An extracellular vesicle of a bulbous plant, a preparation method thereof, and an application thereof

Abstract

The present invention provides an extracellular vesicle of a bulbous plant, a preparation method thereof and an application thereof, belonging to the technical field of extracellular vesicle preparation. The preparation method of the extracellular vesicle of the bulbous plant provided by the present invention comprises the following steps: S1, mixing plant tissues with a buffer solution, pulverizing and filtering to obtain a plant stock solution; S2, treating the plant stock solution obtained in step S1 to obtain a plant clear solution; S3, centrifuging the plant clear solution obtained in step S2 at 3-5°C and 110000-130000 g for 80-100 min, retaining the precipitate, resuspending the precipitate with a buffer solution, and repeating 1-2 times to obtain a resuspended solution; S4, filtering the resuspended solution obtained in step S3, and concentrating the filtrate to obtain extracellular vesicles of the plant. This method can efficiently extract extracellular vesicles from viscous fresh lily and lycoris tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of extracellular vesicle preparation, and relates to an extracellular vesicle of a bulbous plant, a preparation method thereof, and an application thereof. Background Art

[0002] Extracellular vesicles are a class of vesicles with a lipid membrane coating, which contain active macromolecules such as proteins, lipids, and nucleic acids, are secreted by cells into the apoplast, and are crucial for cell - to - cell communication. Plant extracellular vesicles are rich in bioactive substances such as nucleic acids and proteins. Compared with animal - derived extracellular vesicles, plant extracellular vesicles are non - toxic, have a high yield, good biocompatibility, and are non - immunogenic because they are derived from edible plants. Research shows that extracellular vesicles extracted from a variety of edible plants have multiple therapeutic effects such as anti - inflammation, anti - cancer, anti - virus, and anti - oxidation, and have the medicinal potential to be developed into pharmaceutical preparations for the efficient treatment of a series of diseases. In addition, bioactive plant extracellular vesicles can also be used as carriers to deliver bio - macromolecules such as nucleic acids, polysaccharides, and proteins, for encapsulating drugs and synergistically enhancing their therapeutic effects.

[0003] In the prior art, plant - derived extracellular vesicles are mostly extracted from tissues such as juicy fruits, flowers, and leaves. Perennial plants with enlarged underground storage organs, represented by bulbous plants such as lilies, lycoris, etc., usually have a high starch content. After cell wall breaking, the juice is less and viscous, making it difficult to obtain extracellular vesicles of bulbous plants through conventional extracellular vesicle extraction methods. Moreover, Chinese herbal medicines such as lilies, lycoris, and polygonatum with enlarged underground storage organs usually need to be air - dried and processed before they can exert their curative effects. However, it is not yet clear whether air - drying and processing will cause changes in the purity, particle size, morphology, and composition of the obtained extracellular vesicles. Therefore, there is an urgent need for a simple and efficient method for preparing high - quality extracellular vesicles for perennial plants with enlarged underground storage organs.

[0004] Chinese invention patent CN114540271A provides a method for purifying plant exosomes, including: collecting a plant cell lysate, centrifuging to collect the supernatant; adding a precipitation solution to the supernatant to obtain a mixed solution; centrifuging the mixed solution, discarding the supernatant, collecting the precipitate, and adding PBS to completely dissolve the precipitate to obtain a dissolved solution; performing gel filtration chromatography separation on the dissolved solution to obtain exosomes. This method realizes the separation and purification of large - scale and high - purity exosomes through the combination of PEG - 8000 precipitation and gel filtration chromatography, and can simultaneously ensure the content, purity, and biological activity of exosomes. The separated exosomes have a high yield and high purity; high efficiency, simple operation, and are easy for large - scale production. However, this technology does not provide a method for extracting extracellular vesicles from bulbous plants with a high starch content, less juice, and high viscosity.

[0005] Chinese Invention Patent CN118424819A provides a plant exosome and an extraction method thereof. After breaking the cell walls of plant stems and leaves, plant exosomes are separated through filtration, low-speed centrifugation, microfiltration membrane filtration, ultracentrifugation, resuspension, ultracentrifugation, resuspension, and microfiltration membrane filtration. Using the extraction method of plant exosomes of this technology to extract Epimedium exosomes, the types of reagents used in the extraction process are single, the number of instruments is small, and Epimedium exosomes with a size of about 130 nm can be obtained without a large amount of cell culture medium. Moreover, the yield and purity of Epimedium exosomes are both relatively high. Approximately 5×10 12 exosomes can be extracted from 150 g of Epimedium stems and leaves, and the particle size distribution is uniform, concentrated at 130 nm, which is beneficial for subsequent production or research. However, this technology does not provide an extraction method for extracellular vesicles in the swollen underground storage organs of plants with high starch content, little juice, and high viscosity.

[0006] Chinese Invention Patent CN118546855A provides a plant exosome and an extraction method thereof. The extraction method of the plant exosome includes: (a) adding a protease inhibitor to the plant tissue homogenate to obtain a crude juice; performing continuous centrifugation on the crude juice and taking the supernatant to obtain a pretreated juice; (b) adding polyethylene glycol and dextran to the pretreated juice, then performing shaking treatment and the first centrifugation, and collecting the lower-layer nanoparticle suspension; (c) subjecting the nanoparticle suspension to size exclusion chromatography and collecting the target product; (d) optionally, mixing polyethylene glycol and the target product, then performing incubation and the second centrifugation, and collecting the precipitate product. The extraction method of the plant exosome has universality, is simple and fast, does not require special equipment, is easy to control, can obtain exosomes with uniform size, the obtained exosomes have a high concentration, low cost, and are applicable to large-scale production. However, this technology does not provide an extraction method for extracellular vesicles in bulbous plants with high starch content, little juice, and high viscosity.

[0007] In summary, the existing technology still cannot provide a more efficient extraction method for extracellular vesicles in the swollen underground organs of plants with high starch content, little juice, and high viscosity. Summary of the Invention

[0008] In view of this, aiming at the problem that the existing technology still cannot provide a more efficient extraction method for extracellular vesicles in the swollen underground organs of plants with high starch content, little juice, and high viscosity, the present invention provides a method for preparing extracellular vesicles of bulbous plants and its application.

[0009] To achieve the above invention object, the present invention provides a method for preparing extracellular vesicles of bulbous plants, including the following steps:

[0010] S1. Mix the bulbous plant tissue with a buffer solution, crush it, and filter it to obtain a plant stock solution;

[0011] S2. Process the plant stock solution obtained in step S1 to obtain a plant clear solution;

[0012] S3. Centrifuge the plant clear solution obtained in step S2 at 3 - 5°C and 110,000 - 130,000 g for 80 - 100 min, retain the precipitate, resuspend the precipitate with a buffer solution, and repeat 1 - 2 times to obtain a resuspended solution;

[0013] S4. Filter the resuspended solution obtained in step S3, concentrate the filtrate to obtain extracellular vesicles of bulbous plants;

[0014] Among them, the bulbous plant tissue is selected from lily or lycoris;

[0015] The treatment in step S2 includes the following steps:

[0016] S201. Centrifuge the plant stock solution obtained in step S1 at 3 - 5°C and 900 - 1100 g for 8 - 12 min to obtain supernatant 1;

[0017] S202. Centrifuge supernatant 1 obtained in step S201 at 3 - 5°C and 2900 - 3100 g for 18 - 22 min to obtain supernatant 2;

[0018] S203. Mix supernatant 2 obtained in step S202 with hemicellulase, pectinase, and cellulase to obtain an enzyme mixture, let it stand at room temperature for digestion for 2 - 3 hours to obtain supernatant 3, and centrifuge at 3 - 5°C and 4900 - 5100 g for 100 - 140 min to obtain supernatant 3;

[0019] S204. Filter supernatant 3 obtained in step S203 with a filter membrane to obtain filtrate 1;

[0020] S205. Centrifuge filtrate 1 obtained in step S204 at 3 - 5°C and 19,000 - 21,000 g for 80 - 100 min to obtain supernatant 4;

[0021] S206. Filter supernatant 4 obtained in step S205 with a filter membrane to obtain a plant clear solution.

[0022] Preferably, in step S1, the buffer solution is phosphate - buffered saline PBS, the pH value of the buffer solution is 7.2 - 7.4, and the mass of the bulbous plant tissue to the volume of the buffer solution is 1 - 4:4, with the unit of g:mL.

[0023] Preferably, in step S1, the temperature of the pulverization is 3 - 5°C, the time of the pulverization is 2 - 5 min, and the filtration is carried out using a gauze filter.

[0024] Preferably, the treatment in step S2 includes the following steps:

[0025] S201. Centrifuge the plant stock solution obtained in step S1 at 4°C and 1000 g for 10 min to obtain supernatant 1;

[0026] S202. Centrifuge supernatant 1 obtained in step S201 at 4°C and 3000 g for 20 min to obtain supernatant 2;

[0027] S203. Mix supernatant 2 obtained in step S202 with hemicellulase, pectinase and cellulase to obtain an enzyme mixture, let it stand at room temperature for digestion for 2 - 3 hours, and centrifuge at 4°C and 5000 g for 120 min to obtain supernatant 3;

[0028] S204. Filter supernatant 3 obtained in step S203 successively through a 0.8 - μm filter membrane and a 0.45 - μm filter membrane to obtain filtrate 1;

[0029] S205. Centrifuge filtrate 1 obtained in step S204 at 4°C and 20000 g for 90 min to obtain supernatant 4;

[0030] S206. Filter supernatant 4 obtained in step S205 through a 0.22 - μm filter membrane to obtain the plant clear solution;

[0031] Among them, in step S203, the mass concentration of the hemicellulase in the enzyme mixture is 0.1% - 0.3%, the mass concentration of the pectinase in the enzyme mixture is 0.05% - 0.15%, and the mass concentration of the cellulase in the enzyme mixture is 0.05% - 0.15%.

[0032] More preferably, in step S203, the mass concentration of the hemicellulase in the enzyme mixture is 0.2%, the mass concentration of the pectinase in the enzyme mixture is 0.1%, and the mass concentration of the cellulase in the enzyme mixture is 0.1%.

[0033] Preferably, in step S3, centrifuge the plant clear solution obtained in step S2 at 4°C and 120000 g for 90 min, retain the precipitate, resuspend the precipitate with phosphate - buffered saline PBS, and repeat 1 - 2 times to obtain a resuspended solution.

[0034] More preferably, the resuspending the precipitate with phosphate - buffered saline PBS is: add phosphate - buffered saline PBS to the precipitate, pipette to resuspend, and then add phosphate - buffered saline PBS in portions and mix for dilution.

[0035] More preferably, and as an example of the present invention, the resuspension of the precipitate with phosphate buffer solution PBS is as follows: Add phosphate buffer solution PBS to the precipitate, pipette to resuspend, and then add phosphate buffer solution PBS in two portions. After each addition of phosphate buffer solution PBS, pipette to mix and dilute.

[0036] Preferably, in step S4, the filtration is performed using a 0.22 μm filter membrane, and the filtrate is concentrated by centrifugation using a 100 kDa ultrafiltration tube at 4°C and 3000 g.

[0037] Preferably, in step S4, the concentration ratio of the filtrate is: every 200 g of fresh plant tissue is concentrated into 0.2 - 0.8 mL of concentrated solution; more preferably, every 200 g of fresh plant tissue is concentrated into 0.5 mL of concentrated solution.

[0038] Preferably, after the concentration of the filtrate in step S4, it further includes column chromatography extraction, and the column chromatography extraction includes the following steps:

[0039] Add the concentrated solution obtained after concentrating the filtrate into a chromatography column; after adding the concentrated solution, add phosphate buffer solution PBS with a volume 3.5 - 4.5 times that of the concentrated solution, collect the outflow liquid, then add phosphate buffer solution PBS with a volume 5 - 6 times that of the concentrated solution, collect the outflow liquid and combine them to obtain extracellular vesicles of bulbous plants.

[0040] More preferably, the column chromatography extraction is performed using an IZON qEV ultra-pure chromatography column on the concentrated solution after concentration. In the column chromatography extraction:

[0041] After adding the concentrated solution, add phosphate buffer solution PBS with a volume 4 times that of the concentrated solution, collect the outflow liquid, then add phosphate buffer solution PBS with a volume 5.6 times that of the concentrated solution, collect the outflow liquid and combine them, and concentrate to obtain exosomes of bulbous plants.

[0042] On the other hand, the present invention provides extracellular vesicles of bulbous plants prepared by the above preparation method.

[0043] On the other hand, the present invention provides the application of the above preparation method in the production of plant extracellular vesicles.

[0044] Preferably, the plant extracellular vesicles are extracellular vesicles of bulbous plants.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The present invention incorporates enzymatic hydrolysis into various methods for extracting extracellular vesicles, obtaining a preferred method for preparing extracellular vesicles derived from bulbous plants, and solving the dilemma that it is difficult to obtain extracellular vesicles from the underground storage organs of bulbous plants due to their high starch content, difficulty in breaking the cell wall to obtain juice, or the juice being too viscous. Moreover, the present invention is also applicable to air-dried plant tissues and can be applied to plant medicinal materials that need to be processed to reduce toxicity.

[0047] (2) When using the method for extracting extracellular vesicles of plants of the present invention to extract extracellular vesicles of bulbous plants, the types of reagents used in the extraction process are few and easy to obtain, and the types of instruments are single. It is possible to obtain extracellular vesicles of Lilium brownii with a size of about 160 nm and extracellular vesicles of Lycoris radiata with a size of about 150 nm without a large amount of cell culture medium.

[0048] (3) The extracellular vesicles obtained by the present invention have high purity and yield. The purity of extracellular vesicles of Lilium brownii can reach 6.05×10 11 Particles / μg, and the purity of extracellular vesicles of Lycoris radiata can reach 2.18×10 11 Particles / μg. Description of the Drawings

[0049] Figure 1 It is a transmission electron microscope result diagram of extracellular vesicles of Lilium brownii obtained in Example 1.

[0050] Figure 2 It is a particle size distribution diagram of extracellular vesicles of Lilium brownii obtained in Example 1.

[0051] Figure 3 It is a transmission electron microscope result diagram of extracellular vesicles of Lycoris radiata obtained in Example 4.

[0052] Figure 4 It is a particle size distribution diagram of extracellular vesicles of Lycoris radiata obtained in Example 4. Detailed Embodiments

[0053] Terms and Statements of the Present Invention:

[0054] 1. Articles "a", "an" and "the": Unless otherwise explicitly limited to one (kind) of object, they include plural objects.

[0055] 2. Numerical ranges: Unless otherwise explicitly indicated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, the stated range or ratio of 1 to 30 should be considered to be included between the minimum value of 1 and the maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio composed of integers or decimals including the endpoints.

[0056] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary illustration of the scope claimed by the present invention. Those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes and modifications should also fall within the scope claimed by the present invention.

[0057] The present invention will be further described below by way of specific examples. All kinds of chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are mass contents. Unless otherwise specified, it is understood to be carried out at room temperature.

[0058] In the following examples, the phosphate buffer solution PBS is an aqueous solution mixture of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, and the pH of the solution is 7.4.

[0059] In the following examples, the ultra-pure chromatography column model is: IZON qEV ultra-pure chromatography column, and the column bed volume is 8.5 mL.

[0060] In the following examples, the specific information of the dried lily used is as follows:

[0061] Product name: Dried Lanzhou Sweet Lily. Dried lilies from different batches and different origins have no obvious effect on the effects of each example.

[0062] Example 1

[0063] A method for preparing lily extracellular vesicles, the steps are as follows.

[0064] S1. Take about 200 g of fresh lily bulbs, place the materials in a laminar flow hood, cut them into pieces, and then wash them successively with distilled water and PBS solution with a pH of 7.4; pour 200 mL of PBS solution at 4 °C and a pH of 7.4 into a juicer, and crush it with 200 g of fresh lily bulbs for 4 min to obtain a juice; filter the juice with sterile gauze to obtain the lily stock solution.

[0065] S201. Centrifuge the lily stock solution obtained in step S1 at 4 °C and 1000 g for 10 minutes, and take the supernatant.

[0066] S202. Centrifuge the supernatant obtained in step S201 at 4 °C and 3000 g for 20 minutes, and take the supernatant (solution).

[0067] S203. Add hemicellulase, pectinase and cellulase to the supernatant (solution) obtained in step S202, such that the mass concentration of hemicellulase in the solution is 0.2%, the mass concentration of pectinase in the solution is 0.1%, and the mass concentration of cellulase in the solution is 0.1%. Let it stand at room temperature for digestion for 2 hours, centrifuge at 4°C and 5000g for 2h, and take the supernatant.

[0068] S204. Filter the supernatant obtained in step S203 successively using 0.8μm and 0.45μm filter membranes to obtain a filtrate.

[0069] S205. Centrifuge the filtrate obtained in step S204 at 4°C and 20000g for 1.5h, and take the supernatant.

[0070] S206. Filter the supernatant obtained in step S205 using a 0.22μm filter membrane to obtain a plant clear solution.

[0071] S3. Take the plant clear solution obtained in step S206, centrifuge at 120000g for 90 min at 4°C, discard the supernatant and retain the precipitate. Add 1 mL of PBS solution, gently pipette to resuspend the precipitate, then add 1 mL of PBS solution, gently pipette to mix evenly, and then add 1 mL of PBS solution, gently pipette to mix evenly to obtain a resuspended solution (about 3 mL in total). Repeat the above process once, that is, take the obtained resuspended solution, centrifuge at 120000g for 90 min at 4°C, discard the supernatant, retain the precipitate, add 1 mL of PBS solution, gently pipette to resuspend the precipitate, then add 1 mL of PBS solution, gently pipette to mix evenly, and then add 1 mL of PBS solution, gently pipette to mix evenly to obtain a secondary resuspended solution (about 3 mL in total).

[0072] S4. Filter the secondary resuspended solution obtained in step S3 using a 0.22μm filter membrane to obtain a filtrate, and concentrate it to 0.5 mL by centrifugation at 4°C and 3000g using a 100kDa ultrafiltration tube. According to particle size and morphology analysis, it is the extracellular vesicle.

[0073] Example 2

[0074] A method for preparing lily extracellular vesicles is as follows.

[0075] S1. Take 50 g of air-dried lily bulbs, place the material in a laminar flow hood, cut it into pieces and wash it successively with distilled water and PBS solution with a pH of 7.4; pour 200 mL of PBS solution at 4°C and a pH of 7.4 into a juicer, and juice with 50 g of air-dried lily bulbs for 4 min to obtain a juice; filter the juice using sterile gauze to obtain a lily bulb stock solution.

[0076] S201. Centrifuge the dried lily bulb stock solution obtained in step S1 at 4°C and 1000 g for 10 minutes, and take the supernatant.

[0077] S202. Centrifuge the supernatant obtained in step S201 at 4°C and 3000 g for 20 minutes, and take the supernatant (solution).

[0078] S203. Add hemicellulase, pectinase, and cellulase to the supernatant (solution) obtained in step S202, such that the mass concentration of hemicellulase in the solution is 0.2%, the mass concentration of pectinase in the solution is 0.1%, and the mass concentration of cellulase in the solution is 0.1%. Let it stand at room temperature for digestion for 3 hours, centrifuge at 4°C and 5000 g for 2 h, and take the supernatant.

[0079] S204. Filter the supernatant obtained in step S203 successively with 0.8 μm and 0.45 μm filter membranes to obtain a filtrate.

[0080] S205. Centrifuge the filtrate obtained in step S204 at 4°C and 20000 g for 1.5 h, and take the supernatant.

[0081] S206. Filter the supernatant obtained in step S205 with a 0.22 μm filter membrane to obtain a plant clear solution.

[0082] S3. Take the plant clear solution obtained in step S206, centrifuge at 120000 g for 90 min at 4°C, discard the supernatant and retain the precipitate. Add 1 mL of PBS solution, gently pipette to resuspend the precipitate, then add 1 mL of PBS solution, gently pipette to mix evenly, and then add 1 mL of PBS solution, gently pipette to mix evenly to obtain a resuspended solution (about 3 mL in total). Repeat the above process once, that is, take the obtained resuspended solution, centrifuge at 120000 g for 90 min at 4°C, discard the supernatant, retain the precipitate, add 1 mL of PBS solution, gently pipette to resuspend the precipitate, then add 1 mL of PBS solution, gently pipette to mix evenly, and then add 1 mL of PBS solution, gently pipette to mix evenly to obtain a secondary resuspended solution (about 3 mL in total).

[0083] S4. Filter the secondary resuspended solution obtained in step S3 with a 0.22 μm filter membrane to obtain a filtered solution, and concentrate it to 0.5 mL by centrifugation at 4°C and 3000 g using a 100 kDa ultrafiltration tube. According to particle size and morphology analysis, it is the extracellular vesicles.

[0084] Example 3

[0085] A method for preparing lily extracellular vesicles, the steps are as follows.

[0086] S1. Take about 50 g of air-dried dried lily bulbs. Place the materials in a laminar flow hood, cut them into pieces, and then wash them successively with distilled water and PBS solution with a pH of 7.4. Pour 200 mL of PBS solution at 4 °C with a pH of 7.4 into a juicer, and juice with 50 g of air-dried dried lily bulbs for 4 min to obtain a juice extract. Filter the juice extract with sterile gauze to obtain the original lily bulb solution.

[0087] S201. Centrifuge the original lily bulb solution obtained in step S1 at 4 °C and 1000 g for 10 minutes, and take the supernatant.

[0088] S202. Centrifuge the supernatant obtained in step S201 at 4 °C and 3000 g for 20 minutes, and take the supernatant (solution).

[0089] S203. Add hemicellulase, pectinase and cellulase to the supernatant (solution) obtained in step S202, so that the mass concentration of hemicellulase in the solution is 0.2%, the mass concentration of pectinase in the solution is 0.1%, and the mass concentration of cellulase in the solution is 0.1%. Let it stand at room temperature for digestion for 2.5 hours, centrifuge at 4 °C and 5000 g for 2 h, and take the supernatant.

[0090] S204. Filter the supernatant obtained in step S203 successively with 0.8 μm and 0.45 μm filter membranes to obtain a filtrate.

[0091] S205. Centrifuge the filtrate obtained in step S204 at 4 °C and 20000 g for 1.5 h, and take the supernatant.

[0092] S206. Filter the supernatant obtained in step S205 with a 0.22 μm filter membrane to obtain a plant clear solution.

[0093] S3. Take the plant clear solution obtained in step S206, centrifuge at 120000 g for 90 min at 5 °C, discard the supernatant and retain the precipitate. Add 1 mL of PBS solution, gently pipette to resuspend the precipitate, then add 1 mL of PBS solution, gently pipette and mix evenly, and then add 1 mL of PBS solution, gently pipette and mix evenly to obtain a resuspended solution (about 3 mL in total).

[0094] S4. Filter the resuspended solution obtained in step S3 with a 0.22 μm filter membrane. Concentrate the obtained filtrate to 0.5 mL by centrifugation at 4 °C and 3000 g using a 100 kDa ultrafiltration tube, and then add it to an ultra-pure chromatography column. After all the samples enter the chromatography column, immediately add 2 mL of PBS and collect 2.5 mL of the void volume. After the liquid has flowed out, continue to add 2.8 mL of PBS and collect the liquid of the 8th - 14th fractions (i.e., the eluate), with a total target fraction volume of 2.8 mL, and combine them. Concentrate the combined liquid with a 100 kDa ultrafiltration tube. According to particle size and morphology analysis, it is the extracellular vesicles.

[0095] Example 4

[0096] It is different from Example 1 in that in step S1, 200 g of fresh lily bulbs are replaced with 100 g of fresh lycoris radiata bulbs, and the rest are the same.

[0097] Example 5

[0098] It is different from Example 3 in that in step S1, 50 g of air-dried lilies are replaced with 100 g of fresh lycoris radiata bulbs, and the rest of the steps are the same as those in Example 3.

[0099] Comparative Example 1

[0100] Ultracentrifugation (UC) was used to extract extracellular vesicles from lily.

[0101] Compared with Example 1, the difference is that in step S203, the step of adding enzyme solution and standing for digestion is omitted, and the supernatant (solution) obtained in step S202 is directly centrifuged at 4 °C and 5000 g for 2 h, and then the supernatant is taken, and the rest of the steps are the same as those in Example 1.

[0102] Comparative Example 2

[0103] Ultrafiltration size exclusion chromatography (SEC) was used to purify extracellular vesicles from lily.

[0104] Compared with Example 1, the difference is that in step S203, the step of adding enzyme solution and standing for digestion is omitted, and the supernatant (solution) obtained in step S202 is directly centrifuged at 4 °C and 5000 g for 2 h, and then the supernatant is taken; at the same time, steps S3 and S4 are replaced with the following steps.

[0105] The plant clear liquid obtained in step S206 was concentrated to 0.5 mL by centrifugation at 4 °C and 3000 g using a 100 kDa ultrafiltration tube, and then the concentrated liquid was purified using an IZON qEV ultrafiltration chromatography column with a column bed volume of 8.5 mL. 0.5 mL of the extracellular vesicle concentrated liquid was added to the ultrafiltration chromatography column. After all the samples entered the chromatography column, 2 mL of PBS was immediately added, and the 2.5 mL void volume was collected. After the liquid flowed out, 2.8 mL of PBS was continuously added, and the liquid in the 8th - 14th fractions (i.e., the eluate) was collected, with a total target fraction volume of 2.8 mL, and they were combined. The combined liquid was concentrated using a 100 kDa ultrafiltration tube. According to the particle size and morphology analysis, combined with the protein molecular weight and lipid composition judgment, it was the extracellular vesicle.

[0106] The rest of the steps are the same as those in Example 1.

[0107] Comparative Example 3

[0108] Comparative Example 3 used ultracentrifugation combined with ultra-pure size exclusion chromatography column purification method (UC, SEC) to extract extracellular vesicles from lilies. The difference from Example 1 was that:

[0109] The step of adding the enzyme digestion solution and standing for digestion in step S203 was omitted, and the supernatant obtained in step S202 was directly centrifuged at 4°C and 5000g for 2h, and then the supernatant was taken. Steps S3 and S4 were replaced with the following steps:

[0110] Take the plant clear liquid obtained in step S206, centrifuge at 120000g for 90min at 4°C, discard the supernatant and retain the precipitate, add 1 mL of PBS solution, gently pipette to resuspend the precipitate, then add 1 mL of PBS solution, gently pipette and mix evenly, and then add 1 mL of PBS solution, gently pipette and mix evenly to obtain a resuspended liquid (about 3 mL in total). Filter the obtained resuspended liquid through a 0.22 μm filter membrane, and concentrate the filtrate to 0.5 mL by centrifugation at 4°C and 3000g using a 100 kDa ultrafiltration tube, and then add it to the ultra-pure chromatography column. After all the samples enter the chromatography column, immediately add 2 mL of PBS, and collect 2.5 mL of the void volume. After the liquid has flowed out, continue to add 2.8 mL of PBS, and collect the liquid of the 8th - 14th fractions (i.e., the eluate), with a total target fraction volume of 2.8 mL, and combine them. Concentrate the combined liquid using a 100 kDa ultrafiltration tube. According to the particle size and morphology analysis, combined with the judgment of protein molecular weight and lipid composition, it is the extracellular vesicle.

[0111] All other steps were the same as those in Example 1.

[0112] Comparative Example 4

[0113] Comparative Example 4 used the general ultracentrifugation method (UC) to extract extracellular vesicles from Lycoris radiata. The difference from Example 4 was that:

[0114] The step of adding the enzyme digestion solution and standing for digestion in step S203 was omitted, and the supernatant (solution) obtained in step S202 was directly centrifuged at 4°C and 5000g for 2h, and then the supernatant was taken. All other steps were the same as those in Example 4.

[0115] Effect Example

[0116] 1. Transmission electron microscope (TEM) characterization

[0117] Use a pipette to separately aspirate 10 μL of the extracellular vesicles from lilies that have been separated and purified in each example and comparative example, drop them on the copper grid and precipitate for 1 min, and use filter paper to absorb the floating liquid. Drop 10 μL of uranyl acetate on the copper grid and precipitate for 1 min, and use filter paper to absorb the floating liquid. After drying at room temperature for 5 minutes, perform electron microscope detection and imaging at 80 kV to obtain the transmission electron microscope imaging results. Among them, the TEM photo of the extracellular vesicles from lilies prepared in Example 1 is Figure 1, the TEM image of the extracellular vesicles of Lycoris radiata prepared in Example 4 is Figure 3 .

[0118] As can be seen from the figure, the obtained extracellular vesicles of Lilium brownii are uniform in shape and size under the electron microscope, showing a cup-shaped vesicle-like structure.

[0119] 2. Particle size distribution and concentration determination

[0120] Respectively, 1 μL of the isolated and purified plant extracellular vesicles in each example and each comparative example was taken and diluted with 999 μL of 1×PBS. Using the PARTICLE METRIX PMX120 instrument, a new syringe was used to aspirate the diluted plant extracellular vesicle sample for particle size distribution and concentration detection. The particle size distribution curve of the extracellular vesicles of Lilium brownii prepared in Example 1 is Figure 2 , and the particle size distribution curve of the extracellular vesicles of Lycoris radiata prepared in Example 4 is Figure 4 . In Comparative Example 4, no extracellular vesicles were captured by transmission electron microscopy (TEM) characterization, so particle size distribution and concentration determination were not performed.

[0121] 3. Protein concentration determination

[0122] The Thermo Pierce BCA Protein Assay Kit (product model: 2327) was used to detect the protein concentration in the obtained plant extracellular vesicles. The purity of the extracellular vesicles extracted in the examples can be analyzed by combining the particle concentration. In Comparative Example 4, no extracellular vesicles were captured by transmission electron microscopy (TEM) characterization, so the total protein concentration determination was not performed.

[0123] The average particle size, particle concentration, total protein concentration, and purity of the extracellular vesicles obtained in each example and comparative example are shown in Table 1.

[0124] Table 1

[0125]

[0126] As can be seen from the table, the size of the fresh Lilium brownii extracellular vesicles obtained by enzymatic hydrolysis in Example 1 is about 160 nm, and the size of the air-dried Lilium brownii stem cell extracellular vesicles obtained by enzymatic hydrolysis in Examples 2 and 3 is about 180 nm. For the extracellular vesicles of fresh Lilium brownii obtained by the general extracellular vesicle extraction method without enzymatic hydrolysis in Comparative Examples 1-3, the size is significantly larger, about 240 nm. The size of the fresh Lycoris radiata extracellular vesicles obtained by enzymatic hydrolysis in Example 4 is about 150 nm. During the experiment of Comparative Example 4, the extract of Lycoris radiata bulbs was too viscous, and the general ultracentrifugation method without enzymatic hydrolysis was used to extract extracellular vesicles. Finally, no extracellular vesicles were observed in the extract.

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing extracellular vesicles of bulbous plants, characterized in that, It consists of the following steps: S1. Mix the bulbous plant tissue with a buffer solution, crush it, and filter it to obtain the plant stock solution; S2. Treat the plant stock solution obtained in step S1 to obtain the plant clear solution; S3. Centrifuge the plant clear solution obtained in step S2 at 3 - 5°C and 110000 - 130000 g for 80 - 100 min, retain the precipitate, resuspend the precipitate with the buffer solution, and repeat 1 - 2 times to obtain the resuspended solution; S4. Filter the resuspended solution obtained in step S3, and concentrate the filtrate to obtain the extracellular vesicles of the bulbous plant; Among them, the bulbous plant tissue is dried lily; The treatment in step S2 consists of the following steps: S201. Centrifuge the plant stock solution obtained in step S1 at 4°C and 1000 g for 10 min to obtain the supernatant 1; S202. Centrifuge the supernatant 1 obtained in step S201 at 4°C and 3000 g for 20 min to obtain the supernatant 2; S203. Mix the supernatant 2 obtained in step S202 with hemicellulase, pectinase, and cellulase to obtain an enzyme mixture, let it stand and digest at room temperature for 2 - 3 hours, and centrifuge at 4°C and 5000 g for 100 - 140 min to obtain the supernatant 3; S204. Filter the supernatant 3 obtained in step S203 successively with a 0.8 μm filter membrane and a 0.45 μm filter membrane to obtain the filtrate 1; S205. Centrifuge the filtrate 1 obtained in step S204 at 4°C and 20000 g for 90 min to obtain the supernatant 4; S206. Filter the supernatant 4 obtained in step S205 with a 0.22 μm filter membrane to obtain the plant clear solution; Among them, in step S203, the mass concentration of the hemicellulase in the enzyme mixture is 0.1% - 0.3%, the mass concentration of the pectinase in the enzyme mixture is 0.05% - 0.15%, and the mass concentration of the cellulase in the enzyme mixture is 0.05% - 0.15%.

2. The preparation method according to claim 1, wherein In step S1, the buffer solution is phosphate buffer solution PBS, the pH value of the buffer solution is 7.2 - 7.4, and the mass of the bulbous plant tissue to the volume of the buffer solution is 1 - 4:4, with the unit of g:mL.

3. The preparation method according to claim 1, wherein In step S1, the temperature of the crushing is 3 - 5°C, the time of the crushing is 2 - 5 min, and the filtration is carried out using a gauze filter.

4. The preparation method according to claim 1, characterized in that, In step S203, the mass concentration of the hemicellulase in the enzyme mixture is 0.2%, the mass concentration of the pectinase in the enzyme mixture is 0.1%, and the mass concentration of the cellulase in the enzyme mixture is 0.1%.

5. The preparation method according to claim 1, characterized in that, In step S3, centrifuge the plant clear solution obtained in step S2 at 4°C and 120000 g for 90 min, retain the precipitate, resuspend the precipitate with phosphate buffer solution PBS, and repeat 1 - 2 times to obtain the resuspended solution.

6. The preparation method according to claim 1, characterized in that, In step S4, the filtration is carried out using a 0.22 μm filter membrane, and the filtrate concentration is carried out by centrifugal concentration using a 100 kDa ultrafiltration tube at 4°C and 3000 g.

7. The preparation method according to claim 1, wherein In step S4, the concentration ratio of the filtrate is: every 200 g of fresh plant tissue is concentrated into 0.2 - 0.8 mL of concentrated solution.

8. Extracellular vesicles of bulbous plants prepared by the preparation method according to any one of claims 1-7.

9. Use of the preparation method according to any one of claims 1-7 in the production of plant extracellular vesicles.

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