Plant-derived exosome-like nanoparticles and extraction method thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-16
- Publication Date
- 2026-04-03
AI Technical Summary
Existing methods for extracting plant-derived exosome-like nanoparticles suffer from low purity, low recovery efficiency, and cumbersome operation, making it difficult to meet high-throughput requirements.
A method combining juicing and multi-step centrifugation and ultracentrifugation was adopted, including treatment of plant samples with PBS buffer, cellulase and pectinase, followed by purification by PEG-8000 precipitation and sucrose gradient centrifugation, and finally filtration through a 0.22 μm filter membrane to obtain pure exosome-like nanoparticles.
It significantly improves the purity and extraction efficiency of exosome-like nanoparticles, reduces impurities, simplifies the operation process, and is suitable for large-scale production.
Smart Images
Figure CN121780409A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of exosome extraction technology, and more particularly to the extraction of plant-derived exosome-like nanoparticles. Background Technology
[0002] Plant-derived exosome-like nanoparticles are nanoscale vesicles derived from plant cells. They are not only rich in a variety of bioactive components, but also show significant application potential in fields such as drug delivery, disease treatment, and functional foods.
[0003] Currently, commonly used extraction methods mainly include differential centrifugation, density gradient centrifugation, and ultrafiltration.
[0004] Among them, differential centrifugation removes cell debris and larger organelles by gradually increasing centrifugal force, and finally precipitates exosomes under ultra-high speed conditions; density gradient centrifugation, on the other hand, places the sample in a density gradient medium such as sucrose or iodixanol, and then centrifuges it at ultra-high speed to enrich exosomes in a specific density zone, thereby achieving fine purification; ultrafiltration relies on ultrafiltration membranes with different molecular weight cutoffs to achieve selective separation of exosomes from impurities under external pressure.
[0005] However, all of the above methods have obvious limitations: differential centrifugation is prone to exosome structural damage due to shear force during high-speed centrifugation, and due to the complex composition of plant samples, a large number of impurities often co-precipitate with exosomes, which seriously affects their purity and resuspension effect; density gradient centrifugation can effectively improve the purity of exosomes, but the operation process is cumbersome and time-consuming, and the overall yield is low, making it difficult to meet the high-throughput requirements; ultrafiltration has the advantage of simple operation, but when processing plant samples, membrane pore blockage and non-specific adsorption of samples are prone to occur, resulting in a decrease in recovery rate. At the same time, this method can only remove impurities smaller than the membrane pore size, and its effect on the separation of large particles is limited.
[0006] In summary, existing extraction methods still have significant shortcomings in terms of product purity, recovery efficiency, and ease of operation. There is an urgent need to develop a new extraction strategy that is efficient, mild, and has the potential for large-scale application in order to improve the extraction quality and practical application value of plant exosomes. Summary of the Invention
[0007] This invention addresses the problems existing in the extraction methods of plant-derived exosome-like nanoparticles in the prior art by providing a method for extracting plant-derived exosome-like nanoparticles.
[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for extracting plant-derived exosome-like nanoparticles, comprising the following steps: Step S1: Mix the plant sample with pre-cooled PBS buffer and then juice it. Add cellulase and pectinase to obtain a mixture with a concentration of 0.3-1.0%. Let the mixture stand and filter to obtain a crude extract. Step S2: Centrifuge the crude extract obtained in step S1, and collect the supernatant after processing to obtain the refined extract. Step S3: The refined extract obtained in step S2 is subjected to exosome concentration treatment to obtain exosome polymerized concentrate; Step S4: Purify the exosome polymer concentrate obtained in step S3 to obtain plant-derived exosome-like nanoparticles.
[0009] Preferably, step S1 specifically includes the following steps: Step S11: Mix the plant sample with PBS buffer pre-cooled to 4°C and then juice it. Step S12: Add cellulase and pectinase to obtain a mixture with a concentration of 0.3-1.0%; Step S13: After standing for 10-18 hours, the crude extract is obtained by filtering through 100-mesh gauze.
[0010] Preferably, the centrifugation process in step S2 includes two centrifugation processes: first, low-speed centrifugation is performed at a temperature of 4°C, a centrifugal force of 1000 g, and a centrifugation time of 30 min, and the supernatant is collected; then, the collected supernatant is centrifuged at a temperature of 4°C, a centrifugal force of 10000 g, and a centrifugation time of 60 min, and the supernatant is collected to obtain the refined extract.
[0011] Preferably, step S3 specifically includes the following steps: Step S31: Add PEG-8000 at a concentration of 6-10% to the purified extract; Step S32: Let it stand for 8-14 hours at a temperature of 4℃; Step S33: Centrifuge at 4℃, 3000g, and 10-30min, and collect the precipitate after centrifugation. Step S34: The collected precipitate is resuspended in PBS buffer pre-cooled to 4°C to obtain exosome polymer concentrate.
[0012] Preferably, the weight ratio of the precipitate to the PBS buffer in step S34 is 1:10 to 1:50.
[0013] Preferably, step S4 specifically includes the following steps; Step S41: Place the exosome polymer concentrate in an ultracentrifuge tube, add sucrose solution to the bottom of the tube, and collect the exosome solution suspended in the sucrose layer after processing. Step S42: Mix the collected exosome solution with PBS buffer pre-cooled to 4°C until the density is less than 1.05 g / ml, then centrifuge again and collect the precipitate; Step S43: The precipitate was pre-cooled to 4°C and resuspended in PBS buffer, then filtered to obtain plant-derived exosome-like nanoparticles.
[0014] Preferably, the centrifugation conditions for steps S41 and S42 are both 4°C, 120000g, and 70min.
[0015] Preferably, the sucrose solution in step S41 is a sucrose solution with a density of 1.12-1.18 g / ml prepared with PBS.
[0016] Preferably, the filtration in step S43 is performed through a 0.22 μm filter membrane.
[0017] A plant-derived exosome-like nanoparticle, obtained using the aforementioned extraction method for plant-derived exosome-like nanoparticles.
[0018] This invention, by adopting the above technical solutions, has significant technical effects: This invention provides a method for extracting plant-derived exosome-like nanoparticles, which, compared with the simple ultrafiltration method for extracting plant-derived exosome-like nanoparticles, has the advantages of fewer impurities, higher extraction efficiency, and easier resuspension of the precipitate obtained by ultrafiltration. Attached Figure Description
[0019] Figure 1 This is a transmission electron microscopy (TEM) characterization result of the exosome-like nanoparticles derived from Astragalus membranaceus prepared in Example 1 of this invention.
[0020] Figure 2 This is a graph showing the particle size characterization results of the Astragalus-derived exosome-like nanoparticles prepared in Example 1 of this invention.
[0021] Figure 3 This is a transmission electron microscopy (TEM) characterization result of the exosome-like nanoparticles derived from Poria cocos prepared in Example 2 of this invention.
[0022] Figure 4 This is a graph showing the particle size characterization results of the exosome-like nanoparticles derived from Poria cocos prepared in Example 2 of this invention.
[0023] Figure 5 This is a transmission electron microscopy (TEM) characterization result of the licorice-derived exosome-like nanoparticles prepared in Example 3 of this invention.
[0024] Figure 6 This is a graph showing the particle size characterization results of the licorice-derived exosome-like nanoparticles prepared in Example 3 of this invention.
[0025] Figure 7 This is a transmission electron microscopy (TEM) characterization result of the exosome-like nanoparticles derived from shiitake mushrooms prepared in Example 4 of this invention.
[0026] Figure 8 This is a graph showing the particle size characterization results of the shiitake mushroom-derived exosome-like nanoparticles prepared in Example 4 of this invention.
[0027] Figure 9 This is a transmission electron microscopy (TEM) characterization result of the garlic-derived exosome-like nanoparticles prepared in Example 5 of this invention.
[0028] Figure 10 This is a graph showing the particle size characterization results of garlic-derived exosome-like nanoparticles prepared in Example 5 of this invention.
[0029] Figure 11 This is a transmission electron microscopy (TEM) characterization result of the exosome-like nanoparticles derived from Astragalus membranaceus prepared in Comparative Example 1.
[0030] Figure 12 This is a graph showing the particle size characterization results of the Astragalus-derived exosome-like nanoparticles prepared in Comparative Example 1.
[0031] Figure 13 This is a transmission electron microscopy (TEM) characterization result of the exosome-like nanoparticles derived from Poria cocos prepared in Comparative Example 1.
[0032] Figure 14 This is a graph showing the particle size characterization results of the exosome-like nanoparticles derived from Poria cocos prepared in Comparative Example 1. Detailed Implementation
[0033] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.
[0034] Example 1: Extraction of exosome-like nanoparticles from Astragalus membranaceus Step 1: After washing and cutting the Astragalus membranaceus sample into pieces, mix it with PBS buffer pre-cooled to 4 degrees Celsius and then juice it. Add cellulase and pectinase at a concentration of 0.5%, let it stand at 4 degrees Celsius for 12 hours, and then filter it through 100-mesh gauze to obtain the crude extract. Step 2 involves a two-step centrifugation process on the crude extract: low-speed centrifugation at 4°C and 1000 g for 30 minutes, collecting the supernatant; and high-speed centrifugation at 4°C and 10000 g for 60 minutes, collecting the supernatant to obtain the refined extract. Step 3 involves concentrating the above-mentioned extract with exosomes. PEG-8000 is added to the extract at a concentration of 8%, and the mixture is allowed to stand at 4°C for 12 hours. After centrifugation at 3000g for 30 minutes at 4°C, the precipitate is collected, pre-cooled to 4°C, and resuspended in PBS buffer at a ratio of 1:20 to the precipitate. This yields the exosome polymerized concentrate. Step 4 involves purifying the above exosome polymer concentrate. The exosome polymer concentrate is placed in an ultracentrifuge tube, and a sucrose solution with a density of 1.18 g / ml prepared with PBS is added to the bottom of the tube. The tube is centrifuged at 120,000 g for 70 minutes at 4°C. The exosomes suspended in the sucrose layer are collected. The collected exosome solution is mixed with PBS buffer pre-cooled to 4°C until the density is below 1.05 g / ml. The mixture is then centrifuged at 120,000 g for 70 minutes at 4°C. The precipitate is collected, resuspended in PBS buffer pre-cooled to 4°C, and filtered through a 0.22 μm filter membrane to obtain the Astragalus membranaceus-derived exosome-like nanoparticle sample.
[0035] Step 5: Observe the morphology of exosomes using transmission electron microscopy (TEM) and detect the particle size and concentration of exosomes using a nano-Kurt particle size analyzer.
[0036] Depend on Figure 1 It can be seen that the Astragalus-derived exosome-like nanoparticles prepared in this embodiment have typical hemispherical or saucer-like structures and ideal particle size.
[0037] Figure 2 The particle size characterization results of the Astragalus-derived exosome-like nanoparticles prepared in Example 1 are presented. The prepared Astragalus-derived exosome-like nanoparticles have a uniform particle size distribution, an average particle size of 77 nm, and a concentration of 3.93 × 10⁻⁶. 11 per ml.
[0038] Example 2: Extraction of exosome-like nanoparticles from Poria cocos Step 1: After washing and cutting the Poria cocos sample into pieces, mix it with PBS buffer pre-cooled to 4 degrees Celsius and then juice it. Add cellulase and pectinase at a concentration of 1.0%, let it stand at 4 degrees Celsius for 12 hours, and then filter it through 100-mesh gauze to obtain the crude extract. Step 2 involves a two-step centrifugation process on the crude extract: low-speed centrifugation at 4°C and 1000 g for 30 minutes, collecting the supernatant; and high-speed centrifugation at 4°C and 10000 g for 60 minutes, collecting the supernatant to obtain the refined extract. Step 3: The above-mentioned extract was subjected to exosome concentration treatment. PEG-8000 was added to the extract at a concentration of 10%, and the mixture was allowed to stand at 4°C for 12 hours. After centrifugation at 3000g for 30 minutes at 4°C, the precipitate was collected, pre-cooled to 4°C, and resuspended in PBS buffer. The ratio of precipitate to PBS was 1:40, and the exosome polymerized concentrate was obtained. Step 4 involves purifying the above exosome polymer concentrate. The exosome polymer concentrate is placed in an ultracentrifuge tube, and a sucrose solution with a density of 1.18 g / ml prepared with PBS is added to the bottom of the tube. The tube is centrifuged at 120,000 g for 70 minutes at 4°C. The exosomes suspended in the sucrose layer are collected. The collected exosome solution is mixed with PBS buffer pre-cooled to 4°C until the density is below 1.05 g / ml. The mixture is then centrifuged at 120,000 g for 70 minutes at 4°C. The precipitate is collected, resuspended in PBS buffer pre-cooled to 4°C, and filtered through a 0.22 μm filter membrane to obtain the exosome-like nanoparticle sample derived from Poria cocos.
[0039] Step 5: Observe the morphology of exosomes using transmission electron microscopy (TEM) and detect the particle size and concentration of exosomes using a nano-Kurt particle size analyzer.
[0040] Depend on Figure 3 It can be seen that the exosome-like nanoparticles derived from Poria cocos prepared in this embodiment have typical hemispherical or saucer-like structures and ideal particle size.
[0041] Depend on Figure 4 It can be seen that the exosome-like nanoparticles derived from Poria cocos prepared in this embodiment have a uniform particle size distribution, an average particle size of 68 nm, and a concentration of 6.03*10⁻⁶. 12 per ml.
[0042] Example 3: Extraction of Licorice-derived Exosome-like Nanoparticles Step 1: The cleaned and chopped licorice sample was mixed with PBS buffer pre-cooled to 4 degrees Celsius and then juiced. Cellulase and pectinase were added at a concentration of 1.0%. The mixture was allowed to stand at 4 degrees Celsius for 12 hours and then filtered through 100-mesh gauze to obtain the crude extract. Step 2 involves a two-step centrifugation process on the crude extract: low-speed centrifugation at 4°C and 1000 g for 30 minutes, collecting the supernatant; and high-speed centrifugation at 4°C and 10000 g for 60 minutes, collecting the supernatant to obtain the refined extract. Step 3: The above-mentioned extract was subjected to exosome concentration treatment. PEG-8000 was added to the extract at a concentration of 10%, and the mixture was allowed to stand at 4°C for 12 hours. After centrifugation at 3000g for 30 minutes at 4°C, the precipitate was collected, pre-cooled to 4°C, and resuspended in PBS buffer at a ratio of 1:30 to precipitate. The resulting exosome polymer concentrate was obtained. Step 4 involves purifying the above exosome polymer concentrate. The exosome polymer concentrate is placed in an ultracentrifuge tube, and a sucrose solution with a density of 1.18 g / ml prepared with PBS is added to the bottom of the tube. The tube is centrifuged at 120,000 g for 70 minutes at 4°C. The exosomes suspended in the sucrose layer are collected. The collected exosome solution is mixed with PBS buffer pre-cooled to 4°C until the density is below 1.05 g / ml. The mixture is then centrifuged at 120,000 g for 70 minutes at 4°C. The precipitate is collected, resuspended in PBS buffer pre-cooled to 4°C, and filtered through a 0.22 μm filter membrane to obtain licorice-derived exosome-like nanoparticle samples.
[0043] Step 5: Observe the morphology of exosomes using transmission electron microscopy (TEM) and detect the particle size and concentration of exosomes using a nano-Kurt particle size analyzer.
[0044] Depend on Figure 5 It can be seen that the licorice-derived exosome-like nanoparticles prepared in this embodiment have typical hemispherical or saucer-like structures and ideal particle sizes.
[0045] Depend on Figure 6 It can be seen that the licorice-derived exosome-like nanoparticles prepared in this embodiment have a uniform particle size distribution, an average particle size of 72 nm, and a concentration of 2.34 × 10⁻⁶. 12 per ml.
[0046] Example 4: Extraction of exosome-like nanoparticles from shiitake mushrooms Step 1: The cleaned and chopped shiitake mushroom sample was mixed with PBS buffer pre-cooled to 4 degrees Celsius and then juiced. Cellulase and pectinase were added at a concentration of 0.5%. The mixture was allowed to stand at 4 degrees Celsius for 12 hours and then filtered through 100-mesh gauze to obtain the crude extract. Step 2 involves a two-step centrifugation process on the crude extract: low-speed centrifugation at 4°C and 1000 g for 30 minutes, collecting the supernatant; and high-speed centrifugation at 4°C and 10000 g for 60 minutes, collecting the supernatant to obtain the refined extract. Step 3 involves concentrating the above-mentioned extract with exosomes. PEG-8000 at a concentration of 10% is added to the extract, and the mixture is allowed to stand at 4°C for 12 hours. After centrifugation at 3000g for 30 minutes at 4°C, the precipitate is collected, pre-cooled to 4°C, and resuspended in PBS buffer at a ratio of 1:20 to the precipitate. This yields the exosome polymerized concentrate. Step 4 involves purifying the above exosome polymer concentrate. The exosome polymer concentrate is placed in an ultracentrifuge tube, and a sucrose solution with a density of 1.18 g / ml prepared with PBS is added to the bottom of the tube. The tube is centrifuged at 120,000 g for 70 minutes at 4°C. The exosomes suspended in the sucrose layer are collected. The collected exosome solution is mixed with PBS buffer pre-cooled to 4°C until the density is below 1.05 g / ml. The mixture is then centrifuged at 120,000 g for 70 minutes at 4°C. The precipitate is collected, resuspended in PBS buffer pre-cooled to 4°C, and filtered through a 0.22 μm filter membrane to obtain the exosome-like nanoparticle sample from shiitake mushroom.
[0047] Step 5: Observe the morphology of exosomes using transmission electron microscopy (TEM) and detect the particle size and concentration of exosomes using a nano-Kurt particle size analyzer.
[0048] Depend on Figure 7 It can be seen that the shiitake mushroom-derived exosome-like nanoparticles prepared in this embodiment have typical hemispherical or saucer-like structures and ideal particle sizes.
[0049] Depend on Figure 8 It can be seen that the lentinan-derived exosome-like nanoparticles prepared in this embodiment have a uniform particle size distribution, an average particle size of 64 nm, and a concentration of 5.31 × 10⁻⁶. 11 per ml.
[0050] Example 5: Extraction of garlic-derived exosome-like nanoparticles Step 1: After mixing the peeled garlic sample with PBS buffer pre-cooled to 4 degrees Celsius, the sample was juiced. Cellulase and pectinase were added at a concentration of 0.3%. The mixture was allowed to stand at 4 degrees Celsius for 12 hours. After filtration through 100-mesh gauze, the crude extract was obtained. Step 2 involves a two-step centrifugation process on the crude extract: low-speed centrifugation at 4°C and 1000 g for 30 minutes, collecting the supernatant; and high-speed centrifugation at 4°C and 10000 g for 60 minutes, collecting the supernatant to obtain the refined extract. Step 3: The above-mentioned extract was subjected to exosome concentration treatment. PEG-8000 was added to the extract at a concentration of 6%, and the mixture was allowed to stand at 4°C for 12 hours. After centrifugation at 3000g for 30 minutes at 4°C, the precipitate was collected, pre-cooled to 4°C, and resuspended in PBS buffer at a ratio of 1:30 to precipitate. The resulting exosome polymerized concentrate was obtained. Step 4 involves purifying the above exosome polymer concentrate. The exosome polymer concentrate is placed in an ultracentrifuge tube, and a sucrose solution with a density of 1.18 g / ml prepared with PBS is added to the bottom of the tube. The tube is centrifuged at 120,000 g for 70 minutes at 4°C. The exosomes suspended in the sucrose layer are collected. The collected exosome solution is mixed with PBS buffer pre-cooled to 4°C until the density is below 1.05 g / ml. The mixture is then centrifuged at 120,000 g for 70 minutes at 4°C. The precipitate is collected, resuspended in PBS buffer pre-cooled to 4°C, and filtered through a 0.22 μm filter membrane to obtain garlic-derived exosome-like nanoparticle samples.
[0051] Step 5: Observe the morphology of exosomes using transmission electron microscopy (TEM) and detect the particle size and concentration of exosomes using a nano-Kurt particle size analyzer.
[0052] Depend on Figure 9 It can be seen that the garlic-derived exosome-like nanoparticles prepared in this embodiment have typical hemispherical or saucer-like structures and ideal particle sizes.
[0053] Depend on Figure 10 It can be seen that the garlic-derived exosome-like nanoparticles prepared in this embodiment have a uniform particle size distribution, an average particle size of 63 nm, and a concentration of 4.74 × 10⁻⁶. 13 per ml.
[0054] Comparative Example 1: Extraction of Astragalus-derived exosome-like nanoparticles using ultrafiltration method Step 1: After washing and cutting the Astragalus membranaceus sample into pieces, mix it with PBS buffer pre-cooled to 4 degrees Celsius and then juice it. Add cellulase and pectinase at a concentration of 0.5%, let it stand at 4 degrees Celsius for 12 hours, and then filter it through 100-mesh gauze to obtain the crude extract. Step 2 involves a two-step centrifugation process on the crude extract: low-speed centrifugation at 4°C and 1000 g for 30 minutes, collecting the supernatant; and high-speed centrifugation at 4°C and 10000 g for 60 minutes, collecting the supernatant to obtain the refined extract. Step 3 involves purifying the above-mentioned extract. The extract is placed in an ultracentrifuge tube and centrifuged at 120,000g for 70 minutes at 4°C. The precipitate is collected and resuspended in PBS buffer pre-cooled to 4°C. The precipitate is then centrifuged at 120,000g for 70 minutes at 4°C, collected, and resuspended in PBS buffer pre-cooled to 4°C. The precipitate is then filtered through a 0.22μm filter membrane to obtain Astragalus membranaceus-derived exosome-like nanoparticles.
[0055] Step 4: Observe the morphology of exosomes using transmission electron microscopy (TEM) and detect the particle size and concentration of exosomes using a nano-Kurt particle size analyzer.
[0056] Depend on Figure 11It can be seen that the Astragalus-derived exosome-like nanoparticles prepared in this comparative example have a typical hemispherical or saucer-like structure and an ideal particle size, and contain a large number of impurities.
[0057] Depend on Figure 12 It can be seen that the Astragalus-derived exosome-like nanoparticles prepared in this comparative example have a uniform particle size distribution, an average particle size of 72 nm, and a concentration of 1.87 × 10⁻⁶. 11 per ml.
[0058] Comparative Example 2: Extraction of exosome-like nanoparticles from Poria cocos using ultrafiltration method Step 1: The cleaned and diced Poria cocos sample was mixed with PBS buffer pre-cooled to 4 degrees Celsius and then juiced. Cellulase and pectinase were added at a concentration of 0.5%. The mixture was allowed to stand at 4 degrees Celsius for 12 hours and then filtered through 100-mesh gauze to obtain the crude extract. Step 2 involves a two-step centrifugation process on the crude extract: low-speed centrifugation at 4°C and 1000 g for 30 minutes, collecting the supernatant; and high-speed centrifugation at 4°C and 10000 g for 60 minutes, collecting the supernatant to obtain the refined extract. Step 3 involves purifying the above-mentioned extract. The extract is placed in an ultracentrifuge tube and centrifuged at 120,000g for 70 minutes at 4°C. The precipitate is collected and resuspended in PBS buffer pre-cooled to 4°C. The precipitate is then centrifuged at 120,000g for 70 minutes at 4°C, collected, and resuspended in PBS buffer pre-cooled to 4°C. The precipitate is then filtered through a 0.22μm filter membrane to obtain the Poria cocos-derived exosome-like nanoparticle sample.
[0059] Step 4: Observe the morphology of exosomes using transmission electron microscopy (TEM) and detect the particle size and concentration of exosomes using a nano-Kurt particle size analyzer.
[0060] Depend on Figure 13 It can be seen that the exosome-like nanoparticles derived from Poria cocos prepared in this comparative example have a typical hemispherical or saucer-like structure and an ideal particle size, and contain a large number of impurities.
[0061] Depend on Figure 14 It can be seen that the exosome-like nanoparticles derived from Poria cocos prepared in this comparative example have a uniform particle size distribution, an average particle size of 68 nm, and a concentration of 1.94 × 10⁻⁶. 12 pcs / ml Table 1 shows the concentrations of plant-derived exosome-like nanoparticles prepared in Examples 1, 2, 3, 4, and 5.
[0062] Table 1 Concentrations of plant exosomes prepared in different embodiments
[0063] As shown in Table 1, the method of the present invention can be used to prepare plant-derived exosome-like nanoparticles for different plant materials.
[0064] Table 2 shows the concentrations of plant exosomes prepared in Examples 1 and 2, and Comparative Examples 1 and 2.
[0065] Table 2 Concentrations of plant exosomes prepared in different embodiments
[0066] As shown in Table 2, compared with the ultrafiltration method for extracting exosomes, the method of the present invention significantly improves the concentration and greatly enhances the extraction efficiency.
[0067] It is readily understood that those skilled in the art can combine, split, or reorganize the embodiments provided in this application to obtain other embodiments, none of which exceed the protection scope of this application.
[0068] In summary, the above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be covered by the present invention.
Claims
1. A method for extracting plant-derived exosome-like nanoparticles, characterized in that, Includes the following steps: Step S1: Mix the plant sample with pre-cooled PBS buffer and then juice it. Add cellulase and pectinase to obtain a mixture with a concentration of 0.3-1.0%. Let the mixture stand and filter to obtain a crude extract. Step S2: Centrifuge the crude extract obtained in step S1, and collect the supernatant after processing to obtain the refined extract. Step S3: The refined extract obtained in step S2 is subjected to exosome concentration treatment to obtain exosome polymerized concentrate; Step S4: Purify the exosome polymer concentrate obtained in step S3 to obtain plant-derived exosome-like nanoparticles.
2. The method for extracting plant-derived exosome-like nanoparticles according to claim 1, characterized in that, Step S1 specifically includes the following steps: Step S11: Mix the plant sample with PBS buffer pre-cooled to 4°C and then juice it. Step S12: Add cellulase and pectinase to obtain a mixture with a concentration of 0.3-1.0%; Step S13: After standing for 10-18 hours, the crude extract is obtained by filtering through 100-mesh gauze.
3. The method for extracting plant-derived exosome-like nanoparticles according to claim 1, characterized in that, Step S2 centrifugation includes two centrifugation processes: first, low-speed centrifugation is performed at a temperature of 4℃, a centrifugal force of 1000 g, and a centrifugation time of 30 min, and the supernatant is collected; then, the collected supernatant is centrifuged at a temperature of 4℃, a centrifugal force of 10000 g, and a centrifugation time of 60 min, and the supernatant is collected to obtain the refined extract.
4. The method for extracting plant-derived exosome-like nanoparticles according to claim 1, characterized in that, Step S3 specifically includes the following steps: Step S31: Add PEG-8000 at a concentration of 6-10% to the purified extract; Step S32: Let it stand for 8-14 hours at a temperature of 4℃; Step S33: Centrifuge at 4℃, 3000g, and 10-30min, and collect the precipitate after centrifugation. Step S34: The collected precipitate is resuspended in PBS buffer pre-cooled to 4°C to obtain exosome polymer concentrate.
5. The method for extracting plant-derived exosome-like nanoparticles according to claim 4, characterized in that, In step S34, the weight ratio of the precipitate to the PBS buffer is 1:10 to 1:
50.
6. The method for extracting plant-derived exosome-like nanoparticles according to claim 4, characterized in that, Step S4 specifically includes the following steps; Step S41: Place the exosome polymer concentrate in an ultracentrifuge tube, add sucrose solution to the bottom of the tube, and collect the exosome solution suspended in the sucrose layer after processing. Step S42: Mix the collected exosome solution with PBS buffer pre-cooled to 4°C until the density is less than 1.05 g / ml, then centrifuge again and collect the precipitate; Step S43: The precipitate was pre-cooled to 4°C and resuspended in PBS buffer, then filtered to obtain plant-derived exosome-like nanoparticles.
7. The method for extracting plant-derived exosome-like nanoparticles according to claim 6, characterized in that, The centrifugation conditions for steps S41 and S42 are both 4°C, 120000g, and 70min.
8. The method for extracting plant-derived exosome-like nanoparticles according to claim 6, characterized in that, In step S41, the sucrose solution is a sucrose solution with a density of 1.12-1.18 g / ml prepared with PBS.
9. The method for extracting plant-derived exosome-like nanoparticles according to claim 6, characterized in that, In step S43, filtration is performed through a 0.22 μm filter membrane.
10. A plant-derived exosome-like nanoparticle, characterized in that, It is obtained using the extraction method for plant-derived exosome-like nanoparticles as described in any one of claims 1-9.