CN116751733A

CN116751733A - Extraction method of acanthopanax extracellular vesicles

Info

Publication number: CN116751733A
Application number: CN202311001129.XA
Authority: CN
Inventors: 刘旭杰; 叶慧玲; 何燕; 唐钧泽; 曾耀勋; 袁炯鹏; 叶钊仪; 李玉姗
Original assignee: Guangdong University of Technology
Current assignee: Guangdong University of Technology
Priority date: 2023-08-09
Filing date: 2023-08-09
Publication date: 2023-09-15

Abstract

The application discloses an extraction method of acanthopanax extracellular vesicles, belonging to the technical field of extracellular vesicles extraction; the extraction method comprises the following steps: 1) Stirring the whole plant of acanthopanax, collecting juice, and taking the supernatant after three-step low-speed centrifugation; 2) Filtering the supernatant by a two-stage microporous filter membrane to obtain a filtrate after the suction filtration, adding 8% of PEG8000, and centrifuging overnight to obtain a precipitate to obtain crude extracted extracellular vesicles; 3) Purifying extracellular vesicles using sucrose gradient centrifugation in combination with ultracentrifugation; the technical scheme provided by the application uses a PEG8000 polymer precipitation method to initially extract the extracellular vesicles of acanthopanax, and has the advantages of simple operation and strong reproducibility; before PEG8000 is added to precipitate extracellular vesicles, a two-stage microporous filter membrane is adopted for filtration, so that the extraction purity of extracellular vesicles is ensured as much as possible; is suitable for large-scale extraction of the extracellular vesicles of acanthopanax.

Description

Extraction method of acanthopanax extracellular vesicles

Technical Field

The application belongs to the technical field of extraction of plant-derived extracellular vesicles, and particularly relates to an acanthopanax extracellular vesicle and an extraction method thereof.

Background

Acanthopanax senticosus Acanthopanax senticosus Rupr.& maxim.) belonging to the Araliaceae family as a traditional Chinese medicine, is widely distributed in northeast China. Roots and stems of acanthopanax have long been widely used as tonic, heart nourishing and sedative. A large number of pharmacological and clinical researches have proved that acanthopanax has certain functions of immunoregulation, anti-stress, anti-fatigue and anti-inflammatory. Studies have shown that acanthopanax aqueous extract inhibits the gene expression of Inducible Nitric Oxide Synthase (iNOS) by blocking the activation of kappa-light chain enhanced (NF-kB cells) signaling pathway of nuclear factor activated B cells, thereby inhibiting the production of Nitric Oxide (NO); the acanthopanax can effectively reduce inflammatory parameters (including interleukin-6, tumor necrosis factor alpha, neutrophil density and prostaglandin E2) in a mouse air bag inflammatory model; in an in vitro human nasal mucosa model, the combination of scutellaria baicalensis, acanthopanax senticosus and vitamin C can significantly block early and late mediators of allergy and inhibit the release of proinflammatory cytokines.

Extracellular vesicles (Extracellular Vesicles, EVs) are a collective name of various types of vesicles secreted by cells and having a membrane structure, and the types of vesicles in the current study are mainly EVs with a size of less than 200nrn (ref.1: su Yonghui, xu Shanshan, wang Huan, etc. the development of extracellular vesicles of medicinal plants as novel pharmacodynamic substances [ J ]. Chinese herbal medicines, 2023, 54 (12): 4044-4052 ]) reported in the literature, EVs are disk-like or spherical vesicle-like particles having an intact membrane, which have a typical exosome-like appearance, and have a Zeta potential of-41.83 mV (ref. 2:Zhang X,Pan Z,WangY,et al.Taraxacum officinale-derived exome-like nanovesicles modulate gut metabolites to prevent intermittent hypoxia-induced hypertension [ J ]. Biomedicine & Pharmacotherapy,2023, 161:114572.). EVs are released from cells into the surrounding environment, a biological process that is conserved throughout evolution from bacteria to plants and humans. The secretion of EVs was originally thought to be a means of cell clearance of unwanted molecules of matter, but further studies have found that EVs can serve as information carriers, participating in intercellular communication. EVs can transport information from donor cells to recipient cells, and after being recognized by recipient cells, the information is transferred into cells through EVs surface molecules or endocytic pathways, so that signal transduction in the recipient cells is further activated. These information substances include proteins, lipids, metabolites, sugars, RNA (mRNA, miRNA, siRNA) and even DNA, etc. In clinical medicine, EVs can be used to treat, prevent, and be biomarkers for judging certain diseases. It has also been shown in plants that EVs can be an effective carrier for information communication between plants and pathogenic bacterial cells, or participate in the defensive reaction of plants against pathogens, playing an important role in regulating plant immunity. The extracellular vesicles derived from acanthopanax root are based on Chinese medicinal active substances, contain abundant contents, have the advantage of greenization, and have better biocompatibility and low toxicity and side effects.

At present, no report exists on the extraction method of the acanthopanax extracellular vesicles, and the existing extraction method of the plant extracellular vesicles mainly comprises an ultracentrifugation method, a density gradient centrifugation method, an ultrafiltration centrifugation method, a polymer precipitation method, an immunomagnetic bead method, a kit extraction method and the like. The ultracentrifugation method combined with sucrose density gradient centrifugation is the most commonly used method, but requires multiple ultracentrifugation steps, and the extraction of large-scale outer vesicles is long and has low recovery rate; furthermore, repeated centrifugation may also cause damage to the vesicles, thereby reducing their quality. These all limit the large-scale extraction of extracellular vesicles of acanthopanax. Therefore, the application provides an extraction method of the acanthopanax extracellular vesicles, which is imperative to further improve the utilization rate of the acanthopanax extracellular vesicles.

Disclosure of Invention

The application provides an extraction method of acanthopanax extracellular vesicles, which aims to solve the defects of more centrifugation steps, long consumption time, low product yield, large damage to vesicles and the like in the large-scale extraction of the existing plant-derived extracellular vesicles, and the technical scheme provided by the application adopts PEG8000 to extract the acanthopanax extracellular vesicles, and PEG800 and the acanthopanax extracellular vesicles form a compound in a hydrogen bond form, so that the extracellular vesicles can be precipitated under the lower rotating speed and three-step centrifugation operation; meanwhile, the extracellular vesicles of acanthopanax obtained by extraction are purified by combining sucrose density gradient centrifugation with ultra-high-speed centrifugation, so that the extraction efficiency is high, the repeatability is good, the damage to the extracellular vesicles is small, the integrity of the extracted extracellular vesicles is high, and the method is suitable for industrial mass production.

The technical scheme of the application is as follows:

an extraction method of extracellular vesicles of radix Acanthopanacis Senticosi comprises squeezing radix Acanthopanacis Senticosi, adding PEG8000 solution, incubating overnight, and separating and purifying the incubated liquid.

Further, in the extraction method of the acanthopanax extracellular vesicles, phosphate buffer solution with the mass of 1-3 times of that of the acanthopanax is added when the acanthopanax is juiced.

Further, in the extraction method of the acanthopanax extracellular vesicles, the juice obtained by juicing the acanthopanax is filtered by gauze to collect filtrate, the filtrate is centrifugally extracted to collect supernatant, the supernatant is filtered by a two-stage microporous filter membrane to collect filtrate, and then PEG8000 (polyethylene glycol 8000) is added.

Further, in the extraction method of the acanthopanax extracellular vesicles, the mass concentration of the PEG8000 solution is 8%.

Further, in the extraction method of the acanthopanax extracellular vesicles, the separation and purification are that the liquid after incubation is centrifugally separated, supernatant liquid is removed, PBS buffer solution is added for re-suspension precipitation, and then a filter membrane is adopted for filtering and collecting suspension; adding the collected suspension into an overspeed centrifuge tube which is sequentially filled with 8wt%, 15wt%, 30wt%, 45wt% and 60wt% of sucrose solution from bottom to top, then centrifuging to obtain three layers of layered solution and a wall-attached precipitate at the bottom, discarding the precipitate, absorbing the solution of the middle layered part, placing the solution into the cleaned centrifuge tube, adding PBS buffer until the centrifuge tube is full, uniformly mixing, centrifuging to remove the sucrose solution, collecting the precipitate, re-suspending with PBS buffer, and filtering with a filter membrane to obtain the product.

Further, the extraction method of the acanthopanax extracellular vesicles comprises the following steps in sequence:

1) Cleaning fresh radix Acanthopanacis Senticosi, washing with deionized water, draining off surface water, cutting, adding phosphate buffer solution twice the weight, squeezing with a blender, filtering with gauze, and collecting filtered juice;

the adopted acanthopanax is fresh acanthopanax, the leaves are symbiotic or clustered, the acanthopanax has long handles, the leaves are inverted oval or needle-like, the upper part of the leaf margin is zigzag, and the top is sharp. The root should be brown, fragrant and free from mildew or blackening.

2) Centrifugal extraction: centrifuging 500g-1500g of the juice obtained in the step 1) at 4 ℃ for 5min-15min; then taking the supernatant, and centrifuging at 4 ℃ for 15-25 min at 2000-4000 g; centrifuging 9000g-11000g at 4deg.C for 25min-35min, collecting supernatant;

3) Taking the supernatant in the step 2), filtering the supernatant through a two-stage microporous filter membrane, and then adding 8wt% of PEG8000 solution for overnight incubation at 4 ℃.

4) Centrifuging 13000g-15000g of the liquid after the overnight incubation in 3) at 4 ℃ for 20min-40min, preferably 14000g at 4 ℃ for 30min, discarding supernatant, re-suspending the precipitate with 200 mu L PBS buffer, and then filtering with 3 mu m and 0.22 mu m filter membrane to obtain suspension;

5) Adding sucrose solution into an overspeed centrifuge tube according to the gradient, wherein the concentration gradient is as follows from bottom to top in sequence: 8%, 15%, 30%, 45%, 60%, and adding the resulting precipitate suspension in 4) to the uppermost layer of sucrose solution, and then ultracentrifugating 140000g-160000g for 120min at 4℃and preferably 150000g for 120min at 4 ℃.

6) Obtaining three layers of layered solution and adherent precipitate after centrifugation, collecting the liquid of the middle layer, placing the liquid into a cleaned centrifuge tube, adding PBS buffer until the centrifuge tube is full, uniformly mixing, continuing to perform ultracentrifugation for 120min at 150000g at 4 ℃ to remove sucrose solution, resuspending the obtained precipitate with 500 mu L PBS buffer, filtering with a filter membrane of 0.22 mu m to obtain extracellular vesicles of acanthopanax, and packaging the filtrate and storing at-80 ℃;

7) And (3) carrying out vacuum freeze drying on the filtrate in the step 6) to obtain the acanthopanax extracellular vesicles stored in a freeze-dried form.

Furthermore, in the extraction method of the extracellular vesicles, the juicing is performed in a stirring machine for 20min intermittently, specifically, the juicing is stopped for 1min after each stirring time is 2min, and the juicing is performed for 10 times in a co-circulation mode.

Further, in the above method for extracting extracellular vesicles, the centrifugal extraction in step 2) is: centrifuging the acanthopanax juice obtained in the step 1) at 1000g for 10min, then taking supernatant, centrifuging at 3000g for 20min, taking supernatant, centrifuging at 10000g for 30min, and taking supernatant.

Further, in the method for extracting extracellular vesicles described above, the two-stage microporous membrane in step 3) is performed by using a microporous membrane of 0.8 μm, and then by using a microporous membrane of 0.45 μm.

Furthermore, the method for extracting extracellular vesicles comprises the following steps: temperature: -40 ℃ to-60 ℃ and vacuum degree: 5-10 Pa, time: 24h.

The extracellular vesicles of the acanthopanax senticosus in the two preservation forms obtained in the step 6) and the step 7) are identified by adopting transmission electron microscope detection, zeta potential detection and DLS particle size analysis, identification and detection.

Compared with the prior art, the application has the following beneficial effects:

1. according to the technical scheme provided by the application, PEG8000 is adopted to extract the extracellular vesicles of acanthopanax, and because the PEG8000 has strong hydrophilicity, the PEG8000 and the extracellular vesicles of acanthopanax can form a compound in a hydrogen bond form, so that the extracellular vesicles can be precipitated at a lower rotating speed; the method is favorable for improving the yield of the acanthopanax extracellular vesicles, and compared with the traditional ultracentrifugation method, the method for coarsely extracting the acanthopanax extracellular vesicles by adopting the polymer precipitation method of PEG8000 has simpler operation, can treat large-volume samples, and is suitable for large-scale extraction of the acanthopanax extracellular vesicles.

2. The technical scheme provided by the application avoids the pollution of impurity proteins possibly brought by a PEG precipitation method, and adopts a microporous filter membrane of 0.8 and 0.45 mu m to filter before adding PEG8000 to precipitate extracellular vesicles so as to remove proteins with large particle sizes and ensure the extraction purity of the extracellular vesicles as much as possible.

3. According to the technical scheme provided by the application, the extracellular vesicles of acanthopanax are firstly subjected to crude extraction by adopting a polymer precipitation method, so that the extracellular vesicles with higher yield can be obtained, and the extracellular vesicles of acanthopanax obtained by crude extraction are subjected to relatively short-time purification by adopting sucrose density gradient centrifugation combined with ultracentrifugation, so that the damage caused by continuous ultracentrifugation can be reduced.

4. The extraction method provided by the application avoids the ultra-high speed centrifugation step for a long time, and can avoid the damage of the ultra-high speed centrifugation for a long time to the extracellular vesicle structure. The results of the electron microscope, DLS and Zeta potential detection show that the extracted extracellular vesicles of acanthopanax have obvious vesicle structures and are in the shape of double-layer membrane tea holders, and the extracted extracellular vesicles have no obvious damage signs; the particle size of the acanthopanax extracellular vesicles is about 159.9+/-30.1 nm, the Zeta potential is-8.2+/-4.5 mV, the particle size is consistent with the particle size of the plant extracellular vesicles reported in a plurality of documents in a natural state, and the potential is negative and also accords with the description of the plant extracellular vesicles in a plurality of documents.

Drawings

FIG. 1 is a graph showing the result of electron microscopy of extracellular vesicles of acanthopanax extracted in example 1 of the present application;

FIG. 2 is a graph showing the DLS detection result of extracellular vesicles extracted from Acanthopanax senticosus in example 1 of the present application;

FIG. 3 is a graph showing the Zeta potential detection result of extracellular vesicles extracted from Acanthopanax senticosus in example 1 of the present application;

FIG. 4 is a graph showing the result of electron microscopy of extracellular vesicles of acanthopanax extracted in comparative example 1 of the present application;

FIG. 5 is a graph showing the results of DLS detection of extracellular vesicles of acanthopanax senticosus extracted in comparative example 1 of the present application;

FIG. 6 is a graph showing the results of Zeta potential detection of extracellular vesicles of acanthopanax extracted in comparative example 1 of the present application.

Detailed Description

The following description of the technical solutions in the embodiments of the present application will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Example 1

The extraction method of the acanthopanax extracellular vesicles provided by the embodiment comprises the following steps:

1) Cleaning fresh traditional Chinese medicine acanthopanax, washing with deionized water for three times, slightly draining surface water, shearing, adding PBS buffer solution with the weight twice that of acanthopanax, and carrying out intermittent juicing for 20min by using a stirrer, wherein the method specifically comprises the following steps: stopping for 1min after stirring for 2min, and squeezing juice for 10 times in a common cycle. Folding clean gauze for three times, filtering, and collecting filtered juice;

2) Centrifugal extraction: centrifuging 1000g of the juice obtained in the step 1) for 10min at 4 ℃; taking supernatant, and centrifuging at 4deg.C for 20min at 3000 g; centrifuging 10000g of the supernatant at 4 ℃ for 30min, and collecting the supernatant;

3) Taking the supernatant in the step 2), carrying out suction filtration by using a microporous filter membrane with the diameter of 0.8 mu m, then carrying out suction filtration by using a microporous filter membrane with the diameter of 0.45 mu m, collecting filtrate, and then carrying out suction filtration according to a PEG8000 solution: filtrate = 1:7 volume ratio 8wt% PEG8000 solution was added and incubated overnight at 4 ℃.

4) Centrifuging 14000g of the liquid incubated overnight in 3) at 4 ℃ for 30min, removing the supernatant, re-suspending the precipitate with 200 mu L of PBS buffer, and then passing through a 3 mu m and 0.22 mu m filter membrane to obtain a suspension;

5) 5mL of sucrose solution is added into an overspeed centrifuge tube according to the gradient, and the concentration gradient is as follows: 8wt%, 15wt%, 30wt%, 45wt%, 60wt% of the precipitate suspension obtained in 4) was added to the uppermost layer, followed by ultracentrifugation at 150000g for 120min at 4 ℃;

6) Obtaining three layers of layering and wall-attached precipitation after centrifugation, collecting the liquid of the middle layering, placing the liquid into a cleaned centrifuge tube, adding PBS buffer until the centrifuge tube is full, uniformly mixing, continuing to perform ultracentrifugation for 120min at 150000g at 4 ℃ to remove sucrose solution, re-suspending the obtained precipitate with 500 mu LPBS buffer, filtering with a filter membrane of 0.22 mu m to obtain acanthopanax extracellular vesicles, and subpackaging the filtrate and storing at-80 ℃;

7) Vacuum freeze-drying the filtrate in the step 6), wherein the vacuum freeze-drying conditions are as follows: temperature: -40 ℃ to-60 ℃ and vacuum degree: 5-10 Pa, time: 24h. Finally, the extracellular vesicles of acanthopanax which are stored in a freeze-dried form can be obtained.

Example 2

2) Centrifugal extraction: centrifuging 500g of the juice obtained in the step 1) at 4 ℃ for 5min; subsequently taking the supernatant, and centrifuging 2000g at 4 ℃ for 15min; n; then taking supernatant, centrifuging 9000g at 4 ℃ for 25min, and collecting supernatant;

3) Taking the supernatant in the step 2), carrying out suction filtration by using a microporous filter membrane with the diameter of 0.8 mu m, and then carrying out suction filtration by using a microporous filter membrane with the diameter of 0.45 mu m. Followed by PEG8000 solution: filtrate = 1:7 volume ratio 8wt% PEG8000 solution was added and incubated overnight at 4 ℃.

4) Centrifuging 13000g of the liquid incubated overnight in 3) at 4 ℃ for 20min, removing the supernatant, re-suspending the precipitate with 200 mu L of PBS buffer, and then passing through a 3 mu m and 0.22 mu m filter membrane to obtain a suspension;

5) 5mL of sucrose solution is added into an overspeed centrifuge tube according to the gradient, and the concentration gradient is as follows: 8wt%, 15wt%, 30wt%, 45wt%, 60wt% of the precipitate suspension obtained in 4) was added to the uppermost layer, followed by ultracentrifugation of 140000g at 4℃for 120min;

6) Three layers of layering and wall-attached precipitation can be seen after centrifugation is finished, liquid in the middle layer is collected and placed into a cleaned centrifuge tube, PBS buffer is added until the centrifuge tube is full, the mixture is uniformly mixed, ultracentrifugation is continued for 120min at 150000g at 4 ℃ to remove sucrose solution, the obtained precipitate is resuspended by 500 mu LPBS buffer, and then filtration is carried out by a filter membrane with the thickness of 0.22 mu m to obtain acanthopanax extracellular vesicles, and filtrate is taken for subpackaging and stored at the temperature of minus 80 ℃;

Example 3

2) Centrifugal extraction: centrifuging 1500g of the juice obtained in the step 1) at 4 ℃ for 15min; subsequently taking the supernatant, and centrifuging 4000g at 4 ℃ for 25min; subsequently taking the supernatant, centrifuging 11000g for 35min at 4 ℃, and collecting the supernatant;

4) Centrifuging 15000g of the liquid incubated overnight in 3) at 4 ℃ for 40min, removing the supernatant, re-suspending the precipitate with 200 mu LPBS buffer, and then passing through a 3 mu m and 0.22 mu m filter membrane to obtain a suspension;

5) 5mL of sucrose solution, 8wt%, 15wt%, 30wt%, 45wt%, 60wt% of the precipitate suspension obtained in 4) was added in an ultracentrifuge tube in a gradient, and then ultracentrifuged at 160000g for 120min at 4 ℃;

Comparative example 1

The method for extracting the acanthopanax extracellular vesicles by using the PEG8000 with different concentrations provided by the comparative example comprises the following steps:

3) Taking the supernatant in the step 2), carrying out suction filtration by using a microporous filter membrane with the diameter of 0.8 mu m, then carrying out suction filtration by using a microporous filter membrane with the diameter of 0.45 mu m, collecting filtrate, and then carrying out suction filtration according to a PEG8000 solution: filtrate = 1:7 volume ratio 15wt% PEG8000 solution was added and incubated overnight at 4 ℃.

Test example results:

the extracellular vesicles of acanthopanax obtained by extraction in example 1 are detected by an electron microscope, and as shown in fig. 1, the extracellular vesicles of acanthopanax obtained by preparation have obvious vesicle structures, and the extracellular vesicles obtained by extraction have no obvious damage signs.

The extracellular vesicles of acanthopanax obtained by extraction in comparative example 1 were examined by electron microscopy, as shown in fig. 4, which shows that the extracellular vesicles of acanthopanax obtained by preparation also have vesicle structures, but the extracellular vesicles obtained are broken, fragments are formed around, and the morphology is irregular, and the number of vesicles is small.

The extracellular vesicles of acanthopanax obtained in example 1 were subjected to DLS detection, and the detection results are shown in fig. 2, and the particle size is 159.9±30.1nm.

The extracellular vesicles of acanthopanax obtained by extraction in comparative example 1 were subjected to DLS detection, and the detection result is shown in fig. 5, and the particle size is 192.7+43.1nm, and the particle size is larger.

The Zeta potential of the extracellular vesicles of acanthopanax obtained in example 1 was measured, and the measurement result is shown in fig. 3, and the potential is-8.2±4.5mV.

The Zeta potential of the extracellular vesicles of acanthopanax obtained in comparative example 1 was measured, and the measurement result is shown in fig. 6, and the potential is-14.11+1.09 mv.

By extracting acanthopanax senticosus outer vesicles with 15wt% of PEG8000 solution and 8wt% of PEG8000 solution in the examples and comparative examples, it is known that the concentration of the PEG8000 solution has a great influence on the appearance and purity of outer vesicle extraction, probably because the concentration of polymer solution increases to increase adsorbed impurities in the extraction process, so that the 8wt% of PEG8000 solution is preferable for extraction. According to the experimental data, the technical scheme provided by the application avoids the ultra-high speed centrifugation step for a long time, and can avoid the damage of the ultra-high speed centrifugation to the extracellular vesicle structure. The results of the electron microscope, DLS and Zeta potential detection show that the extracted extracellular vesicles of acanthopanax have obvious vesicle structures and are in the shape of double-layer membrane tea holders, and the extracted extracellular vesicles have no obvious damage signs; and the particle size of the extracellular vesicles of acanthopanax is about 159.9 plus or minus 30.1nm, the Zeta potential is-8.2 plus or minus 4.5mV,

the foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.

Claims

1. An extraction method of an acanthopanax extracellular vesicle is characterized in that the acanthopanax extracellular vesicle is obtained by adding PEG8000 solution after juicing, incubating overnight, and separating and purifying the incubated liquid.

2. The method for extracting extracellular vesicles from acanthopanax according to claim 1, wherein phosphate buffer solution 1-3 times of the mass of acanthopanax is added when the acanthopanax is juiced.

3. The method for extracting extracellular vesicles from acanthopanax according to claim 1 or 2, wherein the juice obtained by squeezing acanthopanax is filtered by gauze to collect filtrate, the filtrate is centrifuged to collect supernatant, the supernatant is filtered by two stages of microporous filters to collect filtrate, and PEG8000 is added.

4. The method for extracting extracellular vesicles from acanthopanax according to claim 1, wherein the mass concentration of the PEG8000 solution is 8%.

5. The method for extracting extracellular vesicles from acanthopanax according to claim 1 or 2, wherein the separation and purification are that the supernatant is discarded after the liquid after incubation is centrifuged, the PBS buffer is added for re-suspending and precipitating, and the suspension is collected after filtration by a filter membrane; adding the collected suspension into an overspeed centrifuge tube which is sequentially filled with 8wt%, 15wt%, 30wt%, 45wt% and 60wt% of sucrose solution from bottom to top, then performing centrifugal separation to obtain three layers of layered and mutually layered solutions, filtering and discarding the precipitate, sucking the solution of the middle layered part into another centrifuge tube, adding PBS buffer until the centrifuge tube is filled, uniformly mixing, performing centrifugal separation to remove the sucrose solution, re-suspending the obtained precipitate with PBS buffer, and filtering with a filter membrane to obtain the product.

6. The method for extracting extracellular vesicles from acanthopanax senticosus according to claim 1 or 2, comprising the following steps in order:

2) Centrifugal extraction: centrifuging 500g-1500g of the juice obtained in the step 1) at 4 ℃ for 5min-15min; then taking the supernatant, and centrifuging at 4 ℃ for 15-25 min at 2000-4000 g; then taking supernatant, centrifuging 9000g-11000g at 4 ℃ for 25min-35min, and collecting supernatant;

4) Centrifuging 13000g-15000g of the liquid incubated in the step 3) at 4 ℃ for 20min-40min, discarding the supernatant, re-suspending the precipitate with a proper amount of PBS buffer, and then passing through a 3 μm and 0.22 μm filter membrane to obtain a suspension;

5) Adding sucrose solution into an overspeed centrifuge tube according to the gradient, wherein the concentration gradient is as follows from bottom to top in sequence: 8wt%, 15wt%, 30wt%, 45wt%, 60wt% and adding the precipitate suspension obtained in 4) to the uppermost layer of sucrose solution, and ultracentrifugation at 140000-160000 g for 120min at 4 ℃;

6) Obtaining three layers of layered solution and adherent precipitate after centrifugation, discarding the precipitate, absorbing the solution of the middle layered part, placing into another centrifuge tube, adding PBS buffer until the centrifuge tube is full, uniformly mixing, continuing ultracentrifugation for 120min at 150000g at 4 ℃ to remove sucrose solution, resuspending the obtained precipitate with 500 mu L PBS buffer, filtering with a filter membrane of 0.22 mu m to obtain extracellular vesicles of acanthopanax, packaging the filtrate, and preserving at-80 ℃;

7. The method for extracting extracellular vesicles according to claim 1, 2 or 6, wherein the squeezing is performed in a stirrer for 20min intermittently, specifically: stopping for 1min after stirring for 2min, and squeezing juice for 10 times in a common cycle.

8. The method of extracting extracellular vesicles according to claim 6, wherein the centrifugal extraction in step 2) is: centrifuging the acanthopanax juice obtained in the step 1) at 1000g for 10min, then taking supernatant, centrifuging at 3000g for 20min, taking supernatant, centrifuging at 10000g for 30min, and taking supernatant.

9. The method according to claim 6, wherein the two-stage microporous membrane in the step 3) is a membrane filter with 0.8 μm and then a membrane filter with 0.45 μm.

10. The method for extracting extracellular vesicles according to claim 6, wherein the vacuum freeze-drying conditions are specifically: temperature: -40 ℃ to-60 ℃ and vacuum degree: 5-10 Pa, time: 24h.