and UC treatment groups were both 1×10 8cells / mL, the concentration of the EE treatment group was 0.025% (v / v). The specific treatment groups have been marked in the figure (con: blank control group; UVA: ultraviolet irradiation group; SH: filtration layer by layer; UC: ultracentrifugation; EE: ethanol extraction); B. Semi-quantitative analysis of the WB results in Figure A; C. Statistical difference analysis of the results of three repeated experiments used two-tailed Student's T test, and the mean and SD values are marked in red. *: P-value < 0.05. After folding, the COLI protein is a heterotrimer composed of two α1 chains and one α2 chain. The antibody used in the WB experiment of the present disclosure specifically recognizes the pro-α2 chain of type I collagen, which is represented by COL1a2.

[0055] Figure 14 Venn diagram analysis showing extracellular vesicles of Dendrobium officinale obtained by different separation methods. A. Different colors in the figure represent differential metabolites in different comparison groups. The overlapping part represents the number of metabolites common to multiple metabolic clusters, and the non-overlapping part represents the number of metabolites unique to that metabolic cluster. The numbers represent the corresponding metabolite numbers; B. The bar chart represents the number of metabolites contained in each metabolic cluster.

[0056] Figure 15 Showing the transdermal permeability study of SH. A. Cumulative permeation amount of the sample of SH at different time points, and the fitting of the regression equation (linear is marked in red); B. Diffusion percentage of the sample of SH at different time points.

[0057] Figure 16 Showing the anti-aging effect of SH on the 3D skin model irradiated with UV. A. Representative tissue sections of the 3D skin model tissue morphology (H&E staining), and each treatment group has been marked in the figure (con: blank control group; UV: negative control; VC+VE: positive control, the treatment concentration of which is VC: 100 μg / mL, VE: 7 μg / mL; SH: extracellular vesicles of Dendrobium officinale obtained by filtration layer by layer, and the total treatment amount is 4×10 8 particles); B. Representative sections of tissue collagen fibers (Masson staining); C. Quantitative analysis of tissue morphology results; D. Quantitative analysis of collagen fiber results; E. Tissue elastic coefficient R2. When using the t-test method for statistical analysis, # represents the difference between the data group and the con (control) group, ##: P-value < 0.01, * represents the difference from the UVA group, **: P-value < 0.01.

[0058] Figure 17Show the effect of SH on collagen production in UV-irradiated 3D skin models. A. Representative immunofluorescence images of type I collagen (COL I); B. Representative immunofluorescence images of type IV collagen (COL IV); C. Quantitative analysis of the relative integrated optical density (IOD) value of COL I; D. Quantitative analysis of the relative integrated optical density (IOD) value of COL IV. Among them, VC+VE is the positive control, and the treatment concentrations are VC: 100 μg / mL and VE: 7 μg / mL; SH represents extracellular vesicles of Dendrobium officinale obtained by layer-by-layer filtration, and the total amount of its treatment is 4×10 8 particles. When performing statistical analysis using the t-test method, # indicates the difference between the data group and the con (control) group, ##: P-value < 0.01, * indicates the difference from the UV group, **: P-value < 0.01.

[0059] Figure 18 Show the effect of SH on the expression of cyclobutane-pyrimidine dimers and hyaluronic acid in UV-irradiated 3D skin models. A. Representative immunohistochemical images of cyclobutane-pyrimidine dimers (CPD); B. Representative immunofluorescence images of hyaluronic acid (HA); C. Statistical results of the positive cell rate of CPD; D. Quantitative analysis of the relative integrated optical density (IOD) value of HA. Among them, VC+VE is the positive control, and the treatment concentrations are VC: 100 μg / mL and VE: 7 μg / mL; SH represents extracellular vesicles of Dendrobium officinale obtained by layer-by-layer filtration, and the total amount of its treatment is 4×10 8 particles. When performing statistical analysis using the t-test method, # indicates the difference between the data group and the con (control) group, ##: P-value < 0.01, * indicates the difference from the UV group, *: P-value < 0.05, **: P-value < 0.01.

[0060] Figure 19 Show the effect of SH on basement membrane structural proteins in UV-irradiated 3D skin models. A. Representative immunofluorescence images of laminin 5 (LN5); B. Representative immunofluorescence images of nidogen; C. Quantitative analysis of the relative integrated optical density (IOD) value of LN5; D. Quantitative analysis of the relative integrated optical density (IOD) value of nidogen. Among them, VC+VE is the positive control, and the treatment concentrations are VC: 100 μg / mL and VE: 7 μg / mL; SH represents extracellular vesicles of Dendrobium officinale obtained by layer-by-layer filtration, and the total amount of its treatment is 4×10 8 particles. When performing statistical analysis using the t-test method, # indicates the difference between the data group and the con (control) group, ##: P-value < 0.01, * indicates the difference from the UV group, **: P-value < 0.01.

[0061] Figure 20 Show the effect of SH on connexins in UV-irradiated 3D skin models. A. Representative immunofluorescence of Integrin α6β4; B. Representative immunofluorescence of Plectin; C. Quantitative analysis of the relative integrated optical density (IOD) value of Integrin α6β4; D. Quantitative analysis of the relative integrated optical density (IOD) value of Plectin. Among them, VC+VE is the positive control, and the treatment concentrations are VC: 100 μg / mL and VE: 7 μg / mL; SH represents the extracellular vesicles of Dendrobium officinale obtained by layer-by-layer filtration, and the total amount of its treatment is 4×10 8 particles. When performing statistical analysis using the t-test method, # indicates the difference between the data group and the con (control) group, ##: P-value < 0.01, * indicates the difference from the UV group, **: P-value < 0.01. Detailed description of the invention

[0063] I. Definitions and Terms

[0064] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0065] As used in the context of this disclosure, "vesicle", also known as extracellular vesicle, is a membrane-enclosed structure that is released by cells into the extracellular and intracellular spaces. Extracellular vesicles can contain proteins, lipids, and nucleic acids and can mediate intercellular communication between different cells (including different cell types). Two types of extracellular vesicles are exosomes and microvesicles.

[0066] As used in the context of this disclosure, "exosomes" refers to lipid-bound, cell-secreted small vesicles that are released from cells through the fusion of multivesicular endosomes (MVE) with the plasma membrane and mediate intercellular communication through the intercellular transfer of proteins and RNA. Generally, exosomes range in size from about 30 nm to about 200 nm, sometimes also 30 - 150 nm or 30 - 100 nm, and have a bilayer membrane structure and a saucer-like morphology.

[0067] As used in the context of this disclosure, "plant material" has the meaning commonly understood in the art. Plant raw materials can be in a fresh state or dried. Generally, except for individual plants (such as Lycium barbarum), it is more preferred to produce plant exosomes using fresh plant materials.

[0068] As used in the context of this text, "depth filtration (DF)" means the removal of particles (such as impurities) from a liquid such as a solution using a depth filtration medium, through a combination of size (screening / filtration) and intermolecular interactions (such as electrostatic attraction and hydrophobic interactions between oppositely charged surfaces), to retain particles and impurities throughout the deep portion of the porous structure of the filtration medium. Depth filtration has been successfully used for the primary clarification of bacterial, yeast, insect, and mammalian cell suspensions, either alone or in combination with centrifugation. Depth filtration media can include cellulose (such as cellulose fibers) and / or polypropylene (such as polypropylene fibers), and / or filter aids (such as activated carbon, diatomaceous earth (DE), and / or perlite), and / or resins (such as polymeric resins). In some embodiments, medium-sized particles are removed less by depth filtration; very small particles are effectively removed by Brownian diffusion (plus adsorption) (for example, particles smaller than the voids of the medium can enter the interior of the medium), while very large particles are captured by physical screening or interception (for example, retained and attached to the medium).

[0069] As used herein, the term "depth filter" or "depth capsule filter" achieves filtration within the deep layer of the filter material. Such filters are those that contain a random fiber matrix that combines to form a complex maze of tortuous flow channels. Particle separation in these filters is caused by entrapment or adsorption onto the fiber matrix. The most frequently used depth filter media for bioprocesses of cell culture broths and other feeds include cellulose fibers, filter aids such as DE, and positively charged resin binders. Different from absolute filters, depth filter media retain particles throughout the porous medium, thus allowing retention of particles larger than the pore size. Companies in the current industry that have depth filtration medium products include Sartorius, Merck Millipore, PALL, 3M, etc. Commercially available depth filters include, but are not limited to, the Millistak+Pod depth filter system, XOHC medium (Millipore Corporation), Zeta Plus TM depth filters (3MPurification Inc.). In the present disclosure, depth filtration can be carried out using two or more depth filters arranged in parallel. In this case, commercially available depth filters can include, for example, Millistak+mini DOHC (Millipore Corporation) and XOHC filters (Millipore Corporation) or filters from Pall Corporation (NY), such as Supracap TM Depth Filter Capsules.

[0070] In the context of this disclosure, the terms "cosmetic", "cosmetic composition", "beauty product" or "beauty composition" refer to any chemical industrial product or fine chemical product that is applied, sprayed or otherwise distributed on any part of the human body surface, such as the skin, hair, fingernails, toenails, lips, teeth, etc., for the purpose of cleaning, maintaining, beautifying, modifying and changing appearance, or correcting body odor and maintaining good condition. In some embodiments, the cosmetics, cosmetic compositions, beauty products or beauty compositions of the present disclosure are for non-therapeutic purposes.

[0071] In the context of this disclosure, the term "nutraceutical composition" refers to a product for supplementing the nutrient requirements of the human body, improving physical health and physical functions, which can be administered through the gastrointestinal tract or parenterally, usually through the gastrointestinal tract.

[0072] In the context of this disclosure, "skin" should be understood to include the layers from its outermost or stratum corneum to its innermost or subcutaneous layer (including both the outermost or stratum corneum and the innermost or subcutaneous layer). These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, mast cells, neurons, and / or adipocytes, etc. The term "skin" also includes the scalp. The term "skin" encompasses the skin of mammals and includes human skin. Similarly, the terms "hair, nails and mucosa" encompass the hair, nails and mucosa of mammals (such as humans).

[0073] In the context of this disclosure, the term "treatment" encompasses treatment methods, including methods involving the administration of extracts according to the present disclosure to alleviate or eliminate a disease or disorder or to reduce or eliminate one or more symptoms associated with the disease or disorder. The term "treatment" also encompasses treatment methods involving alleviating or eliminating the physiological consequences of a disease or disorder.

[0074] In the context of this disclosure, the term "care" refers to maintaining the properties of the skin, hair, nails and / or mucosa. The properties are improved or maintained by performing cosmetic treatments and / or care in healthy subjects, in subjects with sensitive skin, and in subjects showing symptoms of the skin, hair, nails and / or mucosa (such as but not limited to ulcers and skin lesions, psoriasis, dermatitis, acne or rosacea, etc.).

[0075] In the context of the present text, when the terms "treatment" and "care" are modified by the modifiers "cosmetic" and / or "non-therapeutic", it means that the purpose of the treatment or care is to contribute to or assist in improving, alleviating or controlling the condition involved, for example, to contribute to or assist in improving or maintaining the cosmetic properties of the skin, hair, nails and / or mucous membranes, which affect the aesthetic appearance of the skin, hair, nails and / or mucous membranes, specifically, for example, to contribute to or assist in repair, anti-inflammation, antioxidant, anti-aging, whitening or moisturizing, etc., to improve the hydration, elasticity, firmness, gloss, tone or texture of the skin, hair, nails and / or mucous membranes.

[0076] In the context of the present text, the term "prevention" refers to the ability of the extracts of the present disclosure to prevent, delay or impede the occurrence or development of a disease or disorder, or to prevent, delay or impede changes in the cosmetic properties of the skin, mucous membranes and / or hair. As used in the present disclosure, the term "prevention" may be used interchangeably with the term "inhibition of occurrence", that is, it refers to the ability of the extracts of the present disclosure to inhibit the occurrence or development of a disease or disorder, or to inhibit changes in the cosmetic properties of the skin, hair, nails and / or mucous membranes.

[0077] In the context of the present text, when used for a specific numerical value or range of values, the term "about" means that the numerical value associated with it fluctuates by ±10%, for example, by ±5%, ±2% or ±1%. For example, as used herein, the expression "about 100" includes 90 and 110 and all values therebetween (such as 90.5, 95, 101, 105, 109.95... etc.). For ratios, the term "about" is used to limit each number of the given ratio. For example, the ratio "about 1:1" means a ratio of (0.9 - 1.1):(0.9 - 1.1). Again, for example, the range of "about n - m" or "about n - about m" means 90%n - 110%n to 90%m - 110%m.

[0078] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, and the methods and materials described below are merely exemplary.

[0079] All patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0080] II. Preparation of Exosomes

[0081] Raw materials

[0082] The raw materials involved in the methods of the present disclosure are plant materials. Preferably, the plant materials can be terrestrial. More preferably, the plant materials are Dendrobium (preferably Dendrobium officinale).

[0083] The plant material may be part or all of a plant, and may be selected from, for example, wood, roots, rhizomes, barks, trunks, flowers, petals, sepals, seeds, fruits, stems, leaves, and / or germ, and mixtures of one or more thereof.

[0084] In some embodiments, the plant material is or includes Dendrobium. The Dendrobium that can be used in the methods of the present disclosure may be dried Dendrobium or fresh Dendrobium, such as fresh or dried Dendrobium stems, or processed traditional Chinese medicine Dendrobium, such as Tiepi Fengdou. The Dendrobium that can be used in the methods of the present disclosure includes, for example but not limited to: Dendrobium officinale, Dendrobium nobile, Dendrobium moniliforme, Dendrobium wardianum, Dendrobium chrysotoxum, Dendrobium fimbriatum, Dendrobium chrysanthum, Dendrobium loddigesii, all or part of Dendrobium phalaenopsis, or its fresh stems, or mixtures of one or more thereof. The methods of the present disclosure may use a mixture of more than one type of Dendrobium as the plant material for exosome extraction.

[0085] Pretreatment

[0086] In some embodiments, the extraction method includes pretreating the plant material (preferably Dendrobium, more preferably Dendrobium officinale). Pretreatment techniques are well known in the art and include physical, chemical, and biological pretreatments, or any combination thereof.

[0087] In some embodiments, the pretreatment includes, but is not limited to, one or more of washing, mixing with an isotonic solution, pulverizing, grinding, stirring, filtering, and centrifuging. Preferably, the pretreatment is carried out in a sterile environment.

[0088] In some embodiments, the washing in the pretreatment can be carried out with an aqueous carrier or other edible or medicinal solvents (including organic solvents or inorganic solvents). Preferably, the cleaning agent is an aqueous carrier. Aqueous carriers are known in the art and include, but are not limited to, sterile water, water for injection, or isotonic solutions. Optionally, the cleaning treatment also contains a certain amount of surfactant in the cleaning solvent. More preferably, the cleaning agent is water or water for injection.

[0089] In some embodiments, the isotonic solution used in the pretreatment includes a solution of an isotonic agent. Isotonic solutions include, but are not limited to, sodium chloride solution, phosphate buffer solution, Ringer's injection, isotonic glucose injection, Ringer's injection of glucose and lactate. Isotonic agents include isotonic agents selected from sodium chloride, mannitol, lactose, glucose (hydrated or anhydrous), sucrose, glycerol, and sorbitol, or solutions of any one of the above. In certain embodiments, sodium chloride is present in an isotonic amount. Preferably, the isotonic solution is a sterile solution or sodium chloride injection containing 9 mg / ml (or 0.9%) sodium chloride.

[0090] In some embodiments, plant materials (preferably Dendrobium, more preferably Dendrobium officinale) are mixed with an isotonic solution in a certain ratio. For example, the plant materials are mixed with the isotonic solution according to the following mass-to-volume ratios (w / v, g / mL, plant mass: isotonic solution volume): about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:20, about 1:30, about 1:50, about 1:100, about 1:200, or about 1:500. Preferably, the range of the mass-to-volume ratio of the plant materials to the isotonic solution is about 1:2 - 1:50, about 1:3 - 1:20, or about 1:5 - 1:20.

[0091] In some embodiments, the comminution in the pretreatment includes any suitable method known in the art, including but not limited to dry grinding, wet grinding, and vibratory ball milling. For example, the comminution includes comminuting the plant or part thereof using powdering technology or cell wall breaking technology. The cell wall breaking technology uses a cell wall breaker.

[0092] In some embodiments, the pretreatment can be carried out at any suitable temperature. For example, the pretreatment can be carried out at the following temperatures: about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 25°C, 30°C, 35°C, or 40°C, or any sub-range within about 0°C to about 40°C (for example, any range between any two of the above temperatures), preferably about 2 - 30°C, more preferably about 2 - 20°C, still more preferably about 2 - 8°C, and most preferably about 4°C. In some embodiments, step (b) of the present disclosure can be carried out at ambient temperature (for example, about 25°C).

[0093] In some embodiments, the operations of stirring, filtering, or centrifuging in the pretreatment can be selected and adjusted by those skilled in the art according to specific needs.

[0094] Layer-by-layer filtration

[0095] The method for extracting plant exosomes of the present disclosure includes the step of layer-by-layer filtration.

[0096] 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 carried out independently or continuously. In some embodiments, the layer-by-layer filtration step includes a depth filtration step and an anti-clogging filtration. Preferably, the anti-clogging filtration may include membrane filtration, such as 0.4 - 0.8 μm membrane filtration; more preferably, the layer-by-layer filtration step includes a depth filtration step, an anti-clogging filtration and a sterilization filtration. Preferably, the anti-clogging filtration may include 0.4 - 0.8 μm membrane filtration and the sterilization filtration includes 0.1 - 0.3 μm membrane filtration.

[0097] The method of the present disclosure can be used to process the plant (preferably Dendrobium, more preferably Dendrobium officinale) at a suitable rate. For example, for one production batch, the rate range for the method of the present disclosure to process the plant is about 30 g / hour, 50 g / hour, 60 g / hour, 70 g / hour, 80 g / hour, 90 g / hour, 100 g / hour, 200 g / hour, 300 g / hour, 400 g / hour, 500 g / hour, 600 g / hour, 700 g / hour, 800 g / hour, 900 g / hour, 1,000 g / hour, or any sub-range within about 10 g / hour to about 1,000 g / hour (such as any range between any two of the above production rates).

[0098] Each filtration step of the layer-by-layer filtration of the disclosed method may be the same or different. For example, the filtration pore sizes are different between different filtration steps. In some embodiments, the layer-by-layer filtration includes 1 filtration step, wherein two different pore size filtration units are used in this filtration step, preferably the two pore sizes decrease sequentially along the direction of the filtrate fluid. For example, the first filtration pore size is about 0.45 μm and the second filtration pore size is about 0.22 μm. In some embodiments, the layer-by-layer filtration includes 2 filtration steps, wherein the pore size in the first filtration step is different from the pore size in the second filtration step. For example, the two pore sizes decrease sequentially along the direction of the filtrate fluid. For example, the pore size in the first filtration step is about 0.45 μm and the pore size in the second filtration step is about 0.22 μm; or for another example, the pore size in the first filtration step is about 2 - 30 μm (such as 30 μm) and the pore size in the second filtration step is about 0.45 μm. In some embodiments, the layer-by-layer filtration includes 3 filtration steps, wherein the pore size in the first filtration step, the pore size in the second filtration step, and the pore size in the third filtration step are not completely the same or at least partially different. Preferably, the three pore sizes decrease sequentially along the direction of the filtrate fluid. 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.

[0099] In the method of the present disclosure, the size of the pore diameter in the filtration step can be about 1000 μm, about 500 μm, about 400 μm, about 300 μm, about 200 μm, about 150 μm, about 100 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, about 15 μm, about 10 μm, about 9 μm, about 8 μm, about 7 μm, about 6 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, about 1 μm, 0.9 μm, about 0.8 μm, about 0.7 μm, about 0.6 μm, about 0.5 μm, about 0.45 μm, about 0.4 μm, about 0.35 μm, about 0.3 μm, about 0.25 μm, about 0.22 μm, about 0.20 μm, about 0.15 μm, about 0.10 μm, about 0.05 μm, about 0.04 μm, about 0.03 μm, about 0.02 μm and about 0.01 μm; the range of the pore diameter in the filtration step can be a range composed of any two of the above pore diameters, such as but not limited to about 0.05 - 2 μm, 0.05 - 1 μm, 0.05 - 0.8 μm, 0.05 - 0.6 μm, 0.05 - 0.4 μm, 0.1 - 2 μm, 0.1 - 1 μm, 0.1 - 0.8 μm, 0.1 - 0.6 μm, 0.1 - 0.4 μm, 0.1 - 0.3 μm, 0.1 - 0.22 μm, 0.2 - 1 μm, 0.2 - 0.8 μm, 0.2 - 0.6 μm, 0.2 - 0.4 μm, 0.3 - 0.8 μm, 0.3 - 0.6 μm, 0.4 - 1.0 μm, 0.4 - 0.8 μm, 0.45 - 0.8 μm, 0.5 - 100 μm, 1 - 100 μm, 1 - 50 μm, 1 - 40 μm, 2 - 50 μm, 2 - 40 μm, 2 - 30 μm, 2 - 20 μm, 2 - 10 μm, 2 - 5 μm, 2 - 4 μm, 3 - 50 μm, 3 - 40 μm, 3 - 30 μm, 3 - 20 μm, 3 - 10 μm, 3 - 5 μm, 3 - 4 μm, 4 - 50 μm, 4 - 40 μm, 4 - 30 μm, 4 - 20 μm, 4 - 10 μm, 4 - 5 μm, 5 - 50 μm, 5 - 40 μm, 5 - 30 μm, 5 - 20 μm, 5 - 10 μm, 6 - 50 μm, 6 - 40 μm, 6 - 30 μm, 6 - 20 μm, 6 - 10 μm, 7 - 50 μm, 7 - 40 μm, 7 - 30 μm, 7 - 20 μm, 7 - 10 μm, 8 - 50 μm, 8 - 40 μm, 8 - 30 μm, 8 - 20 μm, 8 - 10 μm, 9 - 50 μm, 9 - 40 μm, 9 - 30 μm, 9 - 20 μm, 9 - 10 μm. Preferably, the pore diameters for layer-by-layer filtration can be respectively selected from about 1 - 50 μm, 2 - 30 μm, 2 - 20 μm, 6 - 30 μm, 0.05 - 1 μm, 0.1 - 0.8 μm, 0.1 - 0.22 μm, 0.1 - 0.3 μm, 0.2 - 0.8 μm, 0.2 - 0.6 μm, 0.2 - 0.4 μm, 0.3 - 0.8 μm, 0.4 - 1.0 μm, 0.4 - 0.8 μm, 0.45 - 0.8 μm. In some embodiments, more preferably, the pore size in the filtration step can be selected from about 1 - 50 μm, 0.4 - 0.8 μm, and 0.1 - 0.3 μm, or combinations thereof, such as about 2 - 30 μm, about 0.45 - 0.8 μm, or about 0.1 - 0.22 μm, such as about 6 - 30 μm, about 0.45 μm, or about 0.22 μm.

[0100] In the method of the present disclosure, the filtration step in the layer-by-layer filtration can be carried out in one device, where the device includes a plurality of individual units with filtration functions. In some embodiments, the layer-by-layer filtration can be carried out in two or more devices, where each device includes one or more individual units with filtration functions. In some embodiments, the layer-by-layer filtration can be carried out in one device, where the device includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, and 500 individual units with filtration functions.

[0101] In the disclosed method, the layer-by-layer filtration includes the use of solvents such as organic solvents and inorganic solvents for conventional extraction, such as water, isotonic aqueous solutions of sodium chloride, ethanol, acetone, diethyl ether, petroleum ether, ethyl acetate, or mixtures thereof. In some embodiments, the 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, for example, 1 mL / minute, 2 mL / minute, 3 mL / minute, 4 mL / minute, 5 mL / minute, 6 mL / minute, 7 mL / minute, 8 mL / minute, 9 mL / minute, 10 mL / minute, 15 mL / minute, 20 mL / minute, 25 mL / minute, 30 mL / minute, 35 mL / minute, 40 mL / minute, 45 mL / minute, 50 mL / minute, 60 mL / minute, 70 mL / minute, 80 mL / minute, 90 mL / minute, 100 mL / minute, 200 mL / minute, 300 mL / minute, 400 mL / minute, 500 mL / minute, 600 mL / minute, 700 mL / minute, 800 mL / minute, 900 mL / minute, or 1,000 mL / minute, or any sub-range within about 1 mL / minute to about 1,000 mL / minute, such as about 1 mL / minute to about 800 mL / minute, about 1 mL / minute to about 500 mL / minute, about 1 mL / minute to about 200 mL / minute, about 10 mL / minute to about 1,000 mL / minute, about 10 mL / minute to about 800 mL / minute, about 10 mL / minute to about 500 mL / minute, about 10 mL / minute to about 200 mL / minute, about 20 mL / minute to about 1,000 mL / minute, about 20 mL / minute to about 800 mL / minute, about 20 mL / minute to about 500 mL / minute, about 20 mL / minute to about 200 mL / minute, about 50 mL / minute to about 1,000 mL / minute, about 50 mL / minute to about 500 mL / minute, about 50 mL / minute to about 200 mL / minute. In embodiments for large-scale production, the flow rate of the solvent can be as high as about 1 L / minute or even higher. In some embodiments, the flow rate of the solvent in the layer-by-layer filtration of the disclosed method is about 20 mL / minute to about 1,000 mL / minute, such as about 20 mL / minute to about 500 mL / minute, about 20 to about 200 mL / minute, about 30 to about 100 mL / minute.

[0102] The layer-by-layer filtration of the present disclosure can be carried out at any suitable temperature. For example, the layer-by-layer filtration of the present disclosure can be carried out at a suitable temperature, such as about 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 25 °C, 30 °C, 35 °C or 40 °C, or any sub-range within about 0 °C to about 40 °C (for example, any range between any two of the above temperatures), such as about 0 °C to about 35 °C, about 0 °C to about 30 °C, preferably about 0 °C to about 25 °C. In some embodiments, the layer-by-layer filtration of the present disclosure can be carried out at ambient temperature (e.g., 25 °C). In other embodiments, the layer-by-layer filtration of the present disclosure can be carried out at about 4 °C, for example, the depth filtration of the layer-by-layer filtration of the present disclosure at about 4 °C, and the membrane filtration of the layer-by-layer filtration of the present disclosure at about 4 °C.

[0103] Generally speaking, any suitable filtration material can be used for the layer-by-layer filtration of the present disclosure. For example, the material can be a polymer. In some embodiments, the polymer can be a hydrophobic polymer. In some embodiments, the polymer can be a hydrophilic polymer. In some specific embodiments, the polymer includes but is not limited to poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylysine or polyglutamic acid, polyethersulfone (PES) (unmodified), modified polyethersulfone (mPES), polyvinylidene fluoride (PVDF), cellulose acetate, nitrocellulose, MCE (mixed cellulose ester), ultra-high molecular weight polyethylene (UPE), polytetrafluoroethylene (PTFE), nylon, polysulfone, polyacrylonitrile, polypropylene, polyvinyl chloride, polycarbonate, ceramics, diatomaceous earth, glass fiber, resin-bonded glass fiber and combinations thereof. The available forms of the filtration material include but are not limited to microporous membranes, homogeneous membranes, and asymmetric membranes.

[0104] In some embodiments, the layer-by-layer filtration includes using a filter membrane for filtration, and the filter membrane contains the following polymers: nylon, copolymers of acrylic acid, polysulfone, polyvinylidene fluoride, fiber esters, and cellulose esters. The filter membrane is usually made of a polymeric support material, such as PTFE (polytetrafluoroethylene), PES (polyethersulfone), PVP (polyvinylpyrrolidone), PVDF (polyvinylidene fluoride), nylon (polyamide), PP (polypropylene), cellulose (including cellulose esters), PEEK (polyetheretherketone), nitrocellulose, glass fiber, resin-bonded glass fiber, etc. In some embodiments, the polymer can be a hydrophilic polymer. Hydrophilic membranes include but are not limited to Bioassure (from Cuno Inc.); EverLUX TM polyethersulfone; STyLUX TMPolyethersulfone (both from Meissner); Millex GV, Millex HP, Millipak 60, Millipak 200, and Durapore CVGL01TP3 membranes (from Millipore); Fluorodyne TM EX EDF membranes, Supor TM EAV, Supor TM EBV, Supor TM EKV (all from Pall); Sartopore TM (from Sartorius); Hydrophilic PES membranes from Sterlitech; and WFPES PES membranes from Wolftechnik.

[0105] In some other embodiments, the layer-by-layer filtration of the present disclosure, such as depth filtration, may also introduce adsorbents such as silica particles, diatomaceous earth, or carbon particles.

[0106] In some embodiments, the inherent properties of the membrane can be altered by treating the membrane surface. For example, it is known to prepare hydrophilic or hydrophobic membranes by treating with other materials (such as other polymers, graphite, silicon, etc.) to coat the membrane surface. For example, the membranes used in layer-by-layer filtration can be modified with positive charges. For example, the filtration membrane in layer-by-layer filtration can be a charge-modified polyvinylidene fluoride (PVDF) membrane produced by Micropore, or a membrane obtained from Pall using nylon 66 or positively charged polyethersulfone sulfate. The filter membrane can be sterilized (e.g., autoclaved) before use to ensure its sterility.

[0107] Depth filtration

[0108] The present disclosure provides a method for preparing plant exosomes (preferably exosomes of Dendrobium, more preferably exosomes of Dendrobium officinale), which includes a layer-by-layer filtration step having the function or effect of preventing or avoiding clogging. The method of the present disclosure can use any filtration having the function or effect of preventing or avoiding clogging, such as depth filtration (DF), microfiltration (MF), ultrafiltration (UF), sterile filtration, membrane chromatography (MC), and centrifugation.

[0109] In some embodiments, the present disclosure provides a method for preparing plant exosomes (preferably exosomes of Dendrobium, more preferably exosomes of Dendrobium officinale), which includes a layer-by-layer filtration step, and the layer-by-layer filtration step includes depth filtration. Preferably, in some embodiments, the depth filtration is achieved by a depth filter, such as a depth capsule filter.

[0110] In some embodiments, the depth filter is a commercially available depth filter, such as a depth filter produced by Pall Corporation (NY), such as Supracap TM depth filter; it can also be an activated carbon filter such as MilliStak TM series produced by Millipore (Billerica, MA); it can also be other depth filters: Profile star 5μm depth filter (PALL, catalog number BYA050P6), Profile star 3μm depth filter (PALL, catalog number BYA030P6). In some embodiments, the depth filter used in the present disclosure can be a filtration product of Pall Corporation (NY), such as Supracap TM Depth Filter Capsules, and for example, Supracap TM 50 depth filter capsules, or Supracap TM 100 Depth Filter Capsules.

[0111] The loading range for depth filtration that can be used in the methods of the present disclosure is about 50 - 200 L / m 2 , for example 50 - 150 L / m 2 , 40 - 100 L / m 2 .

[0112] For the present disclosure, a depth filter can be any filter having a depth filtration function. In some embodiments, the depth filter can be a filter having any pore size. In some embodiments, the depth filters used in the present disclosure include depth filters having a pore size range of approximately: 0.5 - 100 μm, 1 - 100 μm, 1 - 50 μm, 1 - 40 μm, 2 - 50 μm, 2 - 40 μm, 2 - 30 μm, 2 - 20 μm, 3 - 50 μm, 3 - 40 μm, 3 - 30 μm, 3 - 20 μm, 4 - 50 μm, 4 - 40 μm, 4 - 30 μm, 4 - 20 μm, 5 - 50 μm, 5 - 40 μm, 5 - 30 μm, 5 - 20 μm, 6 - 50 μm, 6 - 40 μm, 6 - 30 μm, 7 - 50 μm, 7 - 40 μm, 7 - 30 μm, 7 - 20 μm, 8 - 50 μm, 8 - 40 μm, 8 - 30 μm, 8 - 20 μm, 9 - 50 μm, 9 - 40 μm, 9 - 30 μm, 9 - 20 μm. Preferably, the pore sizes for depth filtration can be respectively selected from approximately 1 - 50 μm, approximately 2 - 30 μm, approximately 2 - 20 μm, approximately 6 - 30 μm. More preferably, it is a depth filter having a pore size of approximately 2 - 30 μm, such as a depth capsule filter having a pore size of approximately 2 - 30 μm, such as Supracap TM Depth FilterCapsules filter.

[0113] For the present disclosure, the depth filter can be any form of depth filter, such as a filter plate filter, a filter disc filter, a filter element filter, a filter stack filter, a depth capsule filter, or a cartridge filter. In some embodiments, the depth filtration of the present disclosure is performed by a depth capsule filter. Depth capsule filters are typically applied in the clarification filtration of fermentation broth and cell culture medium, the filtration of serum and blood products, the filtration of enzyme preparations, the removal of impurities in chemical drugs, decarbonization filtration, or the filtration of colloidal or viscous materials. The inventors of the present disclosure unexpectedly found that depth capsule filters are also suitable for the extraction of plant materials, especially suitable for the method of extracting plant exosomes of the present disclosure.

[0114] The depth capsule filter generally can include a single-layer membrane structure or a double-layer membrane structure. The depth capsule filter used in the method of the present disclosure can be a single-layer membrane structure, which for example includes cellulose fibers, and / or filter aids (such as diatomaceous earth and perlite) and / or resins. In some embodiments, the depth capsule filter consists of two layers of membranes, where the upper filter plate has a larger pore size to intercept large-sized particles and protect the lower filter plate; the lower filter plate has a smaller pore size to further intercept small particles and ensure the clarity of the filtered liquid. For example, the pore size of the first layer of membrane can be approximately 11 - 30 μm, and the pore size of the second layer of membrane can be approximately 6 - 15 μm; or the pore size of the first layer of membrane is approximately 8 - 20 μm, and the pore size of the second layer of membrane is approximately 2 - 4 μm.

[0115] 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).

[0116] For the present disclosure, any suitable flow rate may be used for the deep filtration. For example, the filtration flow rate for deep filtration that can be used in the method of the present disclosure may range from 100-1000LMH (1m3 / 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 method of the present disclosure can be expressed in mL / min, for example, the depth filtration is performed using a flow rate ranging from 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.

[0117] For purposes of the present disclosure, any suitable pressure may be used for the deep filtration. For example, the deep filtration may be performed using a pressure ranging from 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 above pressures).

[0118] For the present disclosure, the layer-by-layer filtration (such as depth filtration and optional other filtration steps) can be carried out for any suitable time period. For example, for one production batch, the layer-by-layer filtration can be carried out within a time range of about 10 minutes to about 10 hours, such as about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or any sub-range within about 10 minutes to about 10 hours (such as any range between any two of the above time points). Preferably, for example, for 500 ml of crude plant extract obtained by pretreatment, when using layer-by-layer filtration including depth filtration and membrane filtration (such as 0.45 μm membrane filtration and 0.22 μm membrane filtration) defined in the present disclosure, the treatment time is about 0.5 hours - about 2 hours, preferably about 1 hour, while the same crude plant extract takes about 3 hours by ultracentrifugation at 12,000 g for 70 minutes twice.

[0119] The method of the present disclosure can be used to process the plant (preferably Dendrobium, more preferably Dendrobium officinale) at a suitable rate. For example, for one production batch, the rate range for the method of the present disclosure to process the plant, such as crude plant extract, is about 10 g / hour, 30 g / hour, 50 g / hour, 60 g / hour, 70 g / hour, 80 g / hour, 90 g / hour, 100 g / hour, 200 g / hour, 300 g / hour, 400 g / hour, 500 g / hour, 600 g / hour, 700 g / hour, 800 g / hour, 900 g / hour, 1,000 g / hour, or any sub-range within about 10 g / hour to about 1,000 g / hour (such as any range between any two of the above production rates).

[0120] The method for preparing plant exosomes by the layer-by-layer filtration (such as depth filtration and optional other filtration steps) of the present disclosure can obtain about the following number of plant exosomes per 100 g of plant: 1×10 10 individuals, 5×10 10 individuals, 10×10 10 individuals, 11×10 10 individuals, 12×10 10 individuals, 13×10 10 individuals, 14×10 10 individuals, 15×10 10 individuals, 16×10 10 individuals, 17×10 10 individuals, 18×10 10 individuals, 19×10 10 individuals, 20×10 10 individuals, 22×10 10 individuals, 24×10 10pieces, 26, ×10 10 pieces, 28×10 10 pieces, 30×10 10 pieces, 32×10 10 pieces, 34×10 10 pieces, 36×10 10 pieces, 38×10 10 pieces, 40×10 10 pieces, 42×10 10 pieces, 44×10 10 pieces, 46×10 10 pieces, 48×10 10 pieces, 50×10 10 pieces, 52×10 10 pieces, 54×10 10 pieces, 56×10 10 pieces, 58×10 10 pieces, 60×10 10 pieces, 66×10 10 pieces or more. In some embodiments, the layer-by-layer filtration of the present disclosure can obtain about 10×10 10 -30×10 10 pieces, preferably about 10×10 10 -20×10 10 pieces, such as about 12×10 10 pieces of plant exosomes.

[0121] The depth filter described in the present disclosure comprises a depth filtration medium. In some embodiments, the depth filtration medium can be a hydrophilic material with low protein adsorption, such as polyethersulfone (PES), polyvinylidene fluoride (PVDF), polypropylene (PP), etc. (preferably having a pore size range of 1-50 μm). In some embodiments, the depth filtration medium can be in the form of a filter plate. In some embodiments, the depth filtration medium layer is optionally selected from one or more of Supradur P filter plates, K300 filter plates, and BECOPADP 270 filter plates. Generally, common filter plates in the art are generally divided into two types: one is a filter plate made of synthetic fiber and plant fiber materials; the other is a filter plate made of asbestos and pulp. Specifically, the filter plates used in the present disclosure can be produced by companies such as 3M, PALL, and EATON in the United States, and their common models include the SP series filter plates of 3M company, the P series filter plates of PALL company, and the BECO series filter plates of EATON company.

[0122] In some embodiments, the depth filtration medium may be in a gradient distribution or a mixed distribution. In some embodiments, the depth filtration medium may be in a gradient distribution. In some embodiments, the depth capsule filtration device has a bilayer membrane structure with a gradient distribution, and the pore sizes of the two-layer membrane structure gradually decrease along the direction of the filtrate flow. For example, the pore size of the first layer of membrane can be selected to be about 11 - 30 μm, and the pore size of the second layer of membrane can be about 6 - 15 μm; or the pore size of the first layer of membrane can be about 8 - 20 μm, and the pore size of the second layer of membrane can be about 2 - 4 μm.

[0123] In some embodiments, the depth filtration medium may further include a membrane filtration layer. For example, the filtration pore size of the membrane filtration layer is preferably about 0.1 - 10 μm. In some embodiments, the depth filtration medium of the present disclosure can be used in combination with a membrane filtration medium. In some embodiments, the membrane filtration layer is disposed at the liquid outlet end of the depth filtration medium. In some embodiments, the membrane filtration layer is disposed at the liquid inlet end of the depth filtration medium. In some embodiments, the membrane filtration material after depth capsule filtration generally selects a hydrophilic material with low protein adsorption, such as polyethersulfone (PES), polyvinylidene fluoride (PVDF), polypropylene (PP), etc.

[0124] In some embodiments, the depth filtration medium may further include a filter aid. In some embodiments, the filter aid may include diatomaceous earth, perlite, talcum powder, silica gel, activated carbon, asbestos, molecular sieve, clay, etc. In some embodiments, the filter aid may be a filter aid based on mineral sources such as silica, such as perlite, diatomaceous earth or sand, or an activated carbon filter aid optionally derived from natural materials such as wood or coconut shell.

[0125] In some embodiments, the depth filtration described in the present disclosure can use a pump. In some embodiments, the pump may include a peristaltic pump, a diaphragm pump, a gear pump, and a centrifugal drive pump. In some embodiments, the depth filtration can use a peristaltic pump.

[0126] In some embodiments, the flow rate of the pump used for depth filtration can be in the following approximate ranges: 1 - 1000 mL / min, 10 - 1000 mL / min, 10 - 800 mL / min, 10 - 500 mL / min, 15 - 700 mL / min, 16 - 600 mL / min, 17 - 500 mL / min, 18 - 300 mL / min, 19 - 400 mL / min, 20 - 1000 mL / min, 20 - 500 mL / min, 20 - 300 mL / min, 20 - 200 mL / min, 30 - 100 mL / min, 40 - 100 mL / min, or 50 - 100 mL / min. Preferably, the pump used for depth filtration in the present disclosure is a peristaltic pump, and the flow rate is, for example, about 20 - 1000 mL / min, such as about 20 - 500 mL / min, about 20 - 200 mL / min, about 30 - 100 mL / min.

[0127] In some embodiments, an aqueous solution is used to rinse the depth filter in the depth filtration of the present disclosure. Preferably, an isotonic solution is used, more preferably an isotonic sodium chloride solution, and most preferably sodium chloride injection is used to rinse the depth capsule filter. For example, in the depth filtration of the present disclosure, preferably before the start of filtration, sodium chloride injection is used to rinse the depth capsule filter at a flow rate of, for example, about 20 - 1000 mL / min, such as about 20 - 500 mL / min, about 20 - 200 mL / min, about 30 - 100 mL / min.

[0128] In some embodiments, in depth filtration, an aqueous solution is used to clean the depth filter. Preferably, an isotonic solution is used, more preferably an isotonic sodium chloride solution, and most preferably sodium chloride injection is used to clean the depth capsule filter. For example, in the depth filtration of the present disclosure, preferably at the end of depth filtration, sodium chloride injection is used to clean the depth capsule filter at a flow rate of, for example, about 20 - 1000 mL / min, such as about 20 - 500 mL / min, about 20 - 200 mL / min, about 30 - 100 mL / min.

[0129] Membrane filtration

[0130] In some embodiments, layer-by-layer filtration further includes membrane filtration. For example, the layer-by-layer filtration of the present disclosure includes depth filtration and membrane filtration. For example, in the said layer-by-layer filtration, membrane filtration can include anti-clogging membrane filtration and sterilizing membrane filtration. In some embodiments, anti-clogging membrane filtration can be carried out first, and then optionally sterilizing membrane filtration can be carried out. In some embodiments, anti-clogging membrane filtration can be carried out before sterilizing membrane filtration. In some embodiments, anti-clogging membrane filtration can be carried out one or more times, and then sterilizing membrane filtration can be carried out one or more times.

[0131] 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, such as but not limited to about the following ranges: 0.05 - 2 μm, 0.05 - 1 μm, 0.05 - 0.8 μm, 0.05 - 0.6 μm, 0.05 - 0.4 μm, 0.1 - 2 μm, 0.1 - 1 μm, 0.1 - 0.8 μm, 0.1 - 0.6 μm, 0.1 - 0.4 μm, 0.1 - 0.3 μm, 0.1 - 0.22 μm, 0.2 - 1 μm, 0.2 - 0.8 μm, 0.2 - 0.6 μm, 0.2 - 0.4 μm, 0.3 - 0.8 μm, 0.3 - 0.6 μm, 0.4 - 1.0 μm, 0.4 - 0.8 μm, 0.45 - 0.8 μm. In some preferred embodiments, the pore size range of the membrane filtration can be selected from about 0.4 - 0.8 μm and about 0.1 - 0.3 μm or a combination thereof, such as about 0.45 - 0.8 μm and about 0.1 - 0.22 μm.

[0132] In some embodiments, the layer-by-layer filtration of the present disclosure includes membrane filtration after deep filtration. For example, the membrane filtration can include membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm) and membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm). In some embodiments, membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm) can be carried out first, and then optionally membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm) can be carried out. In some embodiments, membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm) can be carried out before membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm). In some embodiments, membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm) can be carried out one or more times, and then membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm) can be carried out one or more times. Preferably, one or more membrane filtrations can be carried out before membrane filtration of about 0.1 - 0.3 μm (such as about 0.22 μm), preferably 1 time of membrane filtration of about 0.4 - 0.8 μm (such as about 0.45 μm), to reduce or prevent the clogging of the 0.22 μm membrane filtration.

[0133] In some embodiments, the membrane filtration includes passing through one or more membrane filters in series. In some embodiments, the number of membrane filters in series ranges from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 membrane filters in series, preferably 1 to 4, more preferably 2 to 3. The cut-off sizes of all the membrane filters in series can be the same or different. In some embodiments, at least 2 of the membrane filters in series have different cut-off sizes. In some embodiments, the size exclusion decreases from large to small along the series of membrane filters. For example, in 3 membrane filters in series, the first filter can be a ~0.45 μm filter, the second filter can be a 0.3 μm filter, and the last filter can be a ~0.22 filter.

[0134] Exemplary membrane filters for the methods of the present disclosure include, but are not limited to, polyethersulfone membrane filters, polyvinylidene fluoride membrane filters, cellulose membrane filters, mixed cellulose ester membrane filters, cellulose acetate membrane filters, nitrocellulose membrane filters, polyamide membrane filters, polycarbonate membrane filters, polytetrafluoroethylene membrane filters, polypropylene membrane filters, nitrated cellulose membrane filters, glass fibers, resin-bonded glass fibers, or bead filters.

[0135] For the methods of the present disclosure, the material of the membrane used for membrane filtration can be any filtration material conventionally used in the art, such as the various filtration materials and membrane materials described in the layer-by-layer filtration section above. In some embodiments, the anti-clogging membrane filtration is a resin-bonded glass fiber membrane, preferably with a pore size of about 0.45 - 0.8 μm. In some embodiments, the sterilizing membrane filtration is a polyethersulfone (PES) membrane, preferably with a pore size of about 0.1 - 0.22 μm.

[0136] In some embodiments, the membrane filtration can be carried out using any suitable pressure. For example, the membrane filtration can be carried out using a feed pressure in the range of about 0.1 psi to about 100 psi, such as about 0.1 psi, 0.5 psi, 1 psi, 2 psi, 3 psi, 4 psi, 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, 10 psi, 20 psi, 30 psi, 40 psi, 50 psi, 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, or any sub-range within about 0.1 psi to about 100 psi (e.g., any range between any two of the above feed pressures). Any suitable permeate-side pressure can be used for the membrane filtration. For example, the membrane filtration can be carried out using a permeate pressure in the range of about 0.1 psi to about 100 psi, such as about 0.1 psi, 0.5 psi, 1 psi, 2 psi, 3 psi, 4 psi, 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, 10 psi, 20 psi, 30 psi, 40 psi, 50 psi, 60 psi, 70 psi, 80 psi, 90 psi, 100 psi, or any sub-range within about 0.1 psi to about 100 psi (e.g., any range between any two of the above pressures).

[0137] In some embodiments, the membrane filtration described in the present disclosure can use a pump. In some embodiments, the pump can include a peristaltic pump, a diaphragm pump, a gear pump, and a centrifugal drive pump. In some embodiments, the membrane filtration can use a peristaltic pump. In some embodiments, the flow rate of the pump used for the membrane filtration can be in the following ranges: 1 - 1000 mL / min, 100 - 1000 mL / min, 200 - 1000 mL / min, 500 - 1000 mL / min, 10 - 800 mL / min, 10 - 500 mL / min, 20 - 1000 mL / min, 20 - 500 mL / min, 20 - 300 mL / min, 20 - 200 mL / min, 30 - 100 mL / min, 40 - 100 mL / min, or 50 - 100 mL / min. Preferably, the flow rate of the pump for the membrane filtration of the present disclosure is about 20 - 1000 mL / min, such as about 500 - 1000 mL / min, about 20 - 500 mL / min, about 20 - 200 mL / min, about 30 - 100 mL / min.

[0138] In some embodiments, an aqueous solution, preferably an aqueous isotonic solution, more preferably an isotonic sodium chloride solution, is used to rinse and / or clean the membrane filter in the membrane filtration described in the present disclosure. Preferably, an injection of sodium chloride is used to clean the membrane filter at a flow rate of about 20 - 1000 mL / min, such as about 20 - 500 mL / min, about 20 - 200 mL / min, about 30 - 100 mL / min.

[0139] In some embodiments, any suitable flow rate can be used for the membrane filtration. For example, a flow rate in the range of about 1 mL / min to about 1,000 mL / min can be used for the membrane filtration, such as about 1 mL / min to about 800 mL / min, about 1 mL / min to about 500 mL / min, about 1 mL / min to about 200 mL / min, about 10 mL / min to about 1,000 mL / min, about 10 mL / min to about 800 mL / min, about 10 mL / min to about 500 mL / min, about 10 mL / min to about 200 mL / min, about 20 mL / min to about 1000 mL / min, about 20 mL / min to about 800 mL / min, about 20 mL / min to about 500 mL / min, about 20 mL / min to about 200 mL / min, about 50 mL / min to about 1000 mL / min, about 50 mL / min to about 500 mL / min, about 50 mL / min to about 200 mL / min. In embodiments of large-scale production, the flow rate of the membrane filtration can be as high as about 1 L / min or even higher. Preferably, the filtration flow rate of the present disclosure is about 20 - 1000 mL / min, such as about 20 - 200 mL / min, about 30 - 100 mL / min.

[0140] In some embodiments, any suitable time limit can be used for the membrane filtration. For example, for one production batch, the membrane filtration can be carried out within a time range of about 10 minutes to about 10 hours, such as about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or any sub-range within about 10 minutes to about 10 hours (for example, any range between any two of the above time points). Preferably, for 500 mL of the crude plant extract obtained by pretreatment, the membrane filtration is carried out for about 1 hour. Preferably, when using layer-by-layer filtration including depth filtration, anti-clogging membrane filtration such as about 0.45 μm filtration and sterilizing membrane filtration such as about 0.22 μm filtration, the treatment time is about 0.5 hours - about 2 hours, preferably about 1 hour.

[0141] In some embodiments, the method of the present disclosure comprising depth filtration and membrane filtration is capable of obtaining the following yields of plant exosomes per 100 g of plant: 1×10 10 particles, 5×10 10 particles, 10×10 10 particles, 11×10 10 particles, 12×10 10 particles, 13×10 10 particles, 14×10 10 particles, 15×10 10 particles, 16×10 10 particles, 17×10 10 particles, 18×10 10 particles, 19×10 10 particles, 20×10 10 particles, 22×10 10 particles, 24×10 10 particles, 26×10 10 particles, 28×10 10 particles, 30×10 10 particles, 32×10 10 particles, 34×10 10 particles, 36×10 10 particles, 38×10 10 particles, 40×10 10 particles, 42×10 10 particles, 44×10 10 particles, 46×10 10 particles, 48×10 10 particles, 50×10 10 particles, 52×10 10 particles, 54×10 10 particles, 56×10 10 particles, 58×10 10 particles, 66×10 10 particles or even more. In some embodiments, the membrane filtration of the present disclosure is capable of obtaining about 10×10 10 - 20×10 10 particles, preferably about 12×10 10 particles of plant exosomes per 100 g of Dendrobium officinale (preferably Dendrobium officinale Kimura et Migo).

[0142] Preferred embodiments

[0143] In one embodiment, the method for preparing plant exosomes of the present disclosure comprises the following steps:

[0144] (a) Obtaining a plant raw material;

[0145] (b) Pretreating the obtained plant raw material to obtain a crude plant extract;

[0146] (c) Perform layer-by-layer filtration on the crude plant extract;

[0147] Among them, the layer-by-layer filtration treatment includes a depth filtration step and an optional membrane filtration step.

[0148] In one embodiment, the method for preparing plant exosomes according to the present disclosure includes the following steps:

[0149] (a) Obtain plant raw materials;

[0150] (b) Pretreat the obtained plant raw materials, preferably by mixing, crushing, filtering, and centrifuging to obtain a crude plant extract; (c) Perform layer-by-layer filtration on the crude plant extract to obtain a filtrate;

[0151] Among them, the layer-by-layer filtration treatment includes a depth filtration step. Preferably, the depth filtration step uses a depth filtration with a pore size of about 1 - 50 μm, more preferably a depth filtration with a pore size of about 2 - 30 μm; and

[0152] (d) Perform membrane filtration on the filtrate in step (c), 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 anti-clogging membrane filtration with a pore size of 0.45 μm and sterilizing membrane filtration with a pore size of 0.22 μm performed in sequence to obtain plant exosomes.

[0153] In the above embodiment, the obtained plant materials are pretreated in step (b), and the pretreatment includes mixing the plant materials with an isotonic solution (preferably sodium chloride injection solution), crushing the plant raw materials using a blender to obtain a plant residue solution, filtering the plant residue solution through a filter screen to obtain a plant juice, and centrifuging the plant juice and collecting the supernatant to obtain a crude plant extract.

[0154] In the above embodiment, large-scale mixing and crushing of the plant materials can be performed in step (b). In some specific embodiments, in step (b), plants with a mass of about 1000 g or even more, such as about 500 - 1000 g, about 50 - 200 g, for example about 100 g, are mixed and crushed. In some embodiments, step (b) is performed at about 4°C.

[0155] In the above embodiments, in step (b), the crushed slag liquid is subjected to stirring treatment. Preferably, the stirring speed is about 50 - 500 rpm, more preferably about 100 - 250 rpm, and even more preferably about 200 ± 20 rpm. In some embodiments, the stirring time is about 10 - 200 min, preferably about 2 - 100 min, and even more preferably about 30 min.

[0156] In some preferred embodiments, the centrifugation in step (b) is carried out at about 1000 - 10000 g. In some preferred embodiments, the centrifugation in step (b) is carried out at at least about 3000 - 6000 g. In some preferred embodiments, the centrifugation time in step (b) is about 5 - 60 min. In some preferred embodiments, the ultracentrifugation time in step (b) is about 15 min, and / or is carried out at about 4 °C.

[0157] In a specific embodiment of the above method for preparing plant exosomes, the plant material is or includes Dendrobium, preferably Dendrobium officinale.

[0158] In a specific embodiment of the above method for preparing plant exosomes, the step of filtration layer by layer includes a deep filtration step, wherein the deep filter is a deep capsule filter with a pore size of about 1 - 50 μm, preferably about 2 - 30 μm, and the loading range is about 50 - 200 L / m 2 , such as 50 - 150 L / m 2 , 40 - 100 L / m 2 ; preferably, the deep filter is a deep filter produced by Pall Corporation (NY), and is Supracap TM Depth Filter Capsules.

[0159] In a specific embodiment of the above method for preparing plant exosomes, in step (d), the filtrate in step (c) is sequentially filtered through a filter of about 0.45 μm and a filter of about 0.22 μm, and the final filtrate is collected to obtain plant exosomes.

[0160] Technical effects

[0161] The method of filtration layer by layer of the present disclosure has the following advantages compared with the ultracentrifugation method:

[0162] (1) Lower time consumption;

[0163] (2) Higher exosome yield or particle concentration;

[0164] (3) It can prevent the filter membrane from being blocked due to excessive impurities, and can ensure the sterility of the product;

[0165] (4) The extracted plant exosomes have excellent metabolomic performance;

[0166] (5) The extracted plant exosomes have a typical exosome-like saucer shape and a particle size in the range of 30 - 200 nm;

[0167] (6) The extracted plant exosomes have anti-inflammatory and immunomodulatory activities.

[0168] The method for preparing the plant exosomes (preferably Dendrobium officinale, more preferably exosomes of Dendrobium officinale var. officinale) of the present disclosure is suitable for small-scale, pilot-scale, and large-scale production. In some embodiments, the method for preparing exosomes of the present disclosure is particularly used for large-scale production, and the amount of plant material that can be processed is, for example, at least about 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9000 g, 10000 g, 50000 g, and even up to 100000 g. Preferably, the method for preparing exosomes of the present disclosure can process plant material within about 1000 g, more preferably within about 10000 g, and most preferably within about 100000 g of plant raw materials.

[0169] In particular, the layer-by-layer filtration treatment of the present disclosure is particularly suitable for large-scale production. For example, based on the deep filtration and membrane filtration preferably used in the present disclosure (as shown in the examples), large-scale production is achieved by increasing the membrane area of the filter. In the layer-by-layer filtration of the present disclosure, by appropriately increasing the membrane area in the layer-by-layer filtration, at least about 100 g, 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, 2000 g, 3000 g, 4000 g, 5000 g, 6000 g, 7000 g, 8000 g, 9000 g, 10000 g, 50000 g, and even 100000 g of plant material or its corresponding pretreatment solution can be filtered.

[0170] III. Exosomes

[0171] The plant exosomes (preferably Dendrobium, more preferably Dendrobium officinale exosomes) prepared according to the method of the present disclosure can have any suitable hydrodynamic particle size or diameter. For example, the plant exosomes can have a hydrodynamic particle size or diameter of about 10 nm to about 10 μm, such as about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm and 10 μm, or any sub-range within about 10 nm to about 10 μm (for example, any range between any two of the above particle sizes). Preferably, the plant exosomes prepared according to the method of the present disclosure have a diameter of about 30 - 450 nm, preferably a diameter of about 30 - 250 nm, and more preferably a diameter of about 30 - 200 nm.

[0172] The plant exosomes can have any suitable shape, including but not limited to saucer shape, spherical shape, disc shape. Preferably, the plant exosomes prepared according to the method of the present disclosure have a saucer shape.

[0173] The plant exosomes prepared according to the method of the present disclosure (preferably exosomes of Dendrobium officinale, more preferably exosomes of Dendrobium officinale var. officinale) have more metabolites in one or more of the following aspects compared to the plant exosomes prepared by the ultracentrifugation method: lipids and lipid-like molecules, organic oxygen compounds, phenylpropanoids and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzene compounds. In some embodiments, the number of lipids in the plant exosomes prepared according to the method of the present disclosure is greater than the number of lipids in the plant exosomes prepared by the ultracentrifugation method. As shown in Table 4 of the examples below, the lipid classification mainly includes fatty acyl (FA), glycerolipids (GL), glycerophospholipids (GP), prenol lipids (PR), sphingolipids (SP), steroids (ST), and saccharolipids (SL). In some embodiments, the number of types of fatty acyl compounds in the plant exosomes prepared according to the method of the present disclosure is 190 - 300, preferably 200 - 250. In some embodiments, the number of types of glycerolipid compounds in the plant exosomes prepared according to the method of the present disclosure is 2 - 10, preferably 3 - 9, more preferably 4 - 8. In some embodiments, the number of types of glycerophospholipid compounds in the plant exosomes prepared according to the method of the present disclosure is 2 - 10, preferably 3 - 8, more preferably 4 - 7. In some embodiments, the number of types of prenol lipid compounds in the plant exosomes prepared according to the method of the present disclosure is 190 - 400, preferably 200 - 350, more preferably 200 - 300. In some embodiments, the number of types of sphingolipid compounds in the plant exosomes prepared according to the method of the present disclosure is 2 - 9, preferably 2 - 5, more preferably 3. In some embodiments, the number of types of steroid compounds in the plant exosomes prepared according to the method of the present disclosure is 10 - 100, preferably 20 - 50, more preferably 20 - 40. In some embodiments, the number of types of saccharolipid compounds in the plant exosomes prepared according to the method of the present disclosure is 0 - 5, preferably 2 - 5, most preferably 2 - 3.

[0174] In some embodiments, the Dendrobium officinale, preferably Dendrobium officinale var. officinale exosomes provided by the present disclosure contain about 1×10 5 to 1×10 10 particles / mL, preferably 1×10 6 to 1×10 9 particles / mL, more preferably 1×10 7 to 1×10 9 particles / mL, for example, about 1×10 8 particles / mL.

[0175] In some embodiments, the Dendrobium officinale exosomes provided by the present disclosure preferably contain one or more of the following metabolites: lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzene compounds. In some embodiments, the Dendrobium officinale exosomes provided by the present disclosure contain at least lipids and lipid-like molecules, and organic oxygen compounds, and the lipids and lipid-like molecules and organic oxygen compounds account for about 40%, 45%, 50%, or more than 60% of the total number of metabolites, for example, about 50% of the total number of metabolites; preferably, the lipids and lipid-like molecules account for 30%-40% of the total number of metabolites, for example, about 35%, and the organic oxygen compounds account for 10%-20% of the total number of metabolites, for example, about 15%.

[0176] In some embodiments, the Dendrobium officinale exosomes provided by the present disclosure contain lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, wherein the lipids and lipid-like molecules account for 30%-40% of the total number of metabolites, for example, about 35%; the organic oxygen compounds account for 10%-20% of the total number of metabolites, for example, about 15%; and the phenylpropanes and polyketides account for 10%-20% of the total number of metabolites, for example, about 14%.

[0177] In some embodiments, the Dendrobium officinale exosomes provided by the present disclosure contain lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, preferably wherein the lipids and lipid-like molecules account for 30%-40% of the total number of metabolites, for example, about 35%; the organic oxygen compounds account for 10%-20% of the total number of metabolites, for example, about 15%; and the phenylpropanes and polyketides account for 10%-20% of the total number of metabolites, for example, about 14%.

[0178] In some embodiments, the Dendrobium officinale exosomes prepared according to the method of the present disclosure contain lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzene compounds, preferably wherein the lipids and lipid-like molecules account for 30%-40% of the total number of metabolites, for example, about 35%; the organic oxygen compounds account for 10%-20% of the total number of metabolites, for example, about 15%; the phenylpropanes and polyketides account for 10%-20% of the total number of metabolites, for example, about 14%; the organic heterocyclic compounds account for 7%-17% of the total number of metabolites, for example, about 12%; the organic acids and their derivatives account for 5%-15% of the total number of metabolites, for example, about 10%; and the benzene compounds account for 4%-10% of the total number of metabolites, for example, about 8%.

[0179] The plant exosomes (preferably Dendrobium officinale exosomes, more preferably Dendrobium officinale exosomes) prepared according to the method of the present disclosure have metabolic upregulation compared with the plant exosomes prepared by the ultracentrifugation method ( Figure 5) For example, tryptophan metabolism, arachidonic acid metabolism, phenylpropanoid biosynthesis, starch and sucrose metabolism, alanine, aspartate and glutamate metabolism, galactose metabolism, ABC transporters, valine, leucine and isoleucine biosynthesis, biosynthesis of cutin, suberin and wax, linoleic acid metabolism, β-alanine metabolism, nucleotide metabolism, histidine metabolism, aminoacyl-tRNA biosynthesis, arginine biosynthesis, biosynthesis of various secondary metabolites.

[0180] Specifically, there are 73 pathways related to differential metabolites between the Dendrobium officinale exosomes prepared by the method according to the present disclosure and those prepared by the ultracentrifugation method, and a total of 20 pathways are significantly enriched. There are 82 pathways related to differential metabolites between the Polygonatum sibiricum exosomes prepared by the method according to the present disclosure and those prepared by the ultracentrifugation method, and a total of 27 pathways are significantly enriched.

[0181] In some embodiments, there are a total of 770 significantly differential metabolites between the Dendrobium officinale exosomes prepared by the method according to the present disclosure and those prepared by the ultracentrifugation method, 537 up-regulated metabolites, and 233 down-regulated metabolites.

[0182] In some embodiments, in the analysis of staining plant exosomes with the lipophilic dye PKH67 in vitro, the positive rate of the Dendrobium officinale exosomes prepared by the method according to the present disclosure is 97.4%. In the detection of the protein content of plant exosomes, the protein content of the Dendrobium officinale exosomes prepared by the method according to the present disclosure is 875.89 μg / mL.

[0183] In some embodiments, the Dendrobium officinale exosomes prepared by the method of the present disclosure show good anti-inflammatory effects, and the TNF-α inhibition rate shown in the examples is above about 60%, even close to 100%.

[0184] IV. Compositions

[0185] The present disclosure provides a plant exosome composition, comprising Dendrobium officinale (preferably Dendrobium officinale Kimura et Migo) exosomes, preferably Dendrobium officinale (preferably Dendrobium officinale Kimura et Migo) exosomes prepared by the method for preparing plant exosomes defined generally or preferably or specifically in the present disclosure.

[0186] Specifically, the plant exosome composition provided by the present disclosure is a pharmaceutical composition, which, in addition to containing Dendrobium (preferably Dendrobium officinale) exosomes (preferably Dendrobium exosomes prepared by the method for preparing plant exosomes defined generally or preferably or specifically in the present disclosure), optionally further contains a pharmaceutically acceptable carrier or other active ingredients. The carriers are usually used in pharmaceutical preparations, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.

[0187] Specifically, the plant exosome composition provided by the present disclosure is a food composition or a nutraceutical composition, which contains Dendrobium (preferably Dendrobium officinale) exosomes, preferably Dendrobium (preferably Dendrobium officinale) exosomes prepared by the method for preparing plant exosomes defined generally or preferably or specifically 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 and used together with other foods or food ingredients according to conventional methods. Generally, when preparing foods or beverages, the food composition or nutraceutical composition of the present disclosure at about 15 parts by weight or less, preferably about 10 parts by weight or less based on the total weight of the composition can be added.

[0188] The nutraceutical composition of the present disclosure itself can be in the form of a nutritional supplement or a health product. In addition to the Dendrobium (preferably Dendrobium officinale) exosomes of the present disclosure, it can also contain other nutrients and / or nutraceutically acceptable excipients or carriers, such as various proteins, fats, vitamins, prebiotics, probiotics, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, carbonates used in carbonated beverages, etc.

[0189] The foods to which the above food composition or nutraceutical composition can be added include but are not limited to meats, sausages, breads, chocolates, sugars, fast foods, cookies, pizzas, ramen, other noodles, chewing gums, dairy products including ice creams, various soups, beverages, tea beverages, alcoholic beverages and vitamin complexes, etc. In some embodiments, the food composition of the present disclosure can be prepared into a functional food.

[0190] The plant exosome composition (pharmaceutical composition / nutraceutical composition / food composition) of the present disclosure can be in different forms for oral administration, such as capsules (including gelatin capsules, soft capsules, hard capsules), tablets (including sugar-coated tablets, tablets, pills, powders, granules, chewing gums), solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, gels or gelatin, and any other forms known to those skilled in the art; it can also be administered through topical or transdermal routes, through any other suitable routes (such as the parenteral route).

[0191] Specifically, the exosome composition provided by the present disclosure is a cosmetic / cosmeceutical composition, which contains Dendrobium (preferably Dendrobium officinale) exosomes, preferably Dendrobium (preferably Dendrobium officinale) exosomes prepared by the method for preparing plant exosomes generally or preferably or specifically defined in the present disclosure. The cosmetic / cosmeceutical composition of the present disclosure usually contains at least one excipient or adjuvant acceptable in cosmetics / cosmeceuticals. The "excipient or adjuvant acceptable in cosmetics / cosmeceuticals" can be selected from: solvents, solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, humectants, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, propellants / aerosols, fragrances, pigments, and other efficacy additives. The forms of the cosmetic composition are, for example but not limited to, soaps, facial soaps, facial cleansers, cleansing foams, cleansing milks, cleansing creams, body washes, softening lotions, skin care gels, skin care lotions, skin care creams, serums, eye creams, face masks, aerosols or sprays, lotions, skin softeners, toners, astringents, emulsions, milk emulsions, moisturizing emulsions, nourishing emulsions, massage creams, nourishing creams, moisturizing creams, hand creams, foundations, serums, nourishing serums, powder compacts, body lotions, and skin cleansing lotions, etc. These forms of the cosmetic composition can be prepared by methods well-known to those skilled in the art.

[0192] When the cosmetic composition of the present disclosure is a paste, cream or gel, animal fibers, plant fibers, waxes, paraffin wax, starches, tragacanth, cellulose derivatives, polyethylene glycols, silica gels, bentonites, silica, talc, zinc oxide, etc. can be used as carrier components. When the cosmetic composition of the present disclosure is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. can be used as carrier components. In particular, when the preparation is a spray, a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether can also be included. When the cosmetic composition of the present disclosure is a solution or emulsion, a solvent, solubilizer or emulsifier can be used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol fatty acid esters, polyethylene glycol or sorbitan fatty acid esters can be used. When the cosmetic composition of the present disclosure is a suspension, a liquid diluent such as water, ethanol or propylene glycol, and a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan anhydride esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc. can be used as carrier components.

[0193] V. Uses

[0194] The exosomes or compositions for any use of the present disclosure may include the exosomes or compositions of the present disclosure generally or specifically, preferably the exosomes of Part III or the compositions of Part IV of the present disclosure. Those skilled in the art will understand that the exosomes for any use of the present disclosure can freely combine all the features disclosed herein. For example, the exosomes of Part III of the present disclosure can be exosomes with any combination of features such as different particle densities, different metabolite species and their different metabolite species percentages as defined.

[0195] Many diseases are accompanied by an inflammatory response, including skin-related inflammations (such as dermatitis, acne, pimples, melasma, etc.). Therefore, reducing the inflammation level will be beneficial to disease treatment and care. Accordingly, in some embodiments, the present disclosure provides the use of a plant exosome (preferably Dendrobium, more preferably Dendrobium officinale exosome) or a composition containing the same for treating or preventing, or for helping / assisting in improving, alleviating or controlling inflammation, such as skin inflammation. In other embodiments, the present disclosure provides the use of a plant exosome (preferably Dendrobium, more preferably Dendrobium officinale exosome) or a composition containing the same for or for helping / assisting in the cosmetic, non-therapeutic treatment and / or care of the skin, hair, nails and / or mucous membranes, preferably for or for helping / assisting in the cosmetic, non-therapeutic treatment and / or care of the inflammation of the skin, hair, nails and / or mucous membranes.

[0196] In some embodiments, the plant exosomes of the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or compositions containing the same can be used in the field of skin for repair, anti-inflammation, antioxidant, anti-aging, whitening, moisturizing, firming and anti-wrinkle and other uses. For example, the plant exosomes or compositions containing the same can be used for anti-inflammatory purposes, preferably for anti-inflammatory purposes in the field of skin. For another example, the plant exosomes or compositions containing the same can be used for antioxidant purposes. For another example, the plant exosomes or compositions containing the same can be used for repair purposes in the field of skin. For another example, the plant exosomes or compositions containing the same can be used for anti-aging purposes. For another example, the plant exosomes or compositions containing the same can be used for whitening purposes. For another example, the plant exosomes or compositions containing the same can be used for moisturizing purposes.

[0197] Therefore, the present disclosure also provides the use of plant exosomes (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or compositions containing the same in the preparation of products (drugs, foods, nutritional health products, beauty / cosmetics). In some embodiments, the drug is used to treat or prevent inflammation, such as dermatitis, acne, pimples, melasma, etc. In some embodiments, the nutritional health product helps or aids in antioxidant, repair, anti-aging, whitening, firming and anti-wrinkle and / or moisturizing.

[0198] The present disclosure also provides a method for treating or preventing inflammation, preferably skin inflammation, or treating or preventing dermatological symptoms, or assisting in improving, alleviating or controlling dermatological symptoms (specifically for repair, anti-inflammation, antioxidant, anti-aging, whitening, firming and anti-wrinkle or moisturizing), which comprises administering to a subject in need a plant exosome (preferably Dendrobium, more preferably Dendrobium officinale exosome) or a composition containing the same (drug composition / food composition / beauty or cosmetic composition / nutritional health composition) prepared by the aforementioned general or preferred defined methods.

[0199] In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same have an anti-aging effect on the skin. In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same have an anti-aging effect on skin cells, such as fibroblasts. In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same have the effect of improving or reducing skin aging caused by ultraviolet irradiation or UV-induced cell aging. In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same have the effect of promoting the expression of type I collagen and / or type IV collagen, reducing cyclobutane-pyrimidine dimers (CPDs), or increasing hyaluronic acid (HA) in skin cells (preferably UV-irradiated skin cells). For example, the Dendrobium officinale exosomes prepared by the present disclosure have the effect of anti-aging or promoting the expression of type I collagen, have the effect of promoting type I collagen (COL I) and / or type IV collagen (COL IV), reducing cyclobutane-pyrimidine dimers (CPDs), or increasing hyaluronic acid (HA).

[0200] In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same have a firming and anti-wrinkle effect on the skin, preferably the skin is irradiated with UV. In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same can increase or restore the thickness of the viable epidermal cell layer or increase collagen fibers. In some embodiments, the Dendrobium officinale exosomes or a composition containing the same provided by the present disclosure can improve the thinning of the viable epidermal cell layer and the reduction of collagen fibers in the skin caused by UV irradiation, and / or maintain skin elasticity. In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same can up-regulate the content of laminin (such as LN 5), nestin, integrin α6β4, and / or reticulin. In some embodiments, the Dendrobium officinale exosomes or a composition containing the same provided by the present disclosure can up-regulate the content of LN 5, nestin, integrin α6β4, and / or reticulin to achieve a firming and anti-wrinkle effect. In some embodiments, the Dendrobium officinale exosomes or a composition containing the same provided by the present disclosure have a firming and anti-wrinkle effect on UV-irradiated skin.

[0201] In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same can improve or reduce UVA-induced cell swelling, or reduce the percentage of swollen cells induced by UVA. In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same have the effects of anti-photoaging, restoring skin elasticity, and removing wrinkles. In some embodiments, the plant exosomes provided by the present disclosure (preferably Dendrobium, more preferably Dendrobium officinale exosomes) or a composition containing the same have transdermal properties, or penetrate through the stratum corneum of the epidermis to exert their active effects.

[0202] In some embodiments, the Dendrobium officinale exosomes provided by the present disclosure or a composition containing the same are used in the preparation of a product / cosmetic / composition / beauty product / drug having antioxidant, repair, anti-aging, whitening, firming and anti-wrinkle, and / or moisturizing effects, preferably having an anti-aging effect or a firming and anti-wrinkle effect on the skin. Optionally, the Dendrobium officinale exosomes or a composition containing the same are the sole active ingredient having antioxidant, repair, anti-aging, whitening, firming and anti-wrinkle, and / or moisturizing effects, preferably having an anti-aging effect or a firming and anti-wrinkle effect on the skin.

[0203] For the above methods and uses of the present disclosure, the plant exosomes or a composition containing the same can be administered orally, topically, or parenterally. In particular, topical or transdermal application can be implemented by iontophoresis, sonophoresis, electroporation, mechanical pressure, osmotic gradient, occlusion therapy, microinjection, microneedles (or microneedle arrays), needle-free injection by pressure, through a microelectrical patch, a facial mask, or any combination thereof. Among them, some techniques for creating channels in the skin (such as microneedles or lasers) themselves as a certain treatment means can be used in combination with the therapy of plant exosomes or a composition containing the same, which helps the plant exosomes or a composition containing the same to be absorbed and reach deeper skin layers, thereby enhancing their active ability. The local area of such combination treatment will be determined by the nature of the condition, disorder, and / or disease to be treated and / or processed.

[0204] The daily dosage and administration frequency of the plant exosomes (preferably Dendrobium (more preferably Dendrobium officinale) exosomes) or a composition containing the same can vary according to various factors, such as the stage of the disease to be treated, age, health status, the presence of complications, etc. On the one hand, the composition can be administered 1 - 3 times a day at a daily dosage of, for example, 1 μg / kg to 200 mg / kg, more specifically 50 μg / kg to 50 mg / kg. However, the dosage does not limit the scope of the present disclosure in any way.

[0205] The plant exosomes of the present disclosure also have uses in treating various diseases in mammals (such as humans). In some embodiments, the plant exosomes have uses in treating or preventing diseases related to the following systems: respiratory system, digestive system, circulatory system, nervous system, endocrine system, urogenital system or musculoskeletal system. In some embodiments, the treatment or prevention of diseases related to the respiratory system includes treating COVID-19, suppressing lung inflammation, treating pulmonary fibrosis or preventing pulmonary fibrosis and lung inflammation. In some embodiments, the treatment or prevention of diseases related to the digestive system includes promoting the recovery of colitis, treating colon cancer, treating acute and chronic colitis, treating ulcerative colitis, treating chronic periodontitis, preventing alcohol-induced liver damage, treating IBD and inhibiting the progression of CAC, preventing colitis, preventing dextran sulfate sodium-induced colitis, treating DSS-induced colitis, inhibiting liver metastasis of colon cancer, inhibiting Clostridium difficile infection, treating liver inflammation, preventing GalN / LPS-induced acute liver injury, inhibiting the proliferation of hepatocellular carcinoma cells or inhibiting the growth of tumor cells. In some embodiments, the treatment or prevention of diseases related to the circulatory system includes improving doxorubicin-induced myocardial injury, preventing damage to the vascular system by various stressors, or preventing oxidative stress of human mesenchymal stromal cells. In some embodiments, the treatment or prevention of diseases related to the nervous system includes treating glioma, treating glioblastoma, inhibiting the development of glioma or stimulating the neural differentiation of stem cells. In some embodiments, the treatment or prevention of diseases related to the endocrine system includes preventing insulin resistance and obesity, or preventing or inhibiting obesity-related gastrointestinal inflammation caused by a high-fat and high-sugar diet. In some embodiments, the treatment or prevention of diseases related to the urogenital system includes inhibiting the occurrence and development of breast cancer in mammals, or treating cervical cancer. In some embodiments, the treatment or prevention of diseases related to the musculoskeletal system includes promoting wound healing. In addition, the plant exosomes of the present disclosure can also be used as a drug delivery tool. In some embodiments, the plant exosomes can be used as a drug delivery tool to transport various drug molecules for treating the above diseases, such as MTX for treating colitis, PTX for treating colon cancer, and DOX for treating glioblastoma. In some embodiments, the plant exosomes can be used as a delivery tool for treating plant diseases caused by fungal infections. In some embodiments, different plant exosomes have different properties, such as changes in size, charge and stability. These properties will inevitably affect the drug loading in the plant exosomes. For example, in certain embodiments, the plant exosomes used as a drug delivery tool are plant exosomes with a smaller size, such as 30-80um, 30-60um or 30-50um.Due to the higher surface area to volume ratio, the plant exosomes with smaller sizes have a larger drug-loading capacity, a faster release rate, and higher stability. In some other embodiments, plant exosomes can be used as drug delivery tools to transport positively charged drug molecules. Due to the presence of phosphate, plant exosomes are usually negatively charged, and thus, under the influence of electrostatic attraction, positively charged molecules are more effectively absorbed and encapsulated by these negatively or neutrally charged plant exosomes. Examples

[0206] The technical solutions of the present disclosure will be further described below in conjunction with specific examples, but the protection scope of the present disclosure is not limited to these examples. Any changes or equivalent substitutions that do not deviate from the concept of the present disclosure are included in the protection scope of the present disclosure.

[0207] For the experimental methods and means without specific conditions noted in the following examples, they are generally carried out according to the conventional conditions of such experimental operations in the art. For the experimental equipment without specific sources and models marked in the following examples, they are all conventional equipment in the art well-known to those skilled in the art and can be routinely determined and operated by those skilled in the art. The experimental materials and reagents used in the following examples can be obtained from commercial channels without further purification and can be directly used unless otherwise specified. Unless otherwise stated, percentages and parts are by weight percentage and weight parts respectively. Unless otherwise stated, the ratio of liquids is by volume ratio, and the temperatures used in the present disclosure are in degrees Celsius (°C).

[0208] 1. Experimental methods

[0209] 1.1 Preparation of crude plant extract

[0210] Wash Dendrobium officinale Kimura et Migo clean and dry it with a clean cloth. Weigh the plant to be processed. According to the mass-volume ratio (m / v, g / mL), plant mass: sodium chloride injection volume = 1:3 - 1:20, mix the weighed plant with sodium chloride injection. Use a blender to crush the plant, and collect the crushed plant residue liquid into a measuring cylinder. Place the measuring cylinder on an ice medium and stir for 30 min at a stirring speed of 200 ± 20 rpm. Use a filter screen to pour in the plant residue liquid to separate the plant residue from the juice, and collect the plant juice. Centrifuge the collected plant juice at 2,000 - 5,000 g for 10 - 35 min at 4°C. In a biosafety cabinet, discard the precipitate and collect the supernatant P1 as the crude plant extract.

[0211] 1.2 Separation and extraction of plant exosomes using the method of layer-by-layer filtration

[0212] S1 Depth filtration: In a biosafety cabinet, connect a peristaltic pump, pump tubing, and a depth capsule filter (Supracap) with a pore size of about 2 - 30 μm TMDepth Filter Capsules, Pall, filtration capacity about 50 - 200 L / m 2 ) are combined and connected together. Rinse the depth filter capsule with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid. Filter P1 at a flow rate of 20 - 200 mL / min and collect the filtrate P2. Remove the filter, clean the pipeline with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid and remove the k pump tube.

[0213] S2 0.45μm filtration: In the biosafety cabinet, connect the peristaltic pump, pump tube and 0.45μm membrane filter (PreFlow membrane filter capsules, Pall) to the peristaltic pump. Rinse the 0.45μm membrane filter with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid. Filter P2 at a flow rate of 20 - 200 mL / min and collect the filtrate P3. Remove the filter, clean the pipeline with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid and remove the pump tube.

[0214] S3 0.22μm filtration: In the biosafety cabinet, connect the peristaltic pump, pump tube and 0.22μm membrane filter (Supor TM EXGrade ECV in Mini Kleenpak TM capsules, Pall) to the peristaltic pump. Rinse the 0.22μm membrane filter with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid. Filter P3 at a flow rate of 20 - 200 mL / min to obtain plant exosomes. Remove the filter, clean the pipeline with sodium chloride injection at a flow rate of 20 - 200 mL / min, and finally drain the liquid and remove the pump tube.

[0215] 1.3 Separate and extract plant exosomes using the method of ultracentrifugation

[0216] Centrifuge the collected crude plant extract P1 at 120000g, 4°C for 70 min using an ultracentrifuge. After centrifugation, discard the supernatant and resuspend the precipitate with sodium chloride injection. After centrifuging again at 120000g, 4°C for 70 min, resuspend the precipitate with sodium chloride injection to obtain plant exosomes.

[0217] 1.4 Transmission electron microscopy detection

[0218] Take 5 μL of each of the above-prepared plant exosome samples and drop them on a copper mesh, and incubate at room temperature for 5 min; after incubation, absorb the excess liquid on one side with blotting paper; drop a drop of 2% uranyl acetate on the copper mesh and incubate at room temperature for 1 min; after incubation, absorb the excess liquid on one side with blotting paper; dry at room temperature for about 20 min. Use a nano transmission electron microscope (FEI, Tecnai G2 Spirit BioTwin) to perform transmission electron microscopy (TEM) detection on the machine.

[0219] 1.5 Nanoparticle size tracking analysis

[0220] Use nanoparticle tracking analysis technology (Nanoparticle Tracking Analysis, NTA) to measure the particle size and concentration of each of the above-prepared plant exosomes: Dilute the standard stock solution to a calibration mother liquor 1000 times (1 μL of the standard stock solution can be formulated into 1 mL of the calibration mother liquor according to the ratio). Take 100 μL of the above-prepared standard mother liquor and add it to 25 mL of pure water to dilute it into a calibration solution 250,000 times. When testing the sample, generally dilute the sample 1000 times first. After calibrating the instrument (Particle Metrix, PMX120) with the calibration mother liquor, inject the test sample into the sample cell with a syringe, measure the sample concentration, and repeat three times. 1.6 PKH67 in vitro labeling of plant exosomes

[0221] Dilute 1 mM of PKH67 (green fluorescent tracer dye) 50 times to 20 μM with Diluent C (universal membrane labeling diluent). Subsequently, take 5 μL of 20 μM PKH67 and 5 μL of the above-prepared plant exosomes and mix them, and incubate at room temperature for 15 min. Use a nano flow cytometer (Flow NanoAnalyzer) to detect the positive rate of PKH67 in the plant exosomes.

[0222] 1.7 Trace protein detection

[0223] After filtering 40% SDS prepared with pure water, dilute it to 2% SDS with pure water as the standard curve diluent. According to Micro BCA TMInstructions for Protein Detection Kit (Thermo Fisher Scientific). Prepare the working reagent WR (Working Reagent, WR) by mixing the reagents MA:MB:MC at a ratio of 25:24:1. Prepare standard curves of 200, 40, 20, 10, 5, 2.5, 1, 0.5, 0 μg / mL by diluting the 2.0 mg / mL BSA stock solution with the diluent. After diluting the plant exosomes with the diluent, add an equal volume of WR, and shake at 100 rpm at 37 °C for 2 hours. Detect the absorbance of the standards and samples at 570 nm using a microplate reader, and calculate the protein concentration of the samples.

[0224] 1.8 Detection of TNF-α Inhibition Rate

[0225] RAW264.7 cells (mouse monocyte macrophage leukemia cells) were cultured in DMEM basal medium supplemented with 10% FBS for 48 h, and then seeded in a 96-well plate at a density of 1.875×10 4 / cm 2 . The cells were cultured in an incubator at 37.0 °C and 5% CO2 for 24 h. After pretreatment with plant exosomes (1×10 9 particles / mL) and dexamethasone (final concentration 1 μg / mL) for 24 h, LPS (final concentration 5 ng / mL) was added for co-treatment for 4 h. The cell supernatant was collected and centrifuged at 500 g at 4 °C for 5 min. The concentration of TNF-α was detected using a Mouse TNF-α ELISA Kit (R&D systems), and the inhibition rate was calculated as follows:

[0226] Inhibition rate (%) = 1 - (average concentration of the experimental group / average concentration of the LPS-treated group).

[0227] 1.9 Sample Preparation for Untargeted Metabolomics Analysis of Plant Exosomes

[0228] Absorb 100 μL of the plant exosome liquid sample into a 1.5 mL centrifuge tube, add 400 μL of the extraction solution (acetonitrile:methanol = 1:1, containing 0.02 mg / mL of the internal standard L-2-chlorophenylalanine), vortex for 30 s, then perform low-temperature ultrasonic extraction for 30 min (5 °C, 40 KHz), let the sample stand at -20 °C for 30 min. Centrifuge at 4 °C, 13000 g for 15 min, transfer the supernatant, dry it with nitrogen, re-dissolve it with 100 μL of the reconstitution solution (acetonitrile:water = 1:1), perform low-temperature ultrasonic extraction for 5 min (5 °C, 40 KHz), centrifuge at 4 °C, 13000 g for 10 min, transfer the supernatant to an injection vial with an inner cannula for on-machine analysis. Take equal volumes of the metabolites of all samples and mix them to prepare a quality control sample (QC). During the instrument analysis process, insert one QC sample into every 5 - 15 samples to examine the repeatability of the entire analysis process.

[0229] 1.10 LC-MS / MS Analysis

[0230] Take equal volumes of the metabolites of all samples and mix them to prepare a quality control sample. During the instrument analysis process, insert one QC sample into every 5 - 15 samples to examine the repeatability of the entire analysis process. The sample mass spectrometry signal acquisition adopts the positive and negative ion scanning modes, and the mass scanning range is 70 - 1050 m / z. The sheath gas flow rate is 50 psi, the auxiliary gas flow rate is 13 psi, the auxiliary gas heating temperature is 425 °C, the positive mode ion spray voltage is set to 3500 V, the negative mode ion spray voltage is set to -3500 V, the ion transfer tube temperature is 325 °C, and the normalized collision energy is 20 - 40 - 60 V cyclic collision energy. The primary mass spectrometry resolution is 60000, the secondary mass spectrometry resolution is 7500, and data is acquired in the DDA mode.

[0231] 1.11 Substance Identification and Analysis

[0232] After the on-machine analysis is completed, import the LC-MS raw data into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Finally, obtain a data matrix of retention time, mass-to-charge ratio, and peak intensity. At the same time, match the MS and MSMS mass spectrometry information with the metabolic public databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) and the self-built library of Meiji to obtain metabolite information.

[0233] Upload the data matrix after database search to the Majorbio Cloud Platform (cloud.majorbio.com) for analysis. First, preprocess the data matrix as follows: The data matrix uses the 80% rule to remove missing values, that is, retain variables with more than 80% non-zero values in at least one group of samples, and then fill in the missing values (fill in the missing values with the minimum value in the original matrix). To reduce the errors caused by sample preparation and instrument instability, use the sum normalization method to normalize the response intensity of the sample mass spectrometry peaks to obtain the normalized data matrix. At the same time, delete variables with a relative standard deviation (RSD) of > 30% in the QC samples and perform log10 logarithmic transformation to obtain the data matrix finally used for subsequent analysis.

[0234] Secondly, use the ropls package (Version 1.6.2) in R language to perform principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) on the preprocessed data matrix, and use 7-fold cross-validation to evaluate the stability of the model. The selection of significantly different metabolites is determined based on the variable importance in the projection (VIP) values obtained from the OPLS-DA model and the p-values of the student's t-test. Metabolites with VIP > 1 and p < 0.05 are significantly different metabolites.

[0235] Annotate the metabolic pathways of the differential metabolites through the KEGG database (Kyoto Encyclopedia of Genes and Genomes, https: / / www.kegg.jp / kegg / pathway.html) to obtain the pathways involved in the differential metabolites. Use the Python software package scipy.stats for pathway enrichment analysis and obtain the biological pathways most relevant to the experimental treatment through Fisher's exact test.

[0236] 2. Experimental Results and Analysis

[0237] 2.1 Screening of Extraction Methods

[0238] For Dendrobium officinale, based on Example 1.1 above, perform the following different filtration treatment combinations and evaluate the final results. The specific extraction methods and corresponding results are shown in the following table:

[0239]

[0240]

[0241] Among them, "+" represents the presence of relevant treatments, and "-" represents the absence of relevant treatments.

[0242] 2.2 Extract plant exosomes (PEN) using the methods of layer-by-layer filtration and ultracentrifugation respectively

[0243] Exosomes were isolated and extracted from Dendrobium officinale using the layer-by-layer filtration method in Examples 1.1-1.2. Specifically, Dendrobium officinale (to 1000 g) was first mixed (adding a solvent sodium chloride injection solution for treatment according to the mass volume ratio (g / mL) shown in Table 1), crushed, and stirred to promote the release of plant exosomes into the solvent sodium chloride injection solution. The plant residue was removed by centrifugation at 4000 g for 15 min to obtain a crude plant extract P1. The larger impurities in P1 were removed by S1 (deep filtration) to obtain P2, and the larger particles in P2 were removed by pre-filtration by S2 (0.45 μm membrane filtration) to obtain P3, and sterilized and filtered by S3 (0.22 μm membrane filtration), and finally the sterile grade exosomes were harvested. It takes about 1 hour on average to produce 500 mL of crude plant extract P1.

[0244] The ultracentrifugation method in Example 1.3 was used to separate and extract exosomes from Dendrobium officinale, mainly by centrifuging the crude plant extract P1 twice at 12000g for 70 min at 4°C. It took about 3 h for 500 mL on average.

[0245] Table 1 Names and tags of PEN separated from Dendrobium officinale by ultracentrifugation and layer-by-layer filtration

[0246] Plant Category Ratio Layer-by-Layer Filtration Ultracentrifugation Dendrobium officinale 1:15 SHV01 DeE01

[0247] The following tests were performed on each plant exosome sample obtained by the above operation. The results showed that the exosomes had the characteristics and effects described in the above detailed description of the invention. As a representative example, the test results of 100g of exosomes obtained by the above treatment of Dendrobium officinale (names and labels are shown in Table 1) are given below.

[0248] 2.3 Physical properties of plant exosomes

[0249] After NTA detection according to Example 1.5, the particle concentration, particle size and final volume of the plant exosomes obtained in the above Example 2.2 were counted.

[0250] As shown in Table 2, the particle size of plant exosomes separated and extracted by layer-by-layer filtration and ultracentrifugation methods is within the range of 30-200nm, but the yield of plant exosomes extracted by layer-by-layer filtration is significantly higher than that of exosomes extracted by ultracentrifugation. The results show that the layer-by-layer filtration method is significantly better than ultracentrifugation in terms of both the efficiency of the exosome separation method and the final yield of the harvest.

[0251] Table 2 Particle size and concentration of plant exosomes

[0252]

[0253]

[0254] 2.4 Qualitative metabolomics analysis of plant exosomes

[0255] 2.4.1 Analysis of unique metabolites

[0256] The plant metabolomics analysis performed according to Examples 1.9 to 1.11 showed that the number of metabolites identified in the exosomes of Dendrobium officinale obtained in Example 2.2 was higher in layer-by-layer filtration than in ultracentrifugation, as shown in Table 3. After analysis using the Venn diagram, the number of metabolites unique to layer-by-layer filtration was also higher than that of ultracentrifugation.

[0257] Table 3 Venn analysis statistics of exosomes extracted from Dendrobium officinale using layer-by-layer filtration and ultracentrifugation

[0258]

[0259] 2.4.2 Compound classification analysis

[0260] Compound classification analysis is to classify metabolites into sugars, amino acids, organic acids, lipids, etc. according to their structure and properties. According to the HDMB compound classification hierarchy Superclass, the metabolites in the exosomes of Dendrobium officinale are mainly concentrated in six categories, namely lipids and lipid-like molecules, organic oxygen compounds, phenylpropanes and polyketides, organic heterocyclic compounds, organic acids and their derivatives, and benzene compounds. Figures 2-3 shown.

[0261] The results showed that 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).

[0262] 2.4.3 Lipid classification analysis

[0263] Lipids and lipid-like molecules rank first in the classification of compounds. The lipid classifications mainly include fatty acyl (FA), glycerolipids (GL), glycerophospholipids (GP), prenol lipids (PR), sphingolipids (SP), steroids (ST) and glycolipids (SP).

[0264] The lipids and lipid-like molecules of the exosomes of Dendrobium officinale were classified and counted, as shown in Table 4. The number of plant exosome lipids extracted by layer-by-layer filtration was greater than that by ultracentrifugation. The differences in the contents of FA and PR between SHV01 and DeE01 were the most significant.

[0265] Table 4 Comparison of lipid classification in exosomes of Dendrobium officinale

[0266]

[0267] 2.5 Analysis of differential metabolites between plant exosomes

[0268] 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].

[0269] like Figure 4 As shown, there are 770 significantly different metabolites between SHV01 and DeE01, including 537 up-regulated metabolites and 233 down-regulated metabolites.

[0270] 2.6 Analysis of differential metabolic pathways among plant exosomes

[0271] Usually, multiple functionally interconnected metabolites constitute a metabolic pathway, and the accumulation of expression differences of multiple metabolites in a metabolic pathway constitutes the expression variation of the entire metabolic pathway. If the proportion of a pathway involved in differential metabolism is much greater than the proportion of background metabolites involved in this pathway, it is considered that the experimental treatment is related to the change of this metabolic pathway. In enrichment analysis, based on the KEGG database, the pathways with significant enrichment were screened out according to the enrichment P value less than 0.05.

[0272] There were 73 differential metabolite-related pathways between SHV01 and DeE01, and 20 significantly enriched pathways.

[0273] like Figure 5 As shown in the figure, 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.

[0274] 2.7 TEM identification of plant exosomes obtained by layer-by-layer filtration separation

[0275] The above analysis of the particle concentration, particle size, yield and plant metabolomics of plant exosomes 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 of exosomes). Therefore, we further performed TEM on the plant exosomes extracted by layer-by-layer filtration.

[0276] The results are as follows Figure 6 As shown in Figure 2, the exosomes of Dendrobium officinale have a typical exosome-like saucer shape, and the particle size is in the range of 30-200 nm (see Figure 7 ), which is consistent with the definition of extracellular vesicles by the International Society for Extracellular Vesicles (ISEV).

[0277] 2.8 Detection of PKH67 in plant exosomes isolated by layer-by-layer filtration

[0278] The plant exosomes prepared in the above Example 2.2 were stained in vitro using the lipophilic dye PKH67, and the PKH67 positive particles were detected and analyzed using a nanoflow detector.

[0279] like Figure 8 The results showed that the positive rate of PKH67 in SHV01 was 97.4%.

[0280] 2.9 Detection of protein content of plant exosomes obtained by layer-by-layer filtration separation

[0281] The protein content of plant exosomes was detected, and it was found that the protein content of Dendrobium officinale exosomes was rich. Figure 9 As shown in A, SHV01 is 875.89 μg / mL.

[0282] 2.10 Detection of anti-inflammatory activity of plant exosomes separated by layer-by-layer filtration

[0283] 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 by RAW264.7.

[0284] like Figure 9 As shown in B, the TNF-α inhibition rate of 1 μg / mL dexamethasone in the positive control group was 91.41%. 9 / mL of Dendrobium officinale exosomes, the TNF-α inhibition rate of SHV01 was 64.40%.

[0285] The results showed that the exosomes extracted from Dendrobium officinale using layer-by-layer filtration had satisfactory anti-inflammatory and immunomodulatory activities.

[0286] 2.11 Scale-up process

[0287] According to the layer-by-layer filtration method in Examples 1.1-1.2, 500 g of Dendrobium officinale was processed to separate and extract exosomes to verify whether layer-by-layer filtration can process plant materials with a mass of about 1000 g or even more. After NTA detection according to Example 1.5, the particle concentration, particle size and final volume of the obtained plant exosomes were counted.

[0288] As shown in Table 5, after the 100g plant raw material for obtaining exosomes was enlarged 5-10 times (e.g., 5 times for Dendrobium officinale), about 5000mL of plant crude extract could be processed by increasing the membrane area in the layer-by-layer filtration method. The particle size of the isolated and extracted plant exosomes was within the range of 30-200nm, and the final yield of exosomes was also considerable.

[0289] Table 5 Particle size and concentration of plant exosomes after the layer-by-layer filtration process was scaled up 5-10 times

[0290]

[0291] 2.12 Isolation and identification of plant extracellular vesicles

[0292] Isolation of plant extracellular vesicles

[0293] We used the layer-by-layer filtration method of Examples 1.1-1.2 to separate extracellular vesicles (named: SH, DH, DG, respectively) from Dendrobium officinale, Rehmannia glutinosa, and Angelica sinensis.

[0294] Identification of plant extracellular vesicles

[0295] The extracellular vesicles isolated from the three plants all have a typical teacup shape ( Figure 10 A, D, G), the size of SH detected by NTA was 163.1±7.8nm ( Figure 10 B), the size of DH is 127.9±5.9nm ( Figure 10 E), the size of DG is 125.1±7.2nm ( Figure 10 H). The extracellular vesicles of three plants were labeled with PKH67 and then detected using NTA. The positive rate of SH PKH67 was 97.4% ( Figure 10 C), the positive rate of DH PKH67 was 34.7% ( Figure 10 F), the positive rate of DG PKH67 was 44.0% ( Figure 10 I). The above results show that a kind of extracellular vesicle-like plant vesicle can be extracted from Dendrobium officinale, Rehmannia glutinosa and Angelica sinensis using the layer-by-layer filtration method.

[0296] 2.13 Anti-aging effects of plant extracellular vesicles on fibroblasts

[0297] Ultraviolet irradiation can affect the gene expression of fibroblasts, reduce the synthesis of collagen, and break the balance between the synthesis and degradation of collagen in the extracellular matrix (ECM). Among the many types of collagen, type I collagen in adult skin accounts for 80%, which is mainly responsible for the support function of the skin. However, the synthesis ability of type I collagen gradually weakens with age or external factors such as ultraviolet rays. Therefore, we used Western Blot technology to detect whether SH, DH, and DG can promote the synthesis of type I collagen in mouse embryonic fibroblasts (3T3 cells, from the Chinese Academy of Sciences Cell Bank, catalog number SCSP-515).

[0298] The experimental results showed that compared with the control group, 8 After culturing 3T3 cells with SH at a concentration of 100 particles / mL for 24 hours, the expression of type I collagen was significantly upregulated, and the overall effect was better than that of 5ng / mL TGF-β (positive drug). DH and DG had a certain effect on promoting the expression of type I collagen, but the effect was not significant ( Figure 11 ).

[0299] After repeating the experiment several times and analyzing the grayscale of the protein bands using ImageJ, we calculated the increase rate of type I collagen expression and found that 1×10 8 At a concentration of 100 particles / mL, SH can promote the expression of type I collagen in 3T3 cells by more than 50% on average, which is significantly better than 5ng / mL positive drugs TGF-β (about 27%), DH (29.33%) and DG (19.27%) (Table 6). In summary, the experimental results show that different plant extracellular vesicles filtered through deep filtration have different anti-aging effects, among which the anti-aging effect of Dendrobium officinale is the most obvious and better than the positive control group.

[0300] Table 6 Promotion rate of plant extracellular vesicles on the background collagen level of 3T3 cells

[0301]

[0302] 2.14 Anti-aging effects of active ingredients from Dendrobium officinale obtained by different extraction processes on fibroblasts

[0303] Preparation of effective components of Dendrobium officinale

[0304] We further compared the anti-aging effects of Dendrobium officinale extracellular vesicles (SH) obtained by layer-by-layer filtration (the extraction method is described in Example 1.12), Dendrobium officinale extracellular vesicles (UC) obtained by ultracentrifugation, and Dendrobium officinale polysaccharides obtained by alcohol extraction (EE, 80% Dendrobium officinale polysaccharides, purchased from Xi'an Ruihe Bioengineering Technology Co., Ltd.) on fibroblasts. Among them, UC was prepared by washing, slicing and crushing Dendrobium officinale in a juicer to collect juice; filtering the juice through a 100-mesh sieve to remove impurities, and collecting the filtrate; centrifuging the filtrate at 2000-4500g for 5-20min, discarding the precipitate, and collecting the supernatant; then centrifuging the supernatant at 8000-11000g for 25-40min, discarding the precipitate, and collecting the supernatant; finally, centrifuging the supernatant at 4°C, 100000-150000g for 70-100min, and collecting the precipitate to obtain Dendrobium officinale extracellular vesicles.

[0305] Detection of anti-aging effects on fibroblasts

[0306] Ultraviolet stimulation can induce cell senescence, and one of the notable characteristics of senescent cells is the change in cell morphology. Senescent cells usually swell, which is often used as a signature molecular event of UV-induced cell senescence. Phalloidin is a toxic cyclic heptapeptide obtained from the poisonous mushroom Amanita phalloides. It can selectively bind to actin in animals and plants and is a staining tool for the cytoskeleton. Under an optical microscope, fluorescently labeled phalloidin can clearly show the morphology and distribution of intracellular microfilaments. Therefore, we used fluorescently labeled phalloidin to detect the effect of UVA irradiation on the morphology of human embryonic lung fibroblasts (HSF cells, purchased from the Kunming Cell Bank of the Chinese Academy of Sciences, item number KCB 200537).

[0307] Experimental Results

[0308] Through modeling, we found that: 2 After 24 hours of stimulation of HSF cells with doses of UVA, the cells showed a flattened and enlarged senescent-like morphology (Fig. 12A-B), which is consistent with the literature reports. Based on this morphological standard, we counted the enlarged cells after UVA irradiation under a fluorescence microscope.

[0309] The experimental results show that 5J / cm 2 After stimulating HSF cells with 5J / cm UVA for 24h, the proportion of enlarged cells increased from 3.78% to 12.57%. 2 UVA model group, 1×10 8After treatment with 500 particles / mL SH, UC, and 0.025% (v / v) EE, the proportion of enlarged cells decreased from 12.57% to 4.82%, 8.13%, and 9.89%, respectively (Fig. 12C-D, Table 7).

[0310] These results indicate that both SH and UC can significantly improve UVA-induced cell swelling, among which SH has the best effect and can basically restore the cell morphology to normal levels.

[0311] Table 7 The proportion of enlarged cells in each experimental group

[0312]

[0313] In addition, Western Blot results showed that compared with the control group, the expression of type I collagen in the SH and UC treatment groups was significantly upregulated after culturing 3T3 cells for 24 hours, among which the effect of SH was better than that of 5ng / mL TGF-β (positive drug). No effect of EE on promoting the expression of type I collagen was observed ( Figure 13 ). After repeating the experiment several times and using ImageJ to analyze the grayscale of the protein bands, we calculated the expression increase rate of type I collagen and found that 5 ng / mL of positive drug TGF-β promoted the expression of type I collagen in 3T3 cells by about 53.7%, while 1×10 8 At a concentration of 100 particles / mL, the average promotion rate of SH on the background expression of type I collagen in 3T3 cells can reach nearly 80%, which is significantly better than positive drugs and UC (54.27%) and EE groups (9.22%) (Table 8). In summary, the experimental results show that compared with different extraction processes, the extracellular vesicles of Dendrobium officinale filtered layer by layer have the best anti-aging effect.

[0314] Table 8 Promotion rate of each experimental group on the synthesis of type I collagen by 3T3 fibroblasts

[0315]

[0316] 2.15 Metabolomic analysis of extracellular vesicles from Dendrobium officinale obtained by different isolation methods

[0317] In order to compare the differences in metabolites contained in the extracellular vesicles of Dendrobium officinale separated by layer-by-layer filtration and ultracentrifugation in Example 2.14, we used non-targeted metabolomics for analysis. After data preprocessing, the number of metabolites identified by SH was 1662, and the number of metabolites identified by UC was 1493. Using Venn diagram analysis, it was found that there were 1394 metabolites shared by SH and UC, 268 metabolites unique to SH, and 99 metabolites unique to UC ( Figure 14 ).

[0318] The expression of total SH metabolites was transformed by Log, and the top 100 metabolites were screened according to the average content of two replicate groups of metabolites. The metabolites were analyzed for cosmetic efficacy according to compound classification (https: / / pubmed.ncbi.nlm.nih.gov / ), and the results are shown in Table 9. Among them, there are 17 metabolites related to the skin, which are classified as hydroxy acids and their derivatives, keto acids and their derivatives, fatty acyl groups, organic oxygen compounds, flavonoids, carboxylic acids and their derivatives, phenols, isoprene lipids, benzene and substituted derivatives. The possible effects in cosmetics are mainly moisturizing, anti-oxidation, promoting collagen synthesis, repairing skin barrier, anti-photoaging, anti-aging, etc. Dendrobium officinale contains trace elements and minerals needed by the skin in the process of metabolism. After being absorbed, these substances can promote the metabolism of the skin, and can also promote the excretion of some metabolites such as oxygen free radicals, which further supports the efficacy of SH in anti-photoaging, restoring skin elasticity, and removing wrinkles.

[0319] Table 9 Representative metabolites of SH (sorted by content)

[0320]

[0321] 2.16SH transdermal properties

[0322] To verify whether SH can penetrate through the stratum corneum and epidermis to exert its active effect, we used a Franz diffusion cell based on suckling pig skin to conduct a transdermal experiment, detected the protein content in the receiving chamber at different time points, and analyzed the cumulative permeation amount and diffusion percentage ( Figure 15 ) calculation.

[0323] The experimental results show that the SH permeation rate is 0.0039 mg / h ( Figure 15 A), the diffusion rate reached 82.55% ( Figure 15 B), that is, penetration occurs.

[0324] 2.17SH's firming and anti-wrinkle effects on 3D full-thickness skin models

[0325] We use a 3D full-thickness skin model ( Guangdong Boxi Biotechnology Co., Ltd.), through UVA (irradiation dose 35J / cm 2 ) to simulate the aging damage of skin caused by UV radiation, using 1.1×10 9 particles / mL SH concentration, 0.9 mL sample volume, total dose 9.9×10 9 Particles were administered subliminally to the skin model, and the changes in tissue structure and collagen fibers under different treatment conditions were observed by section staining.

[0326] The results are as follows Figure 16 As shown in Figure 2, we observed that the epidermis of the model irradiated by UV was significantly thinned by H&E and Masson staining ( Figure 16 A), and the collagen fiber content decreased significantly ( Figure 16 B) By counting the thickness of the epidermal living cell layer, it was found that after SH treatment, the thickness of the epidermal living cell layer increased significantly compared with the UV group, with an increase rate of 37.63% ( Figure 16 C); The collagen fiber content in the SH treatment group increased by 241.07% compared with the UV group ( Figure 16 D).

[0327] Skin elasticity is the ability of the skin to stretch and return to its original shape. DualMPA580) is based on the principle of suction and stretching to determine the elastic properties of the skin. The higher the elastic coefficient R2 (%) value, the better the skin elasticity, and vice versa. Based on the experimental results of tissue structure and collagen fibers, we also tested the skin elasticity of 3D skin.

[0328] The experimental results showed that the R2 value of the skin model treated with SH increased significantly by 41.49% ( Figure 16 E).

[0329] In summary, SH can significantly improve the thinning of the epidermal living cell layer and the reduction of collagen fibers in the 3D full-thickness skin model caused by UV irradiation ( Figure 16 AD), and also plays a significant role in maintaining skin elasticity ( Figure 16 E).

[0330] 2.18SH regulatory effect on collagen expression

[0331] To further study the regulatory effect of SH on collagen expression, we sliced ​​the 3D full-thickness skin model in Example 2.17 and analyzed the expression of type I collagen (COL I, Figure 17 A) and type IV collagen (COL IV, Figure 17 B) Specific immunofluorescence detection was performed, and the protein content was calculated by integrated optical density (IOD). Figure 17 CD). COLⅠ accounts for about 80% of the collagen in the dermis of the skin, and forms collagen fibers with COL III. Promoting COLⅠ can achieve a certain effect of resisting wrinkles and aging, making the skin plump and full; COL IV is the key protein at the junction of the true epidermis and is an important element supporting the skin.

[0332] The results showed that the UV-irradiated model clearly showed the expression of COL I ( Figure 17A) Reduction in COLⅣ at the junction of the epidermis and dermis Figure 17 B), while SH treatment significantly increased the expression of COL I Figure 17 C) and COL IV Figure 17 D), with the promotion rates being 133.90% and 118.00% respectively.

[0333] 2.19 Effects of SH on UV-induced cellular senescence and hyaluronic acid (HA) expression

[0334] Under the influence of internal and external factors, the body will experience DNA damage. For example, exposure to exogenous UV radiation can cause DNA damage, which in turn induces aging of the body. When DNA is irradiated with UV, cyclobutane-pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) are mainly produced. By specifically detecting CPDs through immunohistochemistry, we found that the expression level of CPDs in the UV-irradiated model increased significantly, and SH could significantly reduce the positive cell rate of CPDs Figure 18 A,C).

[0335] HA has the highest content in the skin, providing stability and elasticity to the extracellular matrix by moisturizing and maintaining the extracellular space and interacting with other substances, thus resisting wrinkles, enhancing skin elasticity, and making the skin plump. UV radiation can significantly reduce the HA content in the skin, while SH can significantly increase the HA content Figure 18 B,D).

[0336] 2.20 Exploration of the mechanism of SH in firming and anti-wrinkle effects

[0337] We further explored the mechanism of SH in firming and anti-wrinkle effects. Regarding basement membrane-related proteins, laminin (LN) is an important component protein of the extracellular matrix (ECM) widely distributed in the body and is an important component of the basement membrane. As a multifunctional adhesion molecule, LN participates in the synthesis process of ECM and is an important substance regulating cell proliferation, growth, differentiation, apoptosis, migration, and invasion. Nidogen exists in all basement membranes and is an important structural protein of the basement membrane. It binds to LN in a 1:1 ratio to form a stable complex, promoting the non-covalent molecular connection between LN and COL IV. By detecting LN 5 Figure 19 A,C) and nidogen Figure 19 B,D) in the 3D skin model after UV radiation, we found that SH could significantly upregulate the content of LN 5 and nidogen, thus stabilizing the basement membrane structure and achieving the effect of firming and anti-wrinkle.

[0338] On the other hand, regarding connexins, integrin α6β4 forms the central core of hemidesmosomes, which connect the ECM and the keratinocyte cytoskeleton network within keratinocytes and is an important connexin for the dermo-epidermal junction. Plectin also plays a crucial role in maintaining the integrity of cell structure. It is a component of the desmosome complex that links cytoskeletal proteins and transmembrane molecules. In epithelial cells, plectin connects keratin in hemidesmosomes and integrin α6β4 to the extracellular matrix. We detected integrin α6β4 ( Figure 20 A, C) and plectin ( Figure 20 B, D) in the 3D skin model after UV radiation and found that SH could significantly upregulate the contents of integrin α6β4 and plectin, thereby strengthening the connection between the dermis and epidermis and the binding tightness between cells and the ECM, achieving the effect of firming and anti-wrinkle.

[0339] References

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[0341] [2] Mu J, Zhuang X, Wang Q, Jiang H, Deng ZB, Wang B, Zhang L, Kakar S, Jun Y, Miller D, Zhang HG. Interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles. Mol Nutr Food Res. 2014 Jul;58(7):1561 - 73.

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[0346] [7] CN108384743A.

Claims

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

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

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

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

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

6. A method according to any one of claims 1 to 5, wherein the plant material is selected from Dendrobium, preferably Dendrobium officinale.

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

8. Plant exosomes prepared by the method according to any one of claims 1 to 7 or derived from Dendrobium officinale, preferably Dendrobium officinale.

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

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

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

12. Use of the plant exosomes of claim 8 or 9 or the plant exosome composition of claim 10 or 11 in the preparation of medicines, foods, nutritional supplements, or beauty or cosmetics, wherein the product is used to treat or prevent inflammation, preferably skin inflammation, or helps or assists in improving, relieving or controlling inflammation, preferably skin inflammation, or is used or assists in repair, anti-inflammatory, anti-oxidation, anti-aging, whitening, firming and anti-wrinkle or moisturizing in the field of dermatology.

13. The use of claim 12, wherein the plant exosomes are used for the anti-aging effect of the skin, such as the anti-aging effect of fibroblasts, for example, for improving or reducing the effect of skin aging caused by ultraviolet irradiation or UV-induced cell senescence, or for promoting the expression of type I collagen and / or type IV collagen in skin cells (preferably UV-irradiated skin cells), reducing cyclobutane-pyrimidone dimers (CPD), or increasing hyaluronic acid (HA).

14. The use of claim 12, wherein the plant exosomes are used for tightening and anti-wrinkle effects on the skin, preferably the skin is irradiated with UV, for example, for increasing or restoring the thickness of the epidermal living cell layer or increasing collagen fibers, improving the thinning of the epidermal living cell layer in the skin caused by UV irradiation, the reduction of collagen fibers, and / or maintaining skin elasticity, and for example, for upregulating the content of laminin (such as LN 5), nestin, integrin α6β4 and / or netrin.

15. Use of the plant exosomes of claim 8 or 9 or the plant exosome composition of claim 10 or 11 for treating or preventing inflammation, preferably skin inflammation, or for helping or assisting in improving, alleviating or controlling inflammation, preferably skin inflammation, or for or assisting in repair, anti-inflammatory, anti-oxidation, anti-aging, whitening, firming and anti-wrinkle or moisturizing in the field of dermatology.

16. The use according to claim 15, wherein the plant exosomes are used for the anti-aging effect of the skin, such as the anti-aging effect of fibroblasts, for example, for improving or reducing the effect of skin aging caused by ultraviolet irradiation or UV-induced cell senescence, or for promoting the expression of type I collagen and / or type IV collagen in skin cells (preferably UV-irradiated skin cells), reducing cyclobutane-pyrimidone dimers (CPD), or increasing hyaluronic acid (HA).

17. The use of claim 15, wherein the plant exosomes are used for tightening and anti-wrinkle effects on the skin, preferably the skin is irradiated with UV, for example, for increasing or restoring the thickness of the epidermal living cell layer or increasing collagen fibers, improving the thinning of the epidermal living cell layer in the skin caused by UV irradiation, the reduction of collagen fibers, and / or maintaining skin elasticity, and for example, for upregulating the content of laminin (such as LN 5), nestin, integrin α6β4 and / or netrin.

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

19. A method for dermatological repair, anti-inflammation, anti-oxidation, anti-aging, whitening, firming and anti-wrinkle or moisturizing, comprising applying the beauty or cosmetic composition and / or nutritional health care composition of claim 11 to a subject in need thereof.

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

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