A preparation method for improving antioxidant activity of exosome-like vesicles and application thereof

Abstract

The present application relates to a kind of preparation method and its application of improving exosome-like vesicle antioxidant activity, belong to plant exosome-like vesicle technical field, the present application includes the following steps: obtaining walnut callus, walnut callus is inoculated in MS solid culture medium containing 100-200 μM / L methyl jasmonate, and is induced for 5-10 days;Add pre-cooled phosphate buffer solution, homogenate is broken, 200 mesh screen filter is removed residue, after differential centrifugation, the supernatant is filtered through microporous filter, add equal volume PEG 6000 and mix thoroughly, after 18h again centrifugation, discard supernatant, PBS resuspension precipitate, and obtain walnut exosome-like vesicle crude extract;Take walnut exosome-like vesicle crude extract and add ultrafiltration tube, after centrifugation, take the inner tube solution.The present application is induced by specific adversity to exosome " parent " plant tissue simulation, regulates its metabolic pathway, so that exosome-like vesicle secreted by enrichment high level of antioxidant activity material, then through PEG precipitation method and ultrafiltration extraction process, realize the preparation of high activity vesicle.

Description

Technical Field

[0001] This invention relates to plant exosome-like vesicles, and particularly to a method for preparing exosome-like vesicles with enhanced antioxidant activity and its application. Background Technology

[0002] Exosome-like vesicles are nanoscale, bilayered membrane vesicles actively secreted by cells and serve as important mediators of intercellular communication. Plant exosome-like vesicles, due to their wide availability, high biocompatibility, low immunogenicity, and ability to cross biological barriers, show great potential in drug delivery, disease treatment, and functional foods. In particular, their naturally occurring antioxidant active components (such as phenols, flavonoids, and antioxidant enzymes) give them unique advantages in anti-aging, anti-inflammatory, and prevention and treatment of oxidative stress-related diseases.

[0003] However, exosome-like vesicles extracted from plants generally suffer from unstable activity. Their antioxidant activity is greatly affected by plant growth conditions and extraction processes, exhibiting significant batch-to-batch variations, and the active ingredients are easily degraded. Furthermore, the antioxidant capacity of natural exosome-like vesicles is limited, restricting their application in highly effective antioxidant therapy. Current technologies employ ultracentrifugation and density gradient centrifugation to extract exosomes with antioxidant activity from specific plants, but their activity is dependent on the plant variety, and there is currently no technology for targeted enhancement of antioxidant activity.

[0004] Therefore, developing a stable and efficient method for producing plant exosome-like vesicles with enhanced antioxidant activity has significant industrial value. Summary of the Invention

[0005] To overcome the problems existing in the background technology, the present invention provides a method for preparing exosome-like vesicles with improved antioxidant activity and its application. By simulating specific stress in the exosome "parent" plant tissue, the metabolic pathways of the exosome are regulated, so that the secreted exosome-like vesicles are enriched with high levels of antioxidant active substances. Then, highly active vesicles are prepared by PEG precipitation and ultrafiltration extraction processes.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention provides a method for preparing exosome-like vesicles with enhanced antioxidant activity, specifically comprising the following steps: (1) Obtain walnut callus and transfer the callus to a subculture medium for subculture; (2) The subcultured walnut callus was inoculated into MS solid medium containing 100-200 μM / L methyl jasmonate and induced for 5-10 days; (3) Take the induced walnut callus tissue, add pre-cooled phosphate buffer, homogenize and crush, filter through a 200-mesh sieve to remove the residue, take the supernatant after differential centrifugation and pass it through a microporous filter, add an equal volume of PEG 6000 and mix thoroughly, let stand for 18 hours and then centrifuge again, discard the supernatant, resuspend the precipitate in PBS to obtain the crude extract of walnut exosome-like vesicles. (4) Take the crude extract of the walnut exosome-like vesicles and add it to an ultrafiltration tube. After centrifugation, take the solution from the inner tube to obtain the walnut exosome-like vesicles. Aliquot and store at -80 ℃.

[0007] In the above technical solution, the concentration of methyl jasmonate in step (2) is 200 μM / L, and the induction time is 10 days.

[0008] In the above technical solution, the walnut callus tissue in step (3) is mixed with pre-cooled phosphate buffer solution at a ratio of W:V = 1:1.

[0009] In the above technical solution, the microporous filter in step (3) is a 0.45 μm microporous filter.

[0010] In the above technical solution, the homogenization and crushing conditions in step (3) are: homogenize for 1 minute, pause for 1 minute, and repeat 3 times.

[0011] In the above technical solution, the differential centrifugation conditions in step (3) are: centrifugation for 40 min, followed by centrifugation for 90 min after taking the supernatant, with a centrifugation temperature of 4℃.

[0012] In the above technical solution, the mass concentration of PEG6000 in step (3) is 20%.

[0013] This technical solution also provides an application of walnut exosome-like vesicles in the preparation of antioxidants, anti-aging drugs, health products or cosmetics.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention induces specific stress in the exosome "parent" plant tissue to regulate its metabolic pathway, thereby enriching the secreted exosome-like vesicles with high levels of antioxidant active substances. Subsequently, highly active vesicles are prepared by PEG precipitation and ultrafiltration extraction processes, which have the advantages of high efficiency, safety and time saving. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 These are diagrams illustrating the callus growth state of walnuts induced by different concentrations of methyl jasmonate in Example 1 of this invention. Figure 2 This is a flowchart of the extraction process of walnut exosome-like vesicles in Example 2 of the present invention; Figure 3 This is the ABTS antioxidant Trolox standard curve diagram of Example 3-1 of the present invention; Figure 4 This is the ABTS antioxidant diagram of the present invention; Figure 5 This is the DPPH antioxidant Trolox standard curve diagram of Example 3-2 of the present invention; Figure 6 This is the DPPH antioxidant diagram of the present invention; Figure 7 This is a scatter plot of the correlation between QC samples in this invention; Figure 8 These are principal component analysis diagrams of walnut exosome-like vesicles before and after treatment with methyl jasmonate according to the present invention; A: 2D; B: 3D; Figure 9 This is an OPLS-DA discriminant analysis diagram of walnut exosome-like vesicles treated with methyl jasmonate according to the present invention; A: OPLS-DA score diagram; B: OPLS-DA substitution verification diagram; Figure 10 These are identification diagrams of differential metabolites before and after treatment with methyl jasmonate according to the present invention; A: volcano plot; B: thermogram; Figure 11 This is a classification diagram of the differential metabolites before and after treatment with methyl jasmonate according to the present invention; Figure 12 This is a differential abundance score map of differential metabolites in this invention; Figure 13 This is a classification diagram of differential metabolite pathways in this invention. Detailed Implementation

[0017] To make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present application will be described in further detail below with reference to the accompanying drawings.

[0018] Where the following description relates to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of apparatuses and methods consistent with some aspects of the invention as detailed in the appended claims.

[0019] In the description of this invention, it should be understood that the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Those skilled in the art can understand the specific meaning of these terms in this invention based on the specific circumstances. Furthermore, in the description of this invention, unless otherwise stated, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0021] Example 1: Treatment of walnut callus with methyl jasmonate (1) Preparation of methyl jasmonate stock solution: Take 229 μL of methyl jasmonate solution, add 5 mL of anhydrous ethanol, add sterile water to make up to 10 mL, filter and sterilize in a laminar flow hood, and set aside.

[0022] (2) Treatment of walnut callus with methyl jasmonate: Methyl jasmonate was added to MS solid medium to make the final concentrations 0, 50, 100, 200 and 300 μM / L respectively. Callus tissues with consistent growth and good growth after subculture were selected and cultured in the above medium for 5, 10 and 15 days respectively before sampling.

[0023] When the concentration of methyl jasmonate reached 300 μM / L, the cells showed obvious browning, so the concentration was chosen to be 200 μM / L; after 15 days of culture, all cells except the control showed extensive browning, so 10 days of culture was chosen.

[0024] Example 2: Extraction of Walnut Exosome-like Vesicles Walnut callus tissue cultured in Example 1 was picked and placed in a 50 ml centrifuge tube. Pre-cooled phosphate-buffered saline (PBS) (pH = 7.4) was added. The material and buffer were mixed at a W:V ratio of 1:1. The tissue was homogenized using a homogenizer, with a 1-minute pause between homogenizations to prevent the instrument from overheating. This process was repeated 3 times. The residue was filtered through a 200-mesh sieve, and the filtrate was aliquoted into centrifuge tubes and temporarily placed on ice. The tubes were centrifuged at 4000 g for 40 min, and the supernatant was then centrifuged at 10000 g for 90 min, both at 4°C. The supernatant was then passed through a 0.45 μm microporous filter, and an equal volume of PEG 6000 (20% concentration) was added. The mixture was thoroughly mixed and incubated overnight at 4°C. After 18 h, the tubes were centrifuged at 10000 g for 30 min at 4°C. The supernatant was discarded, and the precipitate was resuspended in PBS to obtain the crude extract of walnut exosome-like vesicles. Take 500 μL of crude extract and slowly add it to an ultrafiltration tube (300 kDa). Centrifuge at 4000 g for 30 min at 4 ℃. Take the solution from the inner tube and aliquot it for storage at -80 ℃.

[0025] Example 3: Antioxidant activity of walnut exosome-like vesicles before and after methyl jasmonate induction. 1. ABTS method for detection A 7.40 mM / L ABTS solution was prepared by mixing equal volumes of distilled water and a 2.60 mM / L K₂S₂O₈ solution. The mixture was then incubated at room temperature in the dark for 12–16 h to obtain ABTS. + The working solution, with an absorbance of 0.70 ± 0.05 at 734 nm as measured by an ELISA reader, was prepared for use. 180 μL of ABTS working solution was mixed with 20 μL of walnut exosome vesicle sample, shaken for 30 s, and incubated in the dark for 6 min. The absorbance at 734 nm was measured using an ELISA reader. Each sample was tested in triplicate, with three replicates per well. ABTS was calculated. + Free radical scavenging rate.

[0026] Where A0 is the absorbance of the control (absorbance of ABTS only), and A is the absorbance after the sample is added.

[0027] Construction of the Trolox standard curve: Accurately weigh approximately 25 mg of water-soluble vitamin E (Trolox) standard powder, dissolve it in an appropriate amount of anhydrous ethanol, and then dilute to 100 mL. Shake well to obtain a 1 mM Trolox solution. Prepare Trolox standard solutions using this solution to form six concentration points: 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, 0.03125 mM, and 0 mM, as detailed in Table 1. Take 180 μL of ABTS... +The working solution was mixed with 20 μL Trolox standard solution, and the absorbance was measured at 734 nm using a microplate reader. The antioxidant activity was calculated based on the standard curve, and the final result was expressed as the number of mM Trolox equivalents per mL of sample (mM / mL).

[0028] Table 1. Preparation of Trolox Standard Solutions 2. DPPH method for detection Prepare 100 mL of 2 mM / L DPPH solution with anhydrous ethanol and store in the dark. Dilute the solution to the required concentration with anhydrous ethanol before use. Then, add 20 μL of the W-ELNs sample and the prepared Trolox standard solution to each well of a 96-well microplate. Five low-concentration spots of the standard solution were prepared: 0.25 mM, 0.125 mM, 0.0625 mM, 0.03125 mM, and 0 mM (see Table 2 for details). Next, add 180 μL of DPPH working solution to each well and mix gently to ensure a complete reaction, maintaining a total reaction volume of 200 μL. After incubating at room temperature in the dark for 30 min, place the microplate in a microplate reader and measure the OD value at a wavelength of 515 nm. Each sample was measured three times, with three replicates per measurement. Calculate the DPPH free radical scavenging rate.

[0029] Where A0 is the control absorbance (absorbance of DPPH only), and A is the absorbance after adding the sample.

[0030] The DPPH radical scavenging rate was calculated based on the standard curve, and the final result was expressed as the number of mM equivalents of Trolox per mL of sample (mM / mL).

[0031] Table 2. Preparation of Trolox Standard Solutions Example 4: Metabolomics analysis of walnut exosome-like vesicles before and after methyl jasmonate induction. 1. Sample preparation (1) Take 100 μL of walnut exosome vesicle sample, add 500 μL of extraction solution containing internal standard (methanol: acetonitrile = 1:1, internal standard concentration 20 mg / L), and vortex mix for 30 s; (2) After sonication for 10 min (ice-water bath), stand at -20℃ for one hour, centrifuge at 12000 rpm for 15 min at 4℃; then carefully take out 500 μL of supernatant into an EP tube and dry the extract in a vacuum concentrator.

[0032] (3) Add 160 μL of extraction buffer (acetonitrile:water = 1:1) to the dried metabolites to reconstitute, vortex for 30 s, and sonicate in an ice-water bath for 10 minutes; then centrifuge at 12000 rpm for 15 min at 4℃; finally, carefully take 120 μL of supernatant into a 2 mL sample bottle, and mix 10 μL of each sample to form a QC sample for instrument detection.

[0033] 2. On-machine testing The liquid chromatography-mass spectrometry (LC-MS) system used for metabolomics analysis consisted of a Waters Acquity I-Class PLUS ultra-high performance liquid chromatography system tandem with a Waters Xevo G2-XS QTOF high-resolution mass spectrometer. The chromatographic column used was a Waters Acquity UPLC HSS T3 column (1.8 μm, 2.1 μm). 100mm). Positive ion mode (POS): Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile; Negative ion mode (NEG): Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile; Injection volume: 2uL. ESI ion source parameters: Capillary voltage: 2500V (positive ion mode) or -2000V (negative ion mode); Cone voltage: 30V; Ion source temperature: 100℃; Desolvation gas temperature: 500℃; Backflush gas flow rate: 50L / h; Desolvation gas flow rate: 800L / h; Mass-to-nucleus ratio (m / z) acquisition range: 50-1200.

[0034] 3. Data Analysis Raw data acquired using MassLynx V4.2 was processed using Progenesis QI software, including peak extraction and alignment. The data was then identified using the online METLIN database (https: / / metlin.scripps.edu / ), KEGG database (http: / / www.genome.jp / kegg / ), HMDB database (https: / / hmdb.ca / ), and Lipidmaps (https: / / lipidmaps.org / ) databases, along with theoretical fragment identification.

[0035] Experimental Analysis I. Antioxidant Results 1. ABTS Antioxidant Experiment The walnut exosome-like vesicles treated with methyl jasmonate exhibited the strongest ABTS Free Radical Scavenging Index (ABTS FI) scavenging ability, exceeding 90%, significantly superior to the other two groups. The scavenging abilities of the walnut callus cell group (CCE) and the control group (Con) were relatively low, less than 60%, with no significant difference between the two groups. When the antioxidant capacity was standardized using the positive control standard Trolox as a reference, the antioxidant capacity of each milliliter of W-ELNs (methyl jasmonate) was equivalent to that of 0.48 μM Trolox; this was approximately 1.8 times higher than the control group. Figure 5 (As shown).

[0036] 2. DPPH Antioxidant Experiment The W-ELNs (MeJA) group exhibited the strongest DPPH free radical scavenging index (DPPH FI) at 52.14%, significantly superior to the other two groups. The callus cell (CCE) group and the W-ELNs (Con) group showed DPPH free radical scavenging capabilities of only around 30%. When its antioxidant capacity was standardized using the positive control standard Trolox, each milliliter of W-ELNs (MeJA) had the equivalent antioxidant capacity of 0.13 μM Trolox, approximately 1.7 times higher than the control group. Figure 7 (As shown).

[0037] II. Metabolomics Results (Data Quality Assessment) 1. Repeatability assessment To investigate the changes in the contents and functional activity of walnut exosome-like vesicles before and after methyl jasmonate treatment, the metabolites carried by the two samples were analyzed. First, the quality control (QC) samples were subjected to repeated injection analysis to assess the stability of the experimental procedure, and the Spearman Rank Correlation (r) between their metabolites was calculated. Figure 7 As shown, the correlation coefficients between all pairs of quality control samples were greater than 0.98, indicating that the entire metabolomics analysis process had excellent repeatability and high precision, the systematic error was effectively controlled, the obtained data were reliable, and it was suitable for subsequent multivariate statistical analysis and differential metabolite identification.

[0038] 2. Principal Component Analysis (PCA) Principal component analysis (PCA) can provide a preliminary understanding of the overall metabolic differences among the samples in each group and the degree of variability within each group. PCA was performed on walnut exosome-like vesicle samples from the methyl jasmonate-treated (M group) and control (C group). The results showed that the first principal component (PC1) score was 56.58%, and the second principal component (PC2) score was 9.68%, indicating small intra-sample differences, good repeatability, and reliable data. Furthermore, the C and M groups were clearly separated along the PC1 direction, showing significant differences between samples, indicating that methyl jasmonate treatment affected the metabolic pathways of walnut exosome-like vesicles.

[0039] 3. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) To maximize the observation of inter-group differences between the methyl jasmonate treatment group and the control group, and to minimize intra-group differences, an orthogonal partial least squares (OPLS-DA) model was established. Figure 9 As shown in Figure A, samples C and M clearly separated into two clusters on the t1 axis, similar to the PCA results, indicating that there are indeed significant differences in the metabolites between the two groups. To ensure the validity of the results, 200 permutation tests were performed on the PLS-DA model (…). Figure 9 B), R 2 Y=1,Q 2 =0.994, all greater than 0.5, verifying the reliability and robustness of the model and eliminating the risk of overfitting.

[0040] 4. Screening of differentially metabolites (1) Identification of differential metabolites A total of 2989 metabolites were identified in the methyl jasmonate-treated and control samples. Based on the triple screening criteria of VIP value ≥1, |log2FC| ≥1, and P < 0.05, 1119 differentially expressed metabolites were screened between the methyl jasmonate-treated and control samples. Among them, 998 metabolites were upregulated and 121 metabolites were downregulated, with significantly more upregulated metabolites than downregulated metabolites. Figure 10 A). Hierarchical clustering analysis was performed on the screened differential metabolites. The heatmap colors showed that the methyl jasmonate treatment group had a significantly higher number of metabolites than the control group. Figure 10 B).

[0041] (2) Classification of differential metabolites like Figure 11As shown, these metabolites belong to 19 classes, with lipids, organic acids, terpenes, flavonoids, polyphenols, and coumarins having the most metabolites, at 141 (12.60%), 86 (7.69%), 76 (6.79%), 68 (6.08%), and 44 (3.93%), respectively. This indicates that these substances are key components in the response to methyl jasmonate-induced metabolites.

[0042] (3) Pathway annotation of differentially metabolites To further clarify the metabolic mechanism of walnut exosome-like vesicles before and after methyl jasmonate treatment, metabolite pathways were analyzed using the KEGG database. (See bubble diagram). Figure 12 Metabolic pathway enrichment analysis based on Differential Abundance Score (DAS) revealed 20 significantly altered metabolic pathways. The vast majority of pathways (19 / 20) showed DAS values ​​of 0.75–1.00, indicating significant upregulation; no pathways showed significant downregulation (DAS ≤ -0.75), suggesting that methyl jasmonate treatment promoted enhanced metabolic flux. Among them, the DAS values ​​of key pathways such as phenylpropanoid biosynthesis, biosynthesis of various plant secondary metabolites, D-amino acid metabolism, arginine and proline metabolism, pyruvate metabolism, phenylalanine metabolism, and tyrosine and tryptophan biosynthesis were all higher than 0.75, indicating that the defense response, amino acid metabolism, and energy supply metabolic network of walnut exosome-like vesicles were activated after methyl jasmonate treatment.

[0043] Meanwhile, the top 20 items in the pathway annotated with the most differentially expressed metabolites were selected. Figure 13The pathways of phenylpropane biosynthesis, phenylalanine metabolism, arginine and proline metabolism, biosynthesis of various alkaloids, and biosynthesis of various plant secondary metabolites are also rich in differential metabolites. Phenylalanine and the metabolism of various amino acids provide the starting amino acid raw materials for the phenylpropane pathway. The main products of the phenylpropane biosynthesis pathway are some flavonoids and coumarins, which are phenylpropane compounds. These compounds have antioxidant activity, and the antioxidant capacity of ABTS and DPPH in walnut exosome-like vesicles was enhanced after methyl jasmonate treatment.

[0044] In the accompanying drawings of this embodiment, the same or similar reference numerals correspond to the same or similar components. In the description of this application, it should be understood that if terms such as "upper," "lower," "left," "right," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting this application. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0045] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing exosome-like vesicles with enhanced antioxidant activity, characterized in that: Specifically, the following steps are included: (1) Obtain walnut callus and transfer the callus to a subculture medium for subculture; (2) The subcultured walnut callus was inoculated into MS solid medium containing 100-200 μM / L methyl jasmonate and induced for 5-10 days; (3) Take the induced walnut callus tissue, add pre-cooled phosphate buffer, homogenize and crush, filter through a 200-mesh sieve to remove the residue, take the supernatant after differential centrifugation and pass it through a microporous filter, add an equal volume of PEG 6000 and mix thoroughly, let stand for 18 hours and then centrifuge again, discard the supernatant, resuspend the precipitate in PBS to obtain the crude extract of walnut exosome-like vesicles. (4) Take the crude extract of the walnut exosome-like vesicles and add it to an ultrafiltration tube. After centrifugation, take the solution from the inner tube to obtain the walnut exosome-like vesicles. Aliquot and store at -80℃.

2. The method for preparing exosome-like vesicles with enhanced antioxidant activity according to claim 1, characterized in that: In step (2), the concentration of methyl jasmonate is 200 μM / L, and the induction time is 10 days.

3. The method for preparing exosome-like vesicles with enhanced antioxidant activity according to claim 1, characterized in that: In step (3), the walnut callus tissue and pre-cooled phosphate buffer solution are mixed at a ratio of W:V = 1:

1.

4. The method for preparing exosome-like vesicles with enhanced antioxidant activity according to claim 1, characterized in that: The microporous filter in step (3) is a 0.45 μm microporous filter.

5. The method for preparing exosome-like vesicles with enhanced antioxidant activity according to claim 1, characterized in that: The homogenization and crushing conditions in step (3) are: homogenize for 1 minute, pause for 1 minute, and repeat 3 times.

6. The method for preparing exosome-like vesicles with enhanced antioxidant activity according to claim 1, characterized in that: The differential centrifugation conditions in step (3) are: centrifuge for 40 min, take the supernatant and centrifuge for 90 min, and the centrifugation temperature is 4℃.

7. The method for preparing exosome-like vesicles with enhanced antioxidant activity according to claim 1, characterized in that: The mass concentration of PEG6000 in step (3) is 20%.

8. The use of walnut exosome-like vesicles prepared by the method according to any one of claims 1 to 7 in the preparation of antioxidants, anti-aging drugs, health products or cosmetics.

Images