Method for extracting plant exosome through combination of density gradient centrifugation and exclusion chromatography

Abstract

The invention relates to the technical field of plant exosome extraction, in particular to a method for extracting a plant exosome by combining density gradient centrifugation with exclusion chromatography. According to the method, the plant exosome is extracted through the combination of density gradient centrifugation and exclusion chromatography, compared with an existing plant exosome extraction method, the cacumen biotae exosome obtained through the method is uniform in particle size and high in vesicle number and protein concentration content, and the method can achieve mass production and is suitable for industrialization; and the method has remarkable practicability and economic benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of plant exosome extraction, and particularly to a method for extracting plant exosomes by combining density gradient centrifugation and size exclusion chromatography. Background Art

[0002] Plant-derived Exosomes are nano-sized vesicles extracted from plant cells, with unique biological functions and broad application potential, including low immunogenicity: Plant-derived exosomes have a relatively high compatibility with human cells and are not easily prone to immune rejection reactions, and safety: Exosomes derived from plants generally have no toxic or side effects and are suitable for use in the fields of medicine and cosmetics. Moreover, plant-derived exosomes contain abundant proteins, lipids, RNA (such as miRNA), and metabolites, and have various biological activities. Plant-derived exosomes can be used as natural carriers to efficiently deliver drugs, RNA, or functional molecules. Plant-derived exosomes can interact with human cells to regulate cell functions, and moreover, by transmitting miRNA or other functional molecules, they can regulate the gene expression and physiological activities of recipient cells. Compared with traditional solvent extraction of plants, the extraction and production process of plant-derived exosomes is environmentally friendly and conforms to the concepts of green chemistry and sustainable development.

[0003] The nano-sized dimension (30 - 150 nm) and natural lipid membrane structure of Platycladus orientalis exosomes make them more easily taken up by cells, improving bioavailability. They can pass through biological barriers (such as the skin barrier, intestinal barrier) to achieve efficient delivery. The macromolecular components in Platycladus orientalis extracts may be difficult to penetrate cell membranes or biological barriers, resulting in low bioavailability. Platycladus orientalis exosomes have natural targeting ability and can interact with specific cells through surface proteins to achieve precise delivery. The targeting ability can be further enhanced through engineering modification (such as surface modification). Platycladus orientalis extracts lack targeting ability, and the components are widely distributed, which may affect the therapeutic effect or increase side effects. Platycladus orientalis exosomes contain abundant functional molecules (such as miRNA, proteins, lipids), and can regulate intercellular communication and gene expression. They have multiple biological activities (such as antioxidant, anti-inflammatory, immunomodulatory). Platycladus orientalis extracts mainly rely on small molecule compounds (such as flavonoids, phenols) to exert their effects, and the functions are relatively single. In terms of safety, Platycladus orientalis exosomes are natural products with high biocompatibility and are not easily prone to immune reactions or toxicity. They are suitable for long-term use. Platycladus orientalis extracts may contain impurities or high-concentration active ingredients, posing a potential risk of toxic or side effects. Generally speaking, Platycladus orientalis exosomes have significant advantages in terms of stability, bioavailability, targeting ability, and functional diversity, and are suitable for the development of modern medicine and functional products, while Platycladus orientalis extracts are more suitable for traditional applications. With the progress of technology, the application prospects of Platycladus orientalis exosomes will be even broader.

[0004] The density gradient centrifugation method is an efficient method for extracting plant exosomes and can obtain exosome vesicles with high purity. However, the current density gradient centrifugation method mostly uses sucrose gradient centrifugation. High-concentration sucrose will affect the function of exosomes, and the existing density gradient centrifugation method has the disadvantages of low vesicle number, low protein concentration, and low exosome purity. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for extracting plant exosomes by combining density gradient centrifugation and size exclusion chromatography. The plant exosomes extracted by the method provided by the present invention have the advantages of a large number of vesicles, high purity, and high protein concentration, and have a strong targeting efficacy on cells.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for extracting plant exosomes by combining density gradient centrifugation and size exclusion chromatography, comprising the following steps:

[0008] Crush the plant tissue to be extracted and mix it with a buffer solution to obtain a mixture;

[0009] After filtering the mixture, collect the filtrate and centrifuge it to obtain a supernatant;

[0010] Using iodixanol as the gradient medium, perform density gradient centrifugation on the supernatant, and collect the iodixanol density layer of 20-30 g / 100 mL to obtain a centrifuged solution containing plant exosomes;

[0011] Ultracentrifuge the centrifuged solution to obtain a precipitate containing plant exosomes;

[0012] Resuspend the precipitate containing plant exosomes and elute it using a size exclusion chromatography column, and collect the eluate containing exosomes; the size exclusion chromatography column is a size exclusion chromatography column for separating particles of 30-500 nm; the elution flow rate is 0.5-1 mL / min.

[0013] Preferably, the plant includes Platycladus orientalis; the plant tissue to be extracted includes Platycladus orientalis leaves.

[0014] Preferably, the filtering method includes: first filtering the mixture using a 100-500 mesh filter screen to obtain a first filtrate; filtering the first filtrate using a 0.22-0.44 μm filter membrane to obtain the filtrate.

[0015] Preferably, the centrifugation speed is 2,000-10,000×g; the time is 10-30 min, and the temperature is 4°C.

[0016] Preferably, the gradient medium comprises iodixanol solutions of 30 g / 100 mL, 20 g / 100 mL, and 10 g / 100 mL.

[0017] Preferably, the gradient medium comprises iodixanol solutions of 40 g / 100 mL, 30 g / 100 mL, 20 g / 100 mL, 10 g / 100 mL, and 5 g / 100 mL.

[0018] Preferably, the centrifugation speed of the density gradient centrifugation is 50,000 - 100,000×g, and the time is 2 - 4 h.

[0019] Preferably, the speed of the ultracentrifugation is 50,000 - 100,000×g, and the time is 1 - 2 h.

[0020] Preferably, the buffer solution comprises phosphate buffer solution; the eluent used for elution comprises phosphate buffer solution; the size - exclusion chromatography column comprises Sepharose CL - 4B.

[0021] The present invention provides the application of the plant exosomes prepared by the method of the above - mentioned technical solution in the preparation of skin care products.

[0022] Beneficial effects:

[0023] 1) The method provided by the present invention combines density gradient centrifugation and size - exclusion chromatography, which is an efficient and high - purity method for extracting plant exosomes. The method provided by the present invention can significantly improve the extraction efficiency and purity of exosomes. The density gradient centrifugation method separates exosomes from other cell debris, proteins and other impurities through gradient media (iodixanol) with different densities, which can effectively remove most non - exosome components and improve the sample purity. The size - exclusion chromatography method separates samples according to molecular size using a porous gel column to further remove residual impurities (such as protein aggregates, lipoproteins, etc.). The combination of the two methods can obtain exosomes with extremely high purity.

[0024] 2) High recovery rate. The density gradient centrifugation method can maximize the recovery of exosomes by optimizing the gradient medium and centrifugation conditions. The high - efficiency separation ability of the chromatography column in the size - exclusion chromatography method can further recover exosomes and reduce sample loss. The combination of the two methods is complementary to ensure a high recovery rate of exosomes.

[0025] 3) The mild centrifugation conditions in the density gradient centrifugation method can avoid the destruction of the exosome structure. The non - denaturing separation conditions in the size - exclusion chromatography method can maintain the natural form and function of exosomes. The combination of the two methods causes no damage to the exosome structure, ensuring that the extracted exosomes have complete biological functions.

[0026] 4) Wide range of applicability. Applicable to the extraction of exosomes from a variety of plant sources (such as fruits, vegetables, herbs, etc.). The sample volume is flexible and can be adjusted according to requirements, suitable for small-scale and large-scale extraction.

[0027] 5) Good reproducibility. The operating conditions of density gradient centrifugation and size exclusion chromatography are easy to standardize, and the experimental results have high reproducibility. Strong stability: The combined method is less affected by sample differences and is suitable for comparative studies between different laboratories.

[0028] 6) Can efficiently remove impurities. Density gradient centrifugation removes large particle impurities (cell debris, cell organelles), and size exclusion chromatography removes small molecule impurities such as proteins and lipids. After combined use, it can comprehensively remove impurities of different sizes to obtain high-purity plant exosomes.

[0029] 7) Technical complementarity. Density gradient centrifugation is suitable for the preliminary separation and concentration of exosomes, and size exclusion chromatography is used for fine purification and impurity removal. The two methods complement each other's advantages and achieve full-process coverage from crude extraction to fine purification. The extraction of plant exosomes by combining density gradient centrifugation and size exclusion chromatography has multiple advantages such as high purity, high recovery rate, and maintaining the integrity of exosomes, and is an efficient and reliable extraction method, especially suitable for the functional research and application development of exosomes.

[0030] 8) The Platycladus orientalis exosomes extracted by using the present invention are protected by a lipid membrane and have higher stability, while the active ingredients in the Platycladus orientalis extracts obtained by existing methods are easily oxidized and degraded.

[0031] 9) The Platycladus orientalis exosomes extracted by using the present invention have high bioavailability, are nanoscale in size, and are easily taken up by cells, while the macromolecules in the Platycladus orientalis extracts obtained by existing methods are difficult to penetrate the barrier.

[0032] 10) The Platycladus orientalis exosomes extracted by using the present invention have higher targeting, natural targeting, while the Platycladus orientalis extracts obtained by existing methods do not.

[0033] 11) The Platycladus orientalis exosomes extracted by using the present invention contain multifunctional efficacy molecules, while the Platycladus orientalis extracts obtained by existing methods mainly rely on small molecule compounds.

[0034] 12) The Platycladus orientalis exosomes extracted by using the present invention have good biocompatibility, while the Platycladus orientalis extracts obtained by existing methods may have toxic and side effects.

[0035] 13) Widely applied, such as in medicine, cosmetics, food, etc., while the extracts are limited to traditional Chinese medicine, health products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0037] Figure 1 Physical map of the exosomes extracted in Example 1;

[0038] Figure 2 NTA particle size distribution and vesicle number of the exosomes extracted in Example 1; among them, the average particle size of D50 is 71 nm, D10: 57 nm; D90: 90 nm, the maximum particle size is 175 nm, the minimum particle size is 56 nm, and the median value is 64 nm;

[0039] Figure 3 TEM microscopic structure image of the exosomes extracted in Example 1; the scale bar is 200 nm;

[0040] Figures 4 to 6 Sequential control microscope images of 30% Platycladus orientalis exosome solution, negative control, and positive control in the chicken embryo irritation experiment in Example 1; Figures 4 to 6 In, the left figure is the toxicity effect result at 0 s of the reaction, and the right figure is the toxicity effect result at 300 s of the reaction;

[0041] Figure 7 Oil red staining map of inhibiting sebum secretion of sebaceous gland cells by the exosomes extracted in Example 1;

[0042] Figure 8 Scratch test map of human primary dermal papilla cells of the exosomes extracted in Example 1. Detailed implementation mode

[0043] The present invention provides a method for extracting plant exosomes by combining density gradient centrifugation and size exclusion chromatography, comprising the following steps:

[0044] Crush the plant tissue to be extracted and mix it with a buffer solution to obtain a mixture;

[0045] After filtering the mixture, collect the filtrate and centrifuge it to obtain a supernatant;

[0046] Using iodixanol as a gradient medium, perform density gradient centrifugation on the supernatant, and collect the iodixanol density layer of 20 - 30 g / 100 mL to obtain a centrifuged solution containing plant exosomes;

[0047] Ultracentrifuge the centrifuged solution to obtain a precipitate containing plant exosomes;

[0048] Resuspend the precipitate containing plant exosomes and elute it using a size exclusion chromatography column, and collect the eluate containing exosomes; the size exclusion chromatography column is a size exclusion chromatography column for separating particles of 30 - 500 nm; the elution flow rate is 0.5 - 1 mL / min.

[0049] The plant tissue to be extracted of the present invention is pulverized and then mixed with a buffer solution to obtain a mixture. As an implementation manner, the plant includes Platycladus orientalis; the plant tissue to be extracted includes Platycladus orientalis leaves. As an implementation manner, the pulverizing method includes grinding with liquid nitrogen. As an implementation manner, the buffer solution includes phosphate buffer solution. As another implementation manner, the buffer solution is a phosphate buffer solution pre-cooled with liquid nitrogen.

[0050] After obtaining the mixture, the mixture is filtered and the filtrate is collected and centrifuged to obtain a supernatant. As an implementation manner, the filtering method includes: performing a first filtration on the mixture using a 100-500 mesh filter screen to obtain a first filtrate; filtering the first filtrate using a 0.22-0.44 μm filter membrane to obtain the filtrate. As an implementation manner, the mesh number of the filter screen is 300-500 mesh. The present invention can remove large particles through the filter screen, and further filter with a filter membrane to remove cell debris and impurities.

[0051] As an implementation manner, the centrifugation speed is 2,000 - 10,000×g; the time is 10 - 30 min, and the temperature is 4°C. As another implementation manner, the centrifugation speed is 5,000 - 8,000×g; the time is 15 - 20 min. The present invention can remove residual cell debris and large particles through appropriate centrifugation conditions.

[0052] After obtaining the supernatant, using iodixanol as a gradient medium, the supernatant is subjected to density gradient centrifugation, and the iodixanol density layer of 20 - 30 g / 100 mL is collected to obtain a centrifugate containing plant exosomes.

[0053] As an implementation manner, the gradient medium includes iodixanol solutions of 30 g / 100 mL, 20 g / 100 mL, and 10 g / 100 mL. As another implementation manner, the gradient medium includes iodixanol solutions of 40 g / 100 mL, 30 g / 100 mL, 20 g / 100 mL, 10 g / 100 mL, and 5 g / 100 mL. As another implementation manner, the gradient medium includes iodixanol solutions of 60 g / 100 mL, 50 g / 100 mL, 40 g / 100 mL, 30 g / 100 mL, 20 g / 100 mL, 10 g / 100 mL, and 5 g / 100 mL.

[0054] As an implementation manner, the centrifugation speed of the density gradient centrifugation is 50,000 - 100,000×g, and the time is 2 - 4 h. As another implementation manner, the centrifugation speed of the density gradient centrifugation is 80,000 - 100,000×g, and the time is 3 - 4 h.

[0055] After obtaining the centrifugate, the present invention performs ultracentrifugation on the centrifugate to obtain a precipitate containing plant exosomes. As an embodiment, the speed of the ultracentrifugation is 50,000 - 100,000×g, and the time is 1 - 2 h. As another embodiment, the speed of the ultracentrifugation is 80,000 - 100,000×g, and the time is 1.5 - 2 h. The present invention can remove the gradient medium by ultracentrifugation.

[0056] After obtaining the precipitate containing plant exosomes, the present invention resuspends the precipitate containing plant exosomes and elutes it using a size exclusion chromatography column, and collects the eluate containing exosomes; the size exclusion chromatography column is a size exclusion chromatography column for separating particles of 30 - 500 nm; the flow rate of the elution is 0.5 - 1 mL / min. As an embodiment, the flow rate of the elution is 0.8 - 1 mL / min. As an embodiment, the eluent used for the elution includes phosphate buffer solution. As an embodiment, the size exclusion chromatography column includes Sepharose CL-4B. As an embodiment, an ultraviolet detector (280 nm) can be used to monitor the elution peak, or a fraction collector can be used to collect the eluate (1 - 2 mL per tube), and exosomes appear in the earlier elution peak (macromolecules are eluted first), and the eluate collected in the 4th - 8th tubes is the eluate containing exosomes.

[0057] The Platycladus orientalis exosomes extracted by the method according to the above technical solution of the present invention not only have high safety and stability, high exosome purity, high vesicle number and protein concentration, but also have strong targeting efficacy on cells.

[0058] Based on the above advantages, the present invention also provides the application of the plant exosomes prepared by the method according to the above technical solution in the preparation of skin care products.

[0059] In order to further illustrate the present invention, the following describes in detail a method for extracting plant exosomes by density gradient centrifugation combined with size exclusion chromatography provided by the present invention with reference to the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0060] In the following examples or comparative examples, the % of the gradient medium is g / 100 mL. The chromatographic buffer is PBS buffer, and the formula is as follows: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4.

[0061] Example 1

[0062] The Platycladus orientalis dry leaves were ground into powder with liquid nitrogen and added to pre-cooled phosphate buffer solution (PBS) for homogenization. The homogenate was filtered through multiple layers of gauze or a filter screen (300 mesh) to remove large particles. It was further filtered through a 0.45 μm filter membrane to remove cell debris and impurities. The filtrate was centrifuged at 50,000 g for 20 min at 4 °C to remove residual cell debris and large particles. Iodixanol (OptiPrep) was selected as the gradient medium, and 40%, 30%, 20%, 10% and 5% gradient medium solutions were added layer by layer from high density to low density in an ultracentrifuge tube. After each layer was added, it was gently placed to avoid mixing. The pretreated sample was carefully added to the top layer of the gradient medium. Using an ultracentrifuge, it was centrifuged at 100,000 g for 3 h at 4 °C. After centrifugation, exosomes would be distributed in a certain layer of the gradient medium according to the density (in the 20%-30% OptiPrep layer), and the target density layer (the layer where exosomes were located) was carefully aspirated using a pipette. The collected exosomes were diluted with PBS and centrifuged again at 100,000 g for 1.5 h at 4 °C to remove the gradient medium. The supernatant was discarded, and the exosome precipitate was resuspended with a small amount of PBS or buffer solution. A suitable size exclusion chromatography column Sepharose CL-4B (suitable for 30-500 nm particles) was selected. The chromatography column was equilibrated with the chromatography buffer until the baseline was stable. The sample was eluted with the chromatography buffer at a constant flow rate of 0.8 mL / min. An ultraviolet detector (280 nm) was used to monitor the elution peak, or a fraction collector was used to collect the eluate (1-2 mL per tube). Exosomes appeared in the earlier elution peak (macromolecules were eluted first, and the eluate in the 4th to 8th tubes was collected). The eluate containing exosomes was collected, and the extracted exosomes were aliquoted ( Figure 1 ), stored at -80 °C for later use, which was convenient for subsequent tests.

[0063] Example 2

[0064] The dry leaves of Platycladus orientalis were ground into powder with liquid nitrogen and added to pre-cooled PBS buffer for homogenization. The homogenate was filtered through multiple layers of gauze or a filter screen (100 mesh) to remove large particles. Further filtration was performed using a 0.22 μm filter membrane to remove cell debris and impurities. The filtrate was centrifuged at 2,000 g for 15 min at 4 °C to remove residual cell debris and large particles. Iodixanol (OptiPrep) was selected as the gradient medium, and 40%, 30%, 20%, 10%, and 5% gradient medium solutions were added layer by layer from high density to low density in an ultracentrifuge tube. After each layer was added, it was gently placed to avoid mixing. The pretreated sample was carefully added to the top layer of the gradient medium. Using an ultracentrifuge, centrifugation was performed at 100,000 g for 3 h at 4 °C. After centrifugation, exosomes would be distributed in a certain layer of the gradient medium according to density (located in the 20%-30% OptiPrep layer), and the target density layer (the layer where exosomes were located) was carefully aspirated using a pipette. The collected exosomes were diluted with PBS and centrifuged again at 100,000 g for 1.5 h at 4 °C to remove the gradient medium. The supernatant was discarded, and the exosome pellet was resuspended with a small amount of PBS or buffer. A suitable size exclusion chromatography column Sepharose CL-4B (applicable to particles of 30-500 nm) was selected. The chromatography column was equilibrated with the chromatography buffer until the baseline was stable. The sample was eluted with the chromatography buffer at a constant flow rate of 0.5 mL / min. An ultraviolet detector (280 nm) was used to monitor the elution peak, or a fraction collector was used to collect the eluate. Exosomes appeared in the earlier elution peak (macromolecules were eluted first). The eluate containing exosomes was collected, the extracted exosomes were aliquoted, and stored at -80 °C for later use to facilitate subsequent tests.

[0065] Example 3

[0066] The Platycladus orientalis dry leaves were ground into powder with liquid nitrogen and added to pre-cooled PBS buffer for homogenization. The homogenate was filtered through multiple layers of gauze or a filter screen (300 mesh) to remove large particles. It was further filtered with a 0.45 μm filter membrane to remove cell debris and impurities. The filtrate was centrifuged at 80,000 g for 15 min at 4 °C to remove residual cell debris and large particles. Iodixanol (OptiPrep) was selected as the gradient medium, and 30%, 25%, 20%, 15%, and 10% gradient medium solutions were added layer by layer from high density to low density in an ultracentrifuge tube. After each layer was added, it was gently placed to avoid mixing. The pretreated sample was carefully added on the top layer of the gradient medium. Using an ultracentrifuge, it was centrifuged at 100,000 g for 3 h at 4 °C. After centrifugation, exosomes would be distributed in a certain layer of the gradient medium according to density (in the 20%-30% OptiPrep layer), and the target density layer (the layer where exosomes were located) was carefully aspirated using a pipette. The collected exosomes were diluted with PBS and centrifuged again at 100,000 g for 1.5 h at 4 °C to remove the gradient medium. The supernatant was discarded, and the exosome precipitate was resuspended with a small amount of PBS or buffer. A suitable size exclusion chromatography column Sepharose CL-4B (applicable to particles of 30-500 nm) was selected. The chromatography column was equilibrated with the chromatography buffer until the baseline was stable. The sample was eluted with the chromatography buffer at a constant flow rate of 0.8 mL / min. An ultraviolet detector (280 nm) was used to monitor the elution peak, or a fraction collector was used to collect the eluate. Exosomes appeared in the earlier elution peak (macromolecules were eluted first). The eluate containing exosomes was collected, the extracted exosomes were aliquoted, and stored at -80 °C for later use to facilitate subsequent tests.

[0067] Example 4

[0068] The Platycladus orientalis dry leaves were ground into powder with liquid nitrogen and added to pre-cooled PBS buffer for homogenization. The homogenate was filtered through multiple layers of gauze or a filter screen (300 mesh) to remove large particles. Further filtration was performed using a 0.22 μm filter membrane to remove cell debris and impurities. The filtrate was centrifuged at 10,000 g for 15 min at 4 °C to remove residual cell debris and large particles. Iodixanol was selected as the gradient medium, and 60%, 50%, 40%, 30%, 20%, 10%, and 5% gradient medium solutions were added layer by layer from high density to low density in an ultracentrifuge tube. After each layer was added, it was gently placed to avoid mixing. The pretreated sample was carefully added to the top layer of the gradient medium. An ultracentrifuge was used to centrifuge at 100,000 g for 3 h at 4 °C. After centrifugation, the exosomes would be distributed in a certain layer of the gradient medium according to their density (located in the 20%-30% OptiPrep layer), and the target density layer (the layer where the exosomes were located) was carefully aspirated using a pipette. The collected exosomes were diluted with PBS and centrifuged again at 100,000 g for 1.5 h at 4 °C to remove the gradient medium. The supernatant was discarded, and the exosome precipitate was resuspended with a small amount of PBS or buffer. A suitable size exclusion chromatography column Sepharose CL-4B (applicable to particles of 30-500 nm) was selected. The chromatography column was equilibrated with the chromatography buffer until the baseline was stable. The sample was eluted with the chromatography buffer at a constant flow rate of 1.0 mL / min. An ultraviolet detector (280 nm) was used to monitor the elution peak, or a fraction collector was used to collect the eluate. The exosomes appeared in the earlier elution peak (macromolecules were eluted first). The eluate containing exosomes was collected, the extracted exosomes were aliquoted, and stored at -80 °C for later use, facilitating subsequent tests.

[0069] Example 5

[0070] The Platycladus orientalis dry leaves were ground into powder with liquid nitrogen and added to pre-cooled PBS buffer for homogenization. The homogenate was filtered through multiple layers of gauze or a filter screen (300 mesh) to remove large particles. Further filtration was performed using a 0.45 μm filter membrane to remove cell debris and impurities. The filtrate was centrifuged at 5,000 g for 15 min at 4 °C to remove residual cell debris and large particles. Iodixanol was selected as the gradient medium, and 40%, 30%, 20%, 10%, and 5% gradient medium solutions were added layer by layer from high density to low density in an ultracentrifuge tube. After each layer was added, it was gently placed to avoid mixing. The pretreated sample was carefully added to the top layer of the gradient medium. An ultracentrifuge was used to centrifuge at 80,000 g for 3 h at 4 °C. After centrifugation, exosomes would be distributed in a certain layer of the gradient medium according to their density (located in the 20%-30% OptiPrep layer), and the target density layer (the layer where exosomes were located) was carefully aspirated using a pipette. The collected exosomes were diluted with PBS and centrifuged again at 80,000 g for 1.5 h at 4 °C to remove the gradient medium. The supernatant was discarded, and the exosome precipitate was resuspended with a small amount of PBS or buffer. A suitable size exclusion chromatography column Sepharose CL-4B (applicable to particles of 30-500 nm) was selected. The chromatography column was equilibrated with the chromatography buffer until the baseline was stable. The sample was eluted with the chromatography buffer at a constant flow rate of 0.8 mL / min. An ultraviolet detector (280 nm) was used to monitor the elution peak, or a fraction collector was used to collect the eluate. Exosomes appeared in the earlier elution peak (macromolecules were eluted first). The eluate containing exosomes was collected, the extracted exosomes were aliquoted, and stored at -80 °C for future use to facilitate subsequent tests.

[0071] Example 6

[0072] The Platycladus orientalis dry leaves were ground into powder with liquid nitrogen and added to pre-cooled PBS buffer for homogenization. The homogenate was filtered through multiple layers of gauze or a filter screen (500 mesh) to remove large particles. It was further filtered with a 0.45 μm filter membrane to remove cell debris and impurities. The filtrate was centrifuged at 5,000 g for 30 min at 4 °C to remove residual cell debris and large particles. Iodixanol was selected as the gradient medium. In an ultracentrifuge tube, 40%, 30%, 20%, 10%, and 5% gradient medium solutions were added layer by layer from high density to low density. After each layer was added, it was gently placed to avoid mixing. The pretreated sample was carefully added to the top layer of the gradient medium. An ultracentrifuge was used to centrifuge at 100,000 g for 4 h at 4 °C. After centrifugation, the exosomes would be distributed in a certain layer of the gradient medium according to the density (located in the 20%-30% OptiPrep layer). The target density layer (the layer where the exosomes were located) was carefully aspirated using a pipette. The collected exosomes were diluted with PBS and centrifuged again at 100,000 g for 2 h at 4 °C to remove the gradient medium. The supernatant was discarded, and the exosome precipitate was resuspended with a small amount of PBS or buffer. A suitable size exclusion chromatography column Sepharose CL-4B (applicable to 30-500 nm particles) was selected. The chromatography column was equilibrated with the chromatography buffer until the baseline was stable. The sample was eluted with the chromatography buffer at a constant flow rate of 0.5 mL / min. An ultraviolet detector (280 nm) was used to monitor the elution peak, or a fraction collector was used to collect the eluate. The exosomes appeared in the earlier elution peak (macromolecules were eluted first). The eluate containing the exosomes was collected, the extracted exosomes were aliquoted, and stored at -80 °C for later use, facilitating subsequent tests.

[0073] Example 7

[0074] The dry leaves of Platycladus orientalis were ground into powder with liquid nitrogen and added to pre-cooled PBS buffer for homogenization. The homogenate was filtered through multiple layers of gauze or a filter screen (500 mesh) to remove large particles. Further filtration was carried out with a 0.45 μm filter membrane to remove cell debris and impurities. The filtrate was centrifuged at 5,000 g for 10 min at 4 °C to remove residual cell debris and large particles. Iodixanol was selected as the gradient medium, and in an ultracentrifuge tube, 40%, 30%, 20%, 10%, and 5% gradient medium solutions were added layer by layer from high density to low density. After each layer was added, it was gently placed to avoid mixing. The pretreated sample was carefully added to the top layer of the gradient medium. Using an ultracentrifuge, centrifugation was carried out at 100,000 g for 2 h at 4 °C. After centrifugation, exosomes would be distributed in a certain layer of the gradient medium according to density (located in the 20%-30% OptiPrep layer), and the target density layer (the layer where exosomes are located) was carefully aspirated using a pipette. The collected exosomes were diluted with PBS and centrifuged again at 100,000 g for 1 h at 4 °C to remove the gradient medium. The supernatant was discarded, and the exosome precipitate was resuspended with a small amount of PBS or buffer. A suitable size exclusion chromatography column Sepharose CL-4B (applicable to particles of 30-500 nm) was selected. The chromatography column was equilibrated with the chromatography buffer until the baseline was stable. The sample was eluted with the chromatography buffer at a constant flow rate of 1.0 mL / min. An ultraviolet detector (280 nm) was used to monitor the elution peak, or a fraction collector was used to collect the eluate. Exosomes appeared in the earlier elution peak (macromolecules were eluted first). The eluate containing exosomes was collected, the extracted exosomes were aliquoted, and stored at -80 °C for future use, facilitating subsequent tests.

[0075] Comparative Example 1 (Size Exclusion Chromatography)

[0076] The dry leaves of Platycladus orientalis were ground into powder with liquid nitrogen and added to pre-cooled PBS buffer for homogenization. The homogenate was filtered through multiple layers of gauze or a filter screen (500 mesh) to remove large particles. Further filtration was carried out with a 0.45 μm filter membrane to remove cell debris and impurities. The filtrate was centrifuged at 5,000 g for 10 min at 4 °C to remove residual cell debris and large particles. Using an ultracentrifuge, centrifugation was carried out at 100,000 g for 2 h at 4 °C. The collected exosomes were diluted with PBS and centrifuged again at 100,000 g for 1 h at 4 °C to remove the gradient medium. The supernatant was discarded, and the exosome precipitate was resuspended with a small amount of PBS or buffer. The extracted exosomes were aliquoted, and stored at -80 °C for future use, facilitating subsequent tests.

[0077] Comparative Example 2 (Density Gradient Method)

[0078] Grind the dry leaves of Platycladus orientalis into powder with liquid nitrogen, add it to pre-cooled PBS buffer solution, and homogenize. Filter the homogenate through multiple layers of gauze or a filter screen (500 mesh) to remove large particles. Further filter with a 0.45μm filter membrane to remove cell debris and impurities. Centrifuge the filtrate at 5,000g for 10 min at 4°C to remove residual cell debris and large particles. Select a suitable size exclusion chromatography column Sepharose CL-4B (suitable for particles with a size of 30 - 500 nm). Equilibrate the chromatography column with chromatography buffer until the baseline is stable. Elute the sample with chromatography buffer at a constant flow rate of 0.8 mL / min. Monitor the elution peak using an ultraviolet detector (280 nm), or collect the eluate using a fraction collector. Exosomes appear in the earlier elution peak (macromolecules are eluted first). Collect the eluate containing exosomes, aliquot the extracted exosomes, and store them at -80°C for later use to facilitate subsequent tests.

[0079] Comparative Example 3

[0080] Commercially available aqueous extract of Platycladus orientalis leaves, with a solid content of 5 - 10% (g / 100 mL).

[0081] Test Example

[0082] Perform the following tests on the samples of Examples 1 - 7 and Comparative Examples 1 - 3:

[0083] 1. NTA particle size and vesicle number test

[0084] Use NTA (nanoparticle tracking analysis) to detect the particle size and vesicle number of exosomes in the samples of Examples 1 - 7 and Comparative Examples 1 - 3. The method is as follows:

[0085] First, dilute the sample to ensure a moderate sample concentration, dilute it to 10 7 -10 9 particles / mL. Then filter using a filter membrane with an appropriate pore size (0.22μm) to remove large particles or impurities. Turn on the machine and preheat: Turn on the NTA instrument and preheat the laser and camera. Calibrate: Use standard particles with a known particle size for calibration to ensure the accuracy of the instrument. Inject the sample: Inject the diluted sample into the sample cell using a syringe or pipette. Avoid air bubbles: Ensure that there are no air bubbles in the sample cell to avoid affecting the measurement. Adjust the laser intensity according to the sample to ensure that the particles are clearly visible. Start the measurement: Start the software and start recording the particle movement. Video recording: Record the video of the Brownian motion of the particles for 30 - 60 s. Finally, perform data analysis. Software analysis: Use NTA software to analyze the video and calculate the particle size distribution. The results are shown in Table 1 and Figure 2 , and the TEM micrograph of the exosomes extracted in Example 1 is shown in Figure 3 .

[0086] Table 1 D50 particle size (nm) and number of vesicles (pcs / mL) in different samples

[0087] Sample Example 1 Example 2 Example 3 Example 4 Example 5 D50 71 82 85 90 82 Vesicle number 5.57E+10 1.71E+10 2.20E+10 3.21E+10 1.98E+10 Sample Example 6 Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 D50 78 79 114 98 / Vesicle number 2.57E+10 4.18E+10 1.98E+8 9.81E+9 /

[0088] Note: E represents 10 to the power of N, where E+10 represents 10 to the power of 10.

[0089] As can be seen from the results, in Example 2, the initial 100 mesh is relatively small, and although some large particles are removed, there are still many impurities left. There is not much difference between the 0.22μm and 0.45μm filter membranes, but because the 100 mesh is relatively small, it is easy to clog, and the filter membrane needs to be replaced many times, which is inconvenient to operate; and the elution speed of the chromatographic column is relatively slow, and there will be a certain loss of vesicles. The centrifugal speed of 2,000g is relatively small, and there are more impurities left, which will cause certain difficulties for subsequent gradient centrifugation and will also affect certain results. In Example 3, the gradient range is relatively narrow, and the purity after elution is lower than that in Example 1. In Example 4, there are more gradients, but too many gradients lead to the loss of target exosome vesicles, and the elution speed is too fast, which may extract some vesicles with larger particle sizes; in Example 5, the centrifugal speed is relatively low, and there is a possibility of large vesicles remaining; in Example 6, the centrifugation and elution time are relatively long, which may destroy the vesicle structure and reduce the number of vesicles; in Example 7, the centrifugation and elution speeds are relatively slow, and there may be impurities remaining, resulting in a purity that is not as high as in Example 1.

[0090] 2. Protein concentration test

[0091] Method: BCA protein assay

[0092] Procedure: Modified Lowry method, using bicinchoninic acid as the reagent. In alkaline medium, the protein converts Cu 2+ Reduction to Cu 1+ , BCA and Cu 1+ The reaction generates a purple complex with a high absorbance. This water-soluble complex exhibits a strong absorbance at 562 nm, and in a wide range of protein concentrations (20-2000 μg / mL), the absorbance has a good linear relationship with the protein concentration. The experimental steps include: mixing the BCA working solution with the protein samples to be tested in Examples 1 to 7 and Comparative Examples 1 to 3, incubating after shaking, measuring the absorbance at 562 nm using a spectrophotometer, and calculating the protein concentration according to the standard curve. The results are shown in Table 2.

[0093] Table 2 Protein concentration in different samples (mg / mL)

[0094] Sample Example 1 Example 2 Example 3 Example 4 Example 5 Protein concentration 1.28 1.13 1.79 1.57 1.21 Sample Example 6 Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 Protein concentration 1.23 1.48 0.57 0.94 0.01

[0095] The results showed that the protein concentration in Example 1 was higher than that in other examples. Among the comparative examples, Comparative Example 2 was better than Comparative Example 1. This was because size exclusion chromatography was more inclined to refined extraction, while density gradient centrifugation tended to crude extraction, and the combination of the two was more excellent. In Comparative Example 3, the main component was water-soluble, and the protein concentration was low, mostly polysaccharides and water-soluble polyphenolic compounds.

[0096] 3. Safety test: Irritation to the chick chorioallantoic membrane

[0097] Test method: The chick chorioallantoic membrane (CAM) assay is a commonly used in vivo model for studying angiogenesis, tumor growth, drug screening, and toxicity testing, etc.

[0098] Source of chick embryos: Fertilized chicken embryos (White Leghorn chicken embryos were selected).

[0099] Incubation of chick embryos: The fertilized chicken embryos were placed in an incubator and incubated until the 9th day (when the CAM blood vessels were well developed). The chicken embryos were turned 2 - 3 times a day to ensure normal embryonic development. At least 6 chicken embryos were used in each group. 0.3 mL of the test substance, an aqueous solution of exosomes at 30% (v / v), was directly dropped onto the surface of the CAM. The reaction was observed, and the time when each toxic effect occurred within 300 s (5 min) was recorded, including bleeding, vasolysis, and coagulation. The negative control and positive control used the stimulation scoring method. The negative control was 0.9% NaCl solution, and the positive control was 0.1 M NaOH solution. The irritation score value IS was calculated using the formula as follows:

[0100] IS = (301 - secH) × 5 / 300 + (301 - secL) × 7 / 300 + (301 - secC) × 9 / 300;

[0101] where secH, secL, and secC represent the average time when bleeding, vasolysis, and coagulation began to occur on the CAM membrane, respectively, with the unit of ss.

[0102] Evaluation of the irritation score results included: IS < 1, no irritation; 1 < IS < 5, mild irritation; 5 ≤ IS < 10, moderate irritation; IS ≥ 10, strong irritation / corrosion

[0103] Test results:

[0104] 1) The IS of the negative control = 0.00, meeting the standard;

[0105] 2) The IS of the positive control = 18.39, meeting the standard.

[0106] The results are shown in Table 3 and Figures 4 to 6 。

[0107] Table 3 IS values in various samples (Samples: Diluted to 30% aqueous solution for testing)

[0108] Sample Example 1 Example 2 Example 3 Example 4 Example 5 IS 0.1 0.3 0.5 0.2 0.1 Sample Example 6 Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 Protein concentration 0.3 0.6 0.7 0.4 4.5

[0109] The results showed that there were almost no changes in the blood vessels and morphology of chicken embryos before and after adding the sample of Example 1, indicating that the sample was non-irritating ( Figure 4 ). Comparing each example, all were non-irritating. Among the comparative examples, except that the Platycladus orientalis leaf extract in Comparative Example 3 was slightly irritating, the rest of the comparative examples were non-irritating. Thus, it can be seen that the plant exosomes extracted by the present invention have relatively high safety compared to the extract of Comparative Example 3.

[0110] 4. Efficacy test: Inhibiting sebum secretion of sebaceous gland cells

[0111] Human cortical cells (SEB-1 cell line) were used to ensure good cell status. The sebaceous gland cells were inoculated into a culture plate (such as a 6-well plate or a 96-well plate), and the density was adjusted according to experimental requirements (to 1×10 5 cells / mL). They were cultured in an incubator at 37 °C and 5% CO2 until the cells reached 70-80% confluence. To reduce the influence of the cell cycle on the experimental results, the cells could be cultured in serum-free medium (DMEM) for 12-24 h to synchronize the cells. Each test sample (example and comparative example) was diluted with serum-free medium into two concentrations of 2% (w / w) and 10% (w / w) respectively. The synchronized cells were divided into a blank control group, a model control group, a 2% diluted aqueous solution sample group, and a 10% diluted aqueous solution sample group, and the specific treatments were as follows:

[0112] Blank control group: After removing the old medium, new medium was added;

[0113] Model control group: After removing the old medium, new medium containing 200 μM oleic acid, 10% fetal bovine serum, and 1% antibiotic (penicillin-streptomycin, 10,000 U / mL) was added;

[0114] 2% diluted aqueous solution sample group: After removing the old medium, serum-free medium containing 2% test sample, 200 μM oleic acid, 10% fetal bovine serum, and 1% antibiotic (penicillin-streptomycin, 10,000 U / mL) was added;

[0115] 10% diluted aqueous solution sample group: After removing the old medium, serum-free medium containing 10% test sample, 200 μM oleic acid, 10% fetal bovine serum, and 1% antibiotic (penicillin-streptomycin, 10,000 U / mL) was added.

[0116] Four groups of cells were cultured at 37 °C under 5% CO2 for 24 h. Before measuring the lipid content, Oil Red O staining method was required. Cell fixation: Fix the cells with 10% formalin for 30 min and wash with PBS. Staining: Add Oil Red O staining solution and incubate at room temperature for 30 min. Washing: Wash with distilled water to remove the unbound dye. Observation: Observe the lipid droplets with an optical microscope, or measure the absorbance (510 nm) with an enzyme-linked immunosorbent assay (ELISA) after extracting the stained dye. Statistically analyze the lipid content of the experimental group and the control group, and calculate the inhibition rate. Each experiment should have at least 3 replicates to ensure the reliability of the data.

[0117] Lipid secretion inhibition rate = (fluorescence intensity of the sample group - fluorescence intensity of the model control group) / fluorescence intensity of the model control group;

[0118] The results are shown in Table 4 and Figure 7 .

[0119] Table 4 Sebaceous gland cell fat secretion inhibition rates of samples at different concentrations

[0120] Sample Example 1 Example 2 Example 3 Example 4 Example 5 2% concentration sample 26％ 21％ 23％ 20％ 22％ 10% concentration sample 37％ 30％ 33％ 33％ 31％ Sample Example 6 Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 2% concentration sample 21％ 24％ 15％ 19％ 9％ 10% concentration sample 34％ 35％ 21％ 25％ 13％

[0121] The results showed that Example 1 had a higher inhibition rate on sebum cell fat secretion, while the inhibition rate of the Platycladus orientalis extract in Comparative Example 3 was lower, probably due to the unclear impurity content resulting in an insignificant effect.

[0122] 5. Efficacy test: Scratch test on human primary dermal papilla cells

[0123] A scratch assay was performed using human primary dermal papilla cells (HDPCs). HDPCs cells (10 6 cells / well) were seeded into 6-well plates and cultured for 1 - 2 days until confluent. Scratch the confluent monolayer of HDPCs cells with the tip of a pipette. Wash with PBS to remove the detached cells or debris. Then, the cells were treated with a 1% (w / w) or 10% (w / w) concentration of the test sample (Example or Comparative Example) in DMEM containing 5% fetal bovine serum. Set only adding 5 μg / mL of minoxidil as the model control group, and the group without adding the test sample as the control group. Images of the scratch area were taken at 0, 12, and 24 h after scratch formation, and the width of the scratch area was measured using ImageJ software. Calculate the percentage of wound closure at each time point based on the remaining size at 0 h. Wound closure rate = (initial scratch width - final scratch width) / initial scratch width × 100%. The results are shown in Table 5 and Figure 8 .

[0124] Table 5 Wound closure rates of different samples (10%) at each time point in the HDPCs cell scratch test

[0125] Sample Example 1 Example 2 Example 3 Example 4 Example 5 0h 0％ 0％ 0％ 0％ 0％ 12h 43％ 38％ 35％ 33％ 39％ 24h 98％ 88％ 93％ 91％ 90％ Sample Example 6 Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 0h 0％ 0％ 0％ 0％ 0％ 12h 36％ 40％ 27％ 33％ 29％ 24h 89％ 94％ 70％ 81％ 61％

[0126] The results showed that: When comparing the wound closure test results in the cell scratch assay, Example 1 recovered faster at 12 and 24 hours, and was basically recovered after 24 hours, with a closure rate reaching 98%. Followed by Example 7. Among the comparative examples, Comparative Example 3 was worse than other comparative examples. It may be that there are certain toxic components in the active substances that slow down the cell closure rate.

[0127] 6. Efficacy test: qPCR test related to hair growth promotion

[0128] Introduction to the tested genes:

[0129] 1) VEGF is an important factor for hair follicle growth, promoting angiogenesis and providing sufficient nutrition and oxygen for hair follicles. The growth promotion of HDPCs is accompanied by an increase in VEGF expression.

[0130] 2) β-catenin is a key molecule in the Wnt signaling pathway, which plays a core role in hair follicle regeneration and hair growth. The growth promotion effect generally activates the Wnt / β-catenin pathway, resulting in an increase in its expression. Wnt / β-Catenin signal transduction has been proven to be crucial for hair follicle development and hair growth. Its activation in HDP cells helps the proliferation and differentiation of hair follicle cells, thus initiating the anagen phase of the hair cycle.

[0131] 3) Transforming growth factor β-1 (TGF-β1) is a hair growth inhibitor. In the case of promoting hair growth, its expression will be down-regulated to reduce the growth inhibitory effect on hair follicles.

[0132] Experimental operation method:

[0133] Extract DNA or RNA from the sample. Use a spectrophotometer or fluorometer to measure the concentration and purity of DNA / RNA. Reaction system configuration: Configure the reaction mixture according to the kit instructions or experimental protocol, including: template DNA / RNA, forward primer and reverse primer, probe (such as using TaqMan probe), dNTPs, Taq DNA polymerase, buffer, MgCl2. Aliquot: Aliquot the reaction mixture into qPCR reaction plates or tubes.

[0134] Table 6 Primer sequences of three genes

[0135] VEGF-F 5’-ATCGAGTACATCTTCAAGCCAT-3’(SEQ ID NO.1) VEGF-R 5’-GTGAGGTTTGATCCGCATAATC-3’(SEQ ID NO.2) β-Catenin-F 5’-TGGATTGATTCGAAATCTTGCC-3’(SEQ ID NO.3) β-Catenin-R 5’-GAACAAGCAACTGAACTAGTCG-3’(SEQ ID NO.4) TGFβ2-F 5’-GCAAAGTTGTGAAAACAAGAGC-3’(SEQ ID NO.5) TGFβ2-R 5’-ATCCCAGGTTCCTGTCTTTATG-3’(SEQ ID NO.6)

[0136] Run on the machine

[0137] Setting procedure: Set the reaction procedure on the qPCR instrument. Place the reaction plate or tube into the qPCR instrument and start the program. Data analysis includes threshold setting: Set the fluorescence threshold in the qPCR software, which is 10 times the standard deviation of the baseline fluorescence signal. Ct value determination: Record the Ct value (cycle threshold) of each sample, that is, the number of cycles when the fluorescence signal reaches the threshold. Quantitative analysis: Calculate the relative or absolute quantification of the target gene according to the standard curve or ΔΔCt method. In result interpretation, standard curve: Draw a standard curve using standard products with known concentrations for quantitative analysis. Relative quantification: Normalize using the reference gene and calculate the relative expression level of the target gene.

[0138] The relative expression level of the gene in the blank control group is set to 1;

[0139] Samples: The exosomes of the examples or comparative examples are diluted to a 10% (w / w) aqueous solution, which is almost non-toxic to cells (cell survival rate > 90%).

[0140] The results are shown in Table 7.

[0141] Table 7 Relative expression levels of each gene in each sample

[0142] Sample Example 1 Example 2 Example 3 Example 4 Example 5 VEGF 2.09 1.83 1.74 1.90 1.74 β-Catenin 1.77 1.64 1.62 1.59 1.71 TGF-β2 0.65 0.73 0.83 0.77 0.79 Sample Example 6 Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 VEGF 1.83 1.91 1.53 1.65 1.90 β-Catenin 1.70 1.73 1.42 1.59 1.62 TGF-β2 0.73 0.69 0.81 0.79 0.65

[0143] The results show that: Among the comparative examples, the effect of Example 1 is the most significant, with VEGF and β-Catenin increasing and TGF-β2 decreasing, and the highest change rate, which may be due to the higher number of vesicles and the concentration of active substances. Among the comparative examples, the effect of Comparative Example 3 is also relatively prominent, probably because of the higher content of active substances in the Platycladus orientalis extract.

[0144] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting plant exosomes by density gradient centrifugation combined with size exclusion chromatography, characterized in that, It includes the following steps: Crush the plant tissue to be extracted and mix it with a buffer solution to obtain a mixture; Filter the mixture, collect the filtrate and centrifuge it to obtain a supernatant; Using iodixanol as a gradient medium, subject the supernatant to density gradient centrifugation, and collect the iodixanol density layer of 20 - 30 g / 100 mL to obtain a centrifugate containing plant exosomes; Ultracentrifuge the centrifugate to obtain a precipitate containing plant exosomes; Resuspend the precipitate containing plant exosomes and elute it using a size exclusion chromatography column, and collect the eluate containing exosomes; the size exclusion chromatography column is a size exclusion chromatography column for separating particles of 30 - 500 nm; the elution flow rate is 0.5 - 1 mL / min.

2. The method according to claim 1, characterized in that, The plant includes Platycladus orientalis; the plant tissue to be extracted includes Platycladus orientalis leaves.

3. The method according to claim 1, wherein The filtering method includes: first filtering the mixture using a 100 - 500 mesh filter screen to obtain a first filtrate; filtering the first filtrate using a 0.22 - 0.44 μm filter membrane to obtain the filtrate.

4. The method according to claim 1, characterized in that The centrifugation speed is 2,000 - 10,000×g; the time is 10 - 30 min, and the temperature is 4°C.

5. The method according to claim 1, characterized in that The gradient medium includes iodixanol solutions of 30 g / 100 mL, 20 g / 100 mL, and 10 g / 100 mL.

6. The method according to claim 1 or 5, characterized in that, The gradient medium includes iodixanol solutions of 40 g / 100 mL, 30 g / 100 mL, 20 g / 100 mL, 10 g / 100 mL, and 5 g / 100 mL.

7. The method according to claim 1, characterized in that, The centrifugation speed of the density gradient centrifugation is 50,000 - 100,000×g, and the time is 2 - 4 h.

8. The method according to claim 1, wherein The ultracentrifugation speed is 50,000 - 100,000×g, and the time is 1 - 2 h.

9. The method according to claim 1, characterized in that, The buffer solution includes phosphate buffer solution; the eluent used for elution includes phosphate buffer solution; the size exclusion chromatography column includes Sepharose CL - 4B.

10. Use of the plant exosomes prepared by the method according to any one of claims 1 - 9 in the preparation of skin care products.

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