Bulbophyte extracellular vesicle as well as preparation method and application thereof

Abstract

The invention provides a bulb plant extracellular vesicle as well as a preparation method and application thereof, and belongs to the technical field of preparation of extracellular vesicles. The preparation method of the bulb plant extracellular vesicles provided by the invention comprises the following steps: S1, mixing plant tissues with a buffer solution, crushing, and filtering to obtain a plant stock solution; s2, treating the plant stock solution obtained in the step S1 to obtain a plant clear solution; s3, centrifuging the plant clear liquid obtained in the step S2 for 80-100 minutes under the conditions that the temperature is 3-5 DEG C and the weight is 110000-130000 g, retaining the precipitate, re-suspending the precipitate by using a buffer solution, and repeating for 1-2 times to obtain a re-suspension liquid; and S4, filtering the resuspension obtained in the step S3, and concentrating the filtrate to obtain the plant extracellular vesicles. The method can be used for efficiently extracting the extracellular vesicles from thick fresh lily and lycoris radiata tissues.

Description

Technical Field

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

[0002] Extracellular vesicles are a type of vesicles coated with a lipid membrane, which contains active macromolecules such as proteins, lipids, and nucleic acids. They are secreted from cells to the apoplast and are essential for intercellular communication. Plant extracellular vesicles are rich in biologically active substances such as nucleic acids and proteins. Compared with animal-derived extracellular vesicles, plant extracellular vesicles are derived from edible plants, so they are not only non-toxic, but also have high yields, good biocompatibility, and are non-immunogenic. Studies have shown that extracellular vesicles extracted from a variety of edible plants have multiple therapeutic effects such as anti-inflammatory, anti-cancer, anti-viral, and antioxidant, and have the potential to be developed into pharmaceutical preparations and effectively treat a series of diseases. In addition, biologically active plant extracellular vesicles can also be used as carriers to deliver biological macromolecules such as nucleic acids, polysaccharides, and proteins, which can be used to encapsulate drugs and synergistically enhance their therapeutic effects.

[0003] In the prior art, plant-derived extracellular vesicles are mostly extracted from juice-rich fruits or flowers, leaves and other tissues. Perennial plants with enlarged underground storage organs, represented by bulbous plants such as lily and Lycoris, usually have high starch content, and the juice is less and viscous after breaking the wall, making it difficult to obtain bulbous plant extracellular vesicles by conventional extracellular vesicle extraction methods. Moreover, Chinese herbal medicines with enlarged underground storage organs such as lily, Lycoris, and Polygonatum usually need to be air-dried and processed before they can exert their therapeutic effects, but whether air-drying and processing will cause changes in the purity, particle size, morphology and composition of the obtained extracellular vesicles is currently unclear. 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 plant cell disruption liquid, centrifuging and collecting supernatant; adding precipitation liquid to the supernatant to obtain a mixed solution; centrifuging the mixed solution, discarding the supernatant, collecting the precipitate, adding PBS to completely dissolve the precipitate to obtain a dissolved solution; subjecting the dissolved solution to gel filtration chromatography separation to obtain exosomes. This method combines PEG-8000 precipitation with gel filtration chromatography to achieve large-scale and high-purity separation and purification of exosomes, which can simultaneously ensure the content, purity, and biological activity of exosomes. The isolated exosomes have high yield and purity; high efficiency, simple operation, and easy large-scale production. However, this technology does not provide a method for extracting extracellular vesicles in bulbous plants with high starch content and low and viscous juice.

[0005] Chinese invention patent CN118424819A provides a plant exosome and its extraction method, wherein the plant stems and leaves are broken and then filtered, centrifuged at low speed, filtered through a microporous membrane, filtered through ultracentrifuged, resuspended, filtered through ultracentrifuged, resuspended, and filtered through a microporous membrane to separate the plant exosomes. This plant exosome extraction method is used to extract epimedium exosomes. The extraction process uses a single type of reagents and a small number of instruments. Epimedium exosomes with a size of about 130nm can be obtained without a large amount of cell culture fluid. In addition, the yield and purity of epimedium exosomes are high. About 5×10 epimedium exosomes can be extracted from 150g of epimedium stems and leaves. 12 The technology does not provide a method for extracting extracellular vesicles from plant underground storage organs with high starch content and low and viscous juice.

[0006] Chinese invention patent CN118546855A provides a plant exosome and an extraction method thereof. The plant exosome extraction method comprises: (a) adding a protease inhibitor to a plant tissue homogenate to obtain a primary juice; subjecting the primary juice to continuous centrifugation, taking the supernatant, and obtaining a pretreated juice; (b) adding polyethylene glycol and dextran to the pretreated juice, shaking and centrifuging for the first time, and collecting the nanoparticle suspension in the lower layer; (c) subjecting the nanoparticle suspension to size exclusion chromatography to collect the target product; (d) optionally, mixing polyethylene glycol and the target product, incubating and centrifuging for the second time, and collecting the precipitated product. The plant exosome extraction method is universal, simple and fast, does not require special equipment, is easy to control, can obtain exosomes of uniform size, and the obtained exosomes have a high concentration, low cost, and are suitable for large-scale production. However, the technology does not provide a method for extracting extracellular vesicles in bulbous plants with high starch content, small amount of viscous juice.

[0007] In summary, the existing technology cannot provide a more efficient method for extracting extracellular vesicles from underground swelling organs of plants with high starch content and low and viscous juice. Summary of the invention

[0008] In view of this, in order to address the problem that the prior art cannot provide a more efficient method for extracting extracellular vesicles from underground swelling organs of plants with high starch content and low and viscous juice, the present invention provides a bulbous plant extracellular vesicle, a preparation method and application thereof.

[0009] To achieve the above-mentioned object of the invention, the present invention provides a method for preparing extracellular vesicles of bulbous plants, comprising the following steps: S1, mixing the bulbous plant tissue with the buffer solution, crushing, filtering, and obtaining the 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 110000-130000g for 80-100 min, retaining the precipitate, resuspending the precipitate with a buffer solution, repeating 1-2 times to obtain a resuspended solution; S4, filtering the resuspension obtained in step S3, and concentrating the filtrate to obtain extracellular vesicles of bulbous plants; Wherein, the bulbous plant tissue is selected from lily or Lycoris radiata; The process in step S2 includes the following steps: S201, centrifuging the plant stock solution obtained in step S1 at 3-5°C and 900-1100g for 8-12 min to obtain supernatant 1; S202, centrifuging the supernatant 1 obtained in step S201 at 3-5°C and 2900-3100g for 18-22 min to obtain supernatant 2; S203, mixing the supernatant 2 obtained in step S202 with hemicellulase, pectinase and cellulase to obtain an enzyme mixture, digesting at room temperature for 2-3 hours to obtain supernatant 3, and centrifuging at 3-5°C and 4900-5100g for 100-140min to obtain supernatant 3; S204, filtering the supernatant 3 obtained in step S203 using a filter membrane to obtain a filtrate 1; S205, centrifuging the filtrate 1 obtained in step S204 at 3-5°C and 19000-21000g for 80-100 min to obtain a supernatant 4; S206, filtering the supernatant 4 obtained in step S205 using a filter membrane to obtain a plant clear solution.

[0010] Preferably, in step S1, the buffer solution is a phosphate buffer solution (PBS), the pH value of the buffer solution is 7.2-7.4, and the mass of the bulbous plant tissue and the volume of the buffer solution are 1-4:4, in units of g:mL.

[0011] Preferably, in step S1, the pulverizing temperature is 3-5°C, the pulverizing time is 2-5 min, and the filtration is performed by filtering with gauze.

[0012] Preferably, the processing in step S2 comprises the following steps: S201, centrifuging the plant stock solution obtained in step S1 at 4°C and 1000 g for 10 min to obtain supernatant 1; S202, centrifuging the supernatant 1 obtained in step S201 at 4°C and 3000g for 20 min to obtain supernatant 2; S203, mixing the supernatant 2 obtained in step S202 with hemicellulase, pectinase and cellulase to obtain an enzyme mixture, digesting at room temperature for 2-3 hours, and centrifuging at 4°C and 5000g for 120 minutes to obtain supernatant 3; S204, filtering the supernatant 3 obtained in step S203 using a 0.8 μm filter membrane and a 0.45 μm filter membrane in sequence to obtain a filtrate 1; S205, centrifuging the filtrate 1 obtained in step S204 at 4°C and 20000g for 90 min to obtain supernatant 4; S206, filtering the supernatant 4 obtained in step S205 using a 0.22 μm filter membrane to obtain a plant clear solution; Wherein, 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%.

[0013] 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%.

[0014] Preferably, in step S3, the plant clear solution obtained in step S2 is centrifuged at 4°C and 120,000 g for 90 min, the precipitate is retained, and the precipitate is resuspended using phosphate buffered saline (PBS), and the process is repeated 1-2 times to obtain a resuspended solution.

[0015] More preferably, the resuspending the precipitate with phosphate buffer solution (PBS) comprises: adding phosphate buffer solution (PBS) to the precipitate, resuspending by pipetting, and then adding phosphate buffer solution (PBS) in portions, mixing and diluting.

[0016] More preferably, and as an example of the present invention, the precipitate is resuspended using phosphate buffer solution (PBS) as follows: add phosphate buffer solution (PBS) to the precipitate, resuspend by pipetting, and then add phosphate buffer solution (PBS) twice, pipetting after each addition of phosphate buffer solution (PBS) to mix and dilute.

[0017] Preferably, in step S4, the filtration is performed by using a 0.22 μm filter membrane, and the filtrate is concentrated by using a 100 kDa ultrafiltration tube for centrifugation at 4° C. and 3000 g.

[0018] Preferably, in step S4, the filtrate is concentrated at a ratio of 0.2-0.8 mL of concentrated solution per 200 g of fresh plant tissue; more preferably, 0.5 mL of concentrated solution per 200 g of fresh plant tissue.

[0019] Preferably, in step S4, after the filtrate is concentrated, column chromatography extraction is further included, and the column chromatography extraction includes the following steps: The concentrated solution obtained by concentrating the filtrate is added to the chromatographic column; after adding the concentrated solution, a phosphate buffer solution PBS with a volume of 3.5-4.5 times the concentrated solution is added, the outflowing liquid is collected, and then a phosphate buffer solution PBS with a volume of 5-6 times the concentrated solution is added, the outflowing liquid is collected and combined to obtain the extracellular vesicles of bulbous plants.

[0020] More preferably, the column chromatography extraction is to use an IZON qEV ultrapure chromatography column to perform column chromatography extraction on the concentrated concentrate, wherein: After adding the concentrate, add phosphate buffer solution PBS with a volume 4 times that of the concentrate, collect the outflowing liquid, then add phosphate buffer solution PBS with a volume 5.6 times that of the concentrate, collect the outflowing liquid, combine and concentrate, and obtain bulbous plant exosomes.

[0021] On the other hand, the present invention provides bulbous plant extracellular vesicles prepared by the above preparation method.

[0022] In another aspect, the present invention provides application of the above preparation method in producing plant extracellular vesicles.

[0023] Preferably, the plant extracellular vesicles are bulbous plant extracellular vesicles.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention incorporates enzymatic hydrolysis into a variety of extracellular vesicle extraction methods, and obtains a preferred method for preparing extracellular vesicles from bulbous plants, which solves the problem that it is difficult to obtain extracellular vesicles from underground storage organs of bulbous plants due to high starch content, difficulty in obtaining juice by breaking the wall, or excessive viscosity of the juice. The present invention is also applicable to air-dried plant tissues and can be applied to plant medicinal materials that need to be processed and detoxified.

[0025] (2) The plant extracellular vesicle extraction method of the present invention is used to extract the extracellular vesicles of bulbous plants. The extraction process uses a small number of reagents that are easy to obtain and a single type of instrument. Without a large amount of cell culture fluid, lily extracellular vesicles with a size of about 160 nm and Lycoris radiata extracellular vesicles with a size of about 150 nm can be obtained.

[0026] (3) The extracellular vesicles obtained by the present invention have high purity and yield. The purity of lily extracellular vesicles can reach 6.05×10 11 Particles / μg, the purity of Lycoris radiata extracellular vesicles can reach 2.18×10 11 Particles / μg. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the transmission electron microscopy result of the lily extracellular vesicles obtained in Example 1.

[0028] Figure 2 This is the particle size distribution diagram of the lily extracellular vesicles obtained in Example 1.

[0029] Figure 3 This is the transmission electron microscopy result of Lycoris radiata extracellular vesicles obtained in Example 4.

[0030] Figure 4 This is the particle size distribution diagram of Lycoris radiata extracellular vesicles obtained in Example 4. DETAILED DESCRIPTION

[0031] Terms and Claims of the Present Invention: 1. The articles “a”, “an” and “the” include plural referents unless expressly limited to one or more referents otherwise.

[0032] 2. Numerical range: Unless otherwise expressly stated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, a range or ratio of 1 to 30 stated should be considered to be included between a minimum value of 1 and a maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio consisting of integers or decimals including the end points.

[0033] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0034] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0035] In the following examples, the phosphate buffer solution PBS is a mixed aqueous solution of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, and the pH of the solution is 7.4.

[0036] In the following examples, the ultrapure chromatographic column model used is: IZON qEV ultrapure chromatographic column, and the column bed volume is 8.5 mL.

[0037] In the following examples, the specific information of the dried lily used is as follows: Product name: Lanzhou sweet lily dried. Dried lilies from different batches and different origins have no significant effect on the effects of each embodiment.

[0038] Example 1 A method for preparing lily extracellular vesicles comprises the following steps.

[0039] S1. Take about 200 g of fresh lily bulbs, place the material in a clean bench, cut into pieces, and wash with distilled water and PBS solution with a pH of 7.4 in turn; 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 minutes to obtain juice; filter the juice with sterile gauze to obtain lily stock solution.

[0040] S201. Centrifuge the lily stock solution obtained in step S1 at 4°C and 1000 g for 10 minutes and collect the supernatant.

[0041] S202. Centrifuge the supernatant obtained in step S201 at 4°C and 3000 g for 20 minutes and take the supernatant (solution).

[0042] 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%. Digest at room temperature for 2 hours, centrifuge at 4°C and 5000g for 2 hours, and take the supernatant.

[0043] S204, filtering the supernatant obtained in step S203 using 0.8 μm and 0.45 μm filter membranes in turn to obtain a filtrate.

[0044] S205. Centrifuge the filtrate obtained in step S204 at 4°C and 20,000 g for 1.5 h, and take the supernatant.

[0045] S206. Filter the supernatant obtained in step S205 using a 0.22 μm filter membrane to obtain a plant clear solution.

[0046] S3. Take the plant clear solution obtained in step S206, centrifuge at 120000g for 90min at 4°C, remove the supernatant and retain the precipitate, add 1mL PBS solution, gently blow to resuspend the precipitate, then add 1mL PBS solution, gently blow to mix evenly, then add 1mL PBS solution, gently blow to mix evenly, and obtain a resuspension (about 3mL in total). Repeat the above process once, that is, take the resuspension obtained, centrifuge at 120000g for 90min at 4°C, remove the supernatant, retain the precipitate, add 1mL PBS solution, gently blow to resuspend the precipitate, then add 1mL PBS solution, gently blow to mix evenly, then add 1mL PBS solution, gently blow to mix evenly, and obtain a secondary resuspension (about 3mL in total).

[0047] S4. The secondary resuspension obtained in step S3 is filtered using a 0.22 μm filter membrane to obtain a filtrate, which is concentrated 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, the filtrate is extracellular vesicles.

[0048] Example 2 A method for preparing lily extracellular vesicles comprises the following steps.

[0049] S1. Take 50 g of air-dried lily bulbs, place the material in a clean bench, cut into pieces, and wash with distilled water and PBS solution with a pH of 7.4 in turn; pour 200 mL of PBS solution at 4°C and a pH of 7.4 into a juicer, and squeeze juice with 50 g of air-dried lily bulbs for 4 minutes to obtain juice; filter the juice with sterile gauze to obtain lily bulb stock solution.

[0050] S201. Centrifuge the dried lily stock solution obtained in step S1 at 4°C and 1000 g for 10 minutes and collect the supernatant.

[0051] S202. Centrifuge the supernatant obtained in step S201 at 4°C and 3000 g for 20 minutes and take the supernatant (solution).

[0052] 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%. Digest at room temperature for 3 hours, centrifuge at 4°C and 5000g for 2 hours, and take the supernatant.

[0053] S204, filtering the supernatant obtained in step S203 using 0.8 μm and 0.45 μm filter membranes in turn to obtain a filtrate.

[0054] S205. Centrifuge the filtrate obtained in step S204 at 4°C and 20,000 g for 1.5 h, and take the supernatant.

[0055] S206. Filter the supernatant obtained in step S205 using a 0.22 μm filter membrane to obtain a plant clear solution.

[0056] S3. Take the plant clear solution obtained in step S206, centrifuge at 120000g for 90min at 4°C, remove the supernatant and retain the precipitate, add 1mL PBS solution, gently blow to resuspend the precipitate, then add 1mL PBS solution, gently blow to mix evenly, then add 1mL PBS solution, gently blow to mix evenly, and obtain a resuspension (about 3mL in total). Repeat the above process once, that is, take the resuspension obtained, centrifuge at 120000g for 90min at 4°C, remove the supernatant, retain the precipitate, add 1mL PBS solution, gently blow to resuspend the precipitate, then add 1mL PBS solution, gently blow to mix evenly, then add 1mL PBS solution, gently blow to mix evenly, and obtain a secondary resuspension (about 3mL in total).

[0057] S4. The secondary resuspension obtained in step S3 is filtered using a 0.22 μm filter membrane to obtain a filtrate, which is concentrated 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, the filtrate is extracellular vesicles.

[0058] Example 3 A method for preparing lily extracellular vesicles comprises the following steps.

[0059] S1. Take about 50 g of air-dried lily bulbs, place the material in a clean bench, cut into pieces, and wash with distilled water and PBS solution with a pH of 7.4 in turn; pour 200 mL of PBS solution at 4°C and a pH of 7.4 into a juicer, and squeeze juice with 50 g of air-dried lily bulbs for 4 minutes to obtain juice; filter the juice with sterile gauze to obtain lily bulb stock solution.

[0060] S201. Centrifuge the dried lily stock solution obtained in step S1 at 4°C and 1000 g for 10 minutes and collect the supernatant.

[0061] S202. Centrifuge the supernatant obtained in step S201 at 4°C and 3000 g for 20 minutes and take the supernatant (solution).

[0062] 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%. Digest at room temperature for 2.5 hours, centrifuge at 4°C and 5000g for 2 hours, and take the supernatant.

[0063] S204, filtering the supernatant obtained in step S203 using 0.8 μm and 0.45 μm filter membranes in turn to obtain a filtrate.

[0064] S205. Centrifuge the filtrate obtained in step S204 at 4°C and 20,000 g for 1.5 h, and take the supernatant.

[0065] S206. Filter the supernatant obtained in step S205 using a 0.22 μm filter membrane to obtain a plant clear solution.

[0066] S3. Take the plant clear solution obtained in step S206, centrifuge at 120000g for 90 min at 5°C, remove the supernatant and retain the precipitate, add 1 mL of PBS solution, gently blow and resuspend the precipitate, then add 1 mL of PBS solution, gently blow and mix evenly, then add 1 mL of PBS solution, gently blow and mix evenly to obtain a resuspension (about 3 mL in total).

[0067] S4, the resuspension obtained in step S3 is filtered using a 0.22 μm filter membrane, and the filtrate is concentrated to 0.5 mL using a 100 kDa ultrafiltration tube at 4 ° C and 3000 g centrifugation, and then added to an ultrapure chromatographic column. After all samples enter the chromatographic column, 2 mL of PBS is added immediately to collect 2.5 mL of void volume. After the liquid flows out, 2.8 mL of PBS is added to collect the liquid (i.e., eluent) of the 8th-14th fraction, a total of 2.8 mL of target fraction volume, and merged. The combined liquid is concentrated with a 100 kDa ultrafiltration tube, and according to particle size and morphology analysis, it is an extracellular vesicle.

[0068] Example 4 The difference from Example 1 is that in step S1, 200 g of fresh lily bulbs are replaced with 100 g of fresh Lycoris bulbs, and the rest are the same.

[0069] Example 5 The difference from Example 3 is that in step S1, 50 g of air-dried lily is replaced by 100 g of fresh Lycoris radiata bulbs, and the remaining steps are consistent with Example 3.

[0070] Comparative Example 1 Extracellular vesicles from lily were extracted by universal ultracentrifugation (UC).

[0071] Compared with Example 1, the difference is that the step of adding enzymatic hydrolysis solution and standing digestion in step S203 is omitted, and the supernatant (solution) obtained in step S202 is directly centrifuged at 4°C and 5000g for 2h and then the supernatant is taken, and the remaining steps are consistent with Example 1.

[0072] Comparative Example 2 Extracellular vesicles from lily were extracted using ultrapure size exclusion chromatography (SEC) column purification.

[0073] Compared with Example 1, the difference is that the step of adding enzymatic hydrolysis solution and standing digestion in step S203 is omitted, and the supernatant (solution) obtained in step S202 is directly centrifuged at 4°C and 5000g for 2h and then the supernatant is taken; at the same time, step S3 and step S4 are replaced by the following steps.

[0074] The plant clear liquid obtained in step S206 was concentrated to 0.5 mL by centrifugation at 4 ° C and 3000g using a 100kDa ultrafiltration tube, and then the concentrate was purified using an IZON qEV ultrapure chromatographic column with a column bed volume of 8.5 mL. Take 0.5 mL of extracellular vesicle concentrate and add it to the ultrapure chromatographic column. After all samples enter the chromatographic column, add 2 mL of PBS immediately and collect 2.5 mL of void volume. After the liquid flows out, continue to add 2.8 mL of PBS, collect the liquid of the 8th-14th fractions (ie, the eluate), a total of 2.8 mL of the target fraction volume, and merge. The combined liquid is concentrated with a 100kDa ultrafiltration tube, and according to the particle size and morphology analysis, combined with the protein molecular weight and lipid composition, it is an extracellular vesicle.

[0075] The remaining steps are consistent with those in Example 1.

[0076] Comparative Example 3 Comparative Example 3 uses ultracentrifugation combined with ultrapure size exclusion chromatography column purification (UC, SEC) to extract lily extracellular vesicles, which is different from Example 1 in that: The step of adding enzymatic solution and standing for digestion in step S203 is omitted, and the supernatant obtained in step S202 is directly centrifuged at 4°C and 5000g for 2h and then the supernatant is taken. Step S3 and step S4 are replaced by the following steps: Take the plant clear liquid obtained in step S206, centrifuge at 120000g for 90min at 4°C, remove the supernatant and retain the precipitate, add 1mL PBS solution, gently blow and resuspend the precipitate, add 1mL PBS solution, gently blow and mix evenly, add 1mL PBS solution, gently blow and mix evenly, and obtain a resuspension (about 3mL in total). The resuspension obtained is filtered using a 0.22μm filter membrane, and the filtrate is concentrated to 0.5 mL by centrifugation using a 100kDa ultrafiltration tube at 4°C and 3000g, and then added to an ultrapure chromatographic column. After all samples enter the chromatographic column, immediately add 2mL PBS and collect 2.5mL of void volume. After the liquid flows out, continue to add 2.8mL PBS, collect the liquid of the 8th to 14th fractions (i.e., the eluent), a total of 2.8mL of the target fraction volume, and merge. The combined liquid was concentrated using a 100 kDa ultrafiltration tube and analyzed based on particle size and morphology, combined with protein molecular weight and lipid composition, to determine that it was an extracellular vesicle.

[0077] The remaining steps are consistent with those in Example 1.

[0078] Comparative Example 4 Comparative Example 4 uses a general ultracentrifugation method (UC) to extract Lycoris radiata extracellular vesicles, which is different from Example 4 in that: The step of adding enzymatic 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. The remaining steps were consistent with Example 4.

[0079] Effect example 1. Transmission electron microscopy (TEM) characterization Use a pipette gun to take 10 μL of the isolated and purified lily extracellular vesicles from each example and comparative example, drop it on a copper mesh and let it settle for 1 minute, and then remove the floating liquid with filter paper. 10 μL of uranyl acetate was dropped on a copper mesh and let it settle for 1 minute, and then remove the floating liquid with filter paper. After drying at room temperature for 5 minutes, electron microscopy was performed at 80 kV to obtain transmission electron microscopy imaging results. Among them, the TEM photo of the lily extracellular vesicles prepared in Example 1 is Figure 1 The TEM image of the Lycoris radiata extracellular vesicles prepared in Example 4 is Figure 3 .

[0080] As can be seen from the figure, the obtained lily extracellular vesicles are uniform in shape and size under electron microscopy and present a cup-shaped vesicle-like structure.

[0081] 2. Particle size distribution and concentration determination 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. The diluted plant extracellular vesicle samples were taken with a new needle using a PARTICLE METRIX PMX120 instrument to detect the particle size distribution and concentration. The particle size distribution curve of the lily extracellular vesicles prepared in Example 1 is as follows: Figure 2 The particle size distribution curve of Lycoris radiata extracellular vesicles prepared in Example 4 is Figure 4 In Comparative Example 4, no extracellular vesicles were captured by transmission electron microscopy (TEM), so the particle size distribution and concentration were not measured.

[0082] 3. Protein concentration determination The protein concentration in the obtained plant extracellular vesicles was detected using the Thermo Pierce BCA protein detection kit (product model: 2327). The purity of the extracellular vesicles extracted in the example can be analyzed in combination with the particle concentration. In comparative example 4, no extracellular vesicles were captured by transmission electron microscopy (TEM), so the total protein concentration was not determined.

[0083] The average particle size, particle concentration, total protein concentration and purity of the extracellular vesicles obtained in each embodiment and comparative example are shown in Table 1.

[0084] Table 1

[0085] As can be seen from the table, the size of the extracellular vesicles of fresh lily obtained by enzymatic hydrolysis in Example 1 is about 160nm, and the size of the extracellular vesicles of air-dried lily stem cells obtained by enzymatic hydrolysis in Example 2 and Example 3 is about 180nm. The extracellular vesicles from fresh lily obtained by the general extracellular vesicle extraction method without enzymatic hydrolysis in Comparative Examples 1-3 are significantly larger, about 240nm. The size of the extracellular vesicles of fresh Lycoris obtained by enzymatic hydrolysis in Example 4 is about 150nm. During the test of Comparative Example 4, the Lycoris radiata bulb extract was too viscous, and the extracellular vesicles were extracted by the general ultracentrifugation method without enzymatic hydrolysis, and no extracellular vesicles were observed in the extract.

[0086] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate 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: The following steps are involved: S1, mixing the bulbous plant tissue with the buffer solution, crushing, filtering, and obtaining the 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 110000-130000g for 80-100 min, retaining the precipitate, resuspending the precipitate with a buffer solution, repeating 1-2 times to obtain a resuspended solution; S4, filtering the resuspension obtained in step S3, and concentrating the filtrate to obtain extracellular vesicles of bulbous plants; Wherein, the bulbous plant tissue is selected from lily or Lycoris radiata; The process in step S2 includes the following steps: S201, centrifuging the plant stock solution obtained in step S1 at 3-5°C and 900-1100g for 8-12 min to obtain supernatant 1; S202, centrifuging the supernatant 1 obtained in step S201 at 3-5°C and 2900-3100g for 18-22 min to obtain supernatant 2; S203, mixing the supernatant 2 obtained in step S202 with hemicellulase, pectinase and cellulase to obtain an enzyme mixture, digesting at room temperature for 2-3 hours, and centrifuging at 3-5°C and 4900-5100g for 100-140 minutes to obtain supernatant 3; S204, filtering the supernatant 3 obtained in step S203 using a filter membrane to obtain a filtrate 1; S205, centrifuging the filtrate 1 obtained in step S204 at 3-5°C and 19000-21000g for 80-100 min to obtain a supernatant 4; S206, filtering the supernatant 4 obtained in step S205 using a filter membrane to obtain a plant clear solution.

2. The preparation method according to claim 1, characterized in that: In step S1, the buffer solution is a phosphate buffer solution (PBS), the pH value of the buffer solution is 7.2-7.4, and the mass of the bulbous plant tissue and the volume of the buffer solution are 1-4:4, in units of g:mL.

3. The preparation method according to claim 1, characterized in that: In step S1, the pulverizing temperature is 3-5°C, the pulverizing time is 2-5min, and the filtering is performed using gauze.

4. The preparation method according to claim 1, characterized in that: The process in step S2 includes the following steps: S201, centrifuging the plant stock solution obtained in step S1 at 4°C and 1000 g for 10 min to obtain supernatant 1; S202, centrifuging the supernatant 1 obtained in step S201 at 4°C and 3000g for 20 min to obtain supernatant 2; S203, mixing the supernatant 2 obtained in step S202 with hemicellulase, pectinase and cellulase to obtain an enzyme mixture, digesting at room temperature for 2-3 hours, and centrifuging at 4°C and 5000g for 100-140 minutes to obtain supernatant 3; S204, filtering the supernatant 3 obtained in step S203 using a 0.8 μm filter membrane and a 0.45 μm filter membrane in sequence to obtain a filtrate 1; S205, centrifuging the filtrate 1 obtained in step S204 at 4°C and 20000g for 90 min to obtain supernatant 4; S206, filtering the supernatant 4 obtained in step S205 using a 0.22 μm filter membrane to obtain a plant clear solution; Wherein, 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%.

5. The preparation method according to claim 4, 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%.

6. The preparation method according to claim 1, characterized in that: In step S3, the plant clear solution obtained in step S2 is centrifuged at 4°C and 120,000 g for 90 min, the precipitate is retained, and the precipitate is resuspended in phosphate buffered saline (PBS), and the process is repeated 1-2 times to obtain a resuspended solution.

7. The preparation method according to claim 1, characterized in that: In step S4, the filtration is performed by using a 0.22 μm filter membrane, and the filtrate is concentrated by using a 100 kDa ultrafiltration tube and centrifuging at 4° C. and 3000 g.

8. The preparation method according to claim 1, characterized in that: In step S4, the filtrate is concentrated at a ratio of 0.2-0.8 mL of concentrated solution per 200 g of fresh plant tissue; in step S4, after the filtrate is concentrated, column chromatography extraction is also included; The column chromatography extraction comprises the following steps: The concentrated solution obtained by concentrating the filtrate is added to the chromatographic column; after adding the concentrated solution, a phosphate buffer solution PBS with a volume of 3.5-4.5 times the concentrated solution is added, the outflowing liquid is collected, and then a phosphate buffer solution PBS with a volume of 5-6 times the concentrated solution is added, the outflowing liquid is collected and combined to obtain the extracellular vesicles of bulbous plants.

9. The extracellular vesicles of bulbous plants obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the preparation method according to any one of claims 1 to 8 in producing plant extracellular vesicles.

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