Extraction method of angelica dahurica exosome-like nanovesicle and application thereof

Abstract

The present application belongs to the technical field of plant extraction, and particularly relates to a method for extracting Angelica dahurica exosome-like nanovesicles and application thereof. Compared with the traditional extraction method, the method of the present application has the characteristics of low equipment requirement, low cost, large processing capacity and high flexibility. The protein and RNA contents in the extracted Angelica dahurica exosome-like nanovesicles can reach 47.9% and 3.12% respectively, which are increased by 15.9% and 16.34% respectively compared with the traditional method, and the number of sneezing and the number of scratching nose of mice stimulated by ovalbumin (OVA) can be effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to a method for extracting exosome-like nanovesicles from Angelica dahurica and its application. Background Technology

[0002] Allergic rhinitis, also known as allergic rhinitis (AR), is a chronic non-infectious inflammation of the nasal mucosa mediated by specific immunoglobulin E (IgE) caused by human contact with allergens. It easily produces a series of reactions such as nasal itching, nasal congestion, tearing, and sneezing.

[0003] Angelica dahurica is a tall, perennial herb belonging to the genus Angelica of the Apiaceae family. Besides its analgesic and antioxidant pharmacological effects, it also possesses anti-inflammatory properties and is frequently used to treat allergic rhinitis. Traditional extraction methods for Angelica dahurica preparations used to treat or alleviate allergic rhinitis typically involve boiling water extraction or ethanol extraction. The active ingredients are generally angelica flavonoids, phenols, and terpenes. However, both water and ethanol extraction methods have limited efficiency; furthermore, proteins, fats, and miRNAs in Angelica dahurica cannot be extracted, resulting in resource waste. Furthermore, there is a lack of literature disclosing the extraction of exosome-like nanovesicles from Angelica dahurica and their specific effects on the human body.

[0004] Therefore, how to extract exosomes from Angelica dahurica and use them in human diseases has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for extracting Angelica dahurica exosome-like nanovesicles and its application. By using differential centrifugation, flocculant-assisted centrifugation, and gradient sucrose centrifugation, Angelica dahurica exosome-like nanovesicles are obtained. The obtained Angelica dahurica exosome-like nanovesicles can effectively relieve allergic rhinitis.

[0006] In a first aspect of the present invention, a method for extracting exosome-like nanovesicles from Angelica dahurica is provided, comprising the following steps:

[0007] S1, After ultra-fine pulverization of Angelica dahurica raw material, homogenization is performed with phosphate buffer solution to obtain Angelica dahurica homogenate;

[0008] S2, the homogenate obtained in S1 is subjected to differential centrifugation to obtain the differential centrifugation pretreatment solution of angelica exosome-like nanovesicles.

[0009] S3, add sodium carboxymethyl cellulose to the differential centrifugation pretreatment solution of Angelica dahurica exosome-like nanovesicles, let stand overnight at 4°C, centrifuge, collect the precipitate, and obtain crude extract of Angelica dahurica exosome-like nanovesicles.

[0010] S4, resuspend the crude extract of Angelica dahurica exosome-like nanovesicles in phosphate buffer solution to obtain a resuspension of the crude extract of Angelica dahurica exosome-like nanovesicles.

[0011] S5, Sucrose density gradient centrifugation purification: Add a 15-60% sucrose solution to a centrifuge tube, add the resuspension obtained in S4 above the sucrose solution and perform ultra-high speed centrifugation. Collect the bands between 30%-45% sucrose solution into a bottle, add phosphate buffer solution and perform ultra-high speed centrifugation again to wash with sucrose, collect the precipitate, and you will get the Angelica dahurica exosome-like nanovesicle extract purified by sucrose density gradient centrifugation.

[0012] Preferably, in S1, the Angelica dahurica raw material is dried Angelica dahurica slices, which are pulverized to 120-200 mesh using an ultra-micro pulverizer; after pulverization, 8-10 times the mass volume of phosphate buffer solution is added and mixed evenly to obtain Angelica dahurica homogenate; the pH of the phosphate buffer solution is 7.2 and the concentration is 0.01M.

[0013] Preferably, in S2, the differential centrifugation is performed as follows: the supernatant is collected after centrifugation at 2000-5000g for 0.5-1 hours, and then the supernatant is collected again after centrifugation at 6000-15000g for 1-2 hours. The supernatant is filtered to obtain the differential centrifugation pretreatment solution of Angelica dahurica exosome-like nanovesicles.

[0014] Preferably, the filtration is performed by passing the supernatant through a 0.45μm aqueous filter membrane.

[0015] Preferably, in S3, the amount of sodium carboxymethyl cellulose added is 0.05-2% of the system mass; and the mixture is centrifuged at 10,000 rpm for 60 minutes.

[0016] Preferably, in step S4, the volume of the phosphate buffer solution is 4 to 6 times the weight of the crude extract of Angelica dahurica exosome-like nanovesicles; the pH of the phosphate buffer solution is 7.2 and the concentration is 0.01M.

[0017] Preferably, in step S5, the sucrose concentrations in the sucrose density gradient centrifugation are 15%, 30%, 45%, and 60% from top to bottom in the centrifuge tube; the volume of the phosphate buffer solution used to wash the sucrose is 15 to 20 times the volume of the strip between 30% and 45% sucrose solution; and centrifugation is performed at 100,000-150,000 g for 1 to 2 hours.

[0018] In a second aspect of the invention, the application of Angelica dahurica exosome-like nanovesicles prepared by the above method in relieving allergic rhinitis is provided.

[0019] Preferably, the Angelica dahurica exosome-like nanovesicles can reduce sneezing and nose scratching symptoms of allergic rhinitis caused by ovalbumin (OVA) stimulation.

[0020] Preferably, the Angelica dahurica exosome-like nanovesicles can reduce the increase in NO, IL-6, and IL-1β levels caused by LPS stimulation of mononuclear macrophage leukemia cells (RAW264.7).

[0021] The beneficial effects of this invention are as follows:

[0022] (1) The method for extracting Angelica dahurica exosome-like nanovesicles provided by the present invention can extract Angelica dahurica exosome-like nanovesicles with protein and RNA contents of 47.9% and 3.12% respectively, which are 15.9% and 16.34% higher than those of traditional methods, greatly improving the protein and RNA contents;

[0023] (2) The present invention uses differential centrifugation combined with sodium carboxymethyl cellulose. Compared with traditional extraction methods, the method of the present invention has the advantages of low equipment requirements, low cost, large processing capacity and high flexibility; it can avoid mechanical damage, protect sample activity and improve precipitation specificity, and is easy to separate.

[0024] (3) The exosome-like nanovesicles extracted by the present invention can effectively reduce the number of sneezes and nose scratching caused by ovalbumin (OVA) stimulation in mice, and can effectively relieve the symptoms of allergic rhinitis. Attached Figure Description

[0025] Figure 1 Transmission electron microscopy results and particle size analysis results of exosome-like nanovesicles of Angelica dahurica;

[0026] Figure 2 The effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on cellular NO levels;

[0027] Figure 3 The effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on the level of the cellular inflammatory factor IL-6;

[0028] Figure 4 The effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on the level of the cellular inflammatory factor IL-1β. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0030] Example 1

[0031] A method for extracting exosome-like nanovesicles from Angelica dahurica, comprising the following steps:

[0032] S1. The dried Angelica dahurica slices are pulverized to about 160 mesh using an ultra-micro pulverizer. After pulverization, 9 times the mass volume of Angelica dahurica is added to a 0.01M phosphate buffer solution (pH=7.2) and mixed well to obtain Angelica dahurica homogenate.

[0033] S2, the homogenate obtained in S1 is subjected to differential centrifugation. The supernatant is collected after centrifugation at 5000g for 1 hour. The supernatant is then collected again after centrifugation at 15000g for 2 hours to obtain the differential centrifugation pretreatment solution of Angelica dahurica exosome-like nanovesicles.

[0034] S3. Add 0.5% sodium carboxymethyl cellulose by mass to the differential centrifugation pretreatment solution of Angelica dahurica exosome-like nanovesicles, let stand overnight at 4°C, then centrifuge at 10000 rpm for 60 minutes, collect the precipitate, and obtain crude extract of Angelica dahurica exosome-like nanovesicles.

[0035] S4. The crude extract of Angelica dahurica exosome-like nanovesicles was resuspended in 5 times the volume of phosphate buffer solution to obtain a resuspension of the crude extract of Angelica dahurica exosome-like nanovesicles.

[0036] S5, Sucrose density gradient centrifugation purification: 15%, 30%, 45%, and 60% sucrose solutions were added sequentially to centrifuge tubes. The resuspension obtained in S4 was added above the sucrose solution. Centrifugation was performed at 150,000g for 1 hour. The band between 30% and 45% sucrose solution was aspirated and collected in a bottle. 20 times the volume of the aspirated band was added to phosphate buffer solution, and centrifugation was performed again at 150,000g for 1 hour for sucrose washing. The precipitate was collected, which yielded the sucrose density gradient centrifugation purified Angelica dahurica exosome-like nanovesicle extract.

[0037] The transmission electron microscopy (TEM) results and particle size analysis results of the obtained Angelica dahurica exosome-like nanovesicle extract are shown in the figure. Figure 1 It can be seen that the detected Angelica dahurica exosome-like nanovesicles have a "teacup-shaped" structure with an average particle size of 258.4 nm, which is consistent with the characteristics of plant exosomes.

[0038] Example 2

[0039] The difference from Example 1 is that in S3, the amount of sodium carboxymethyl cellulose added is 1% of the body mass, while the other steps are the same as in Example 1.

[0040] Example 3

[0041] The difference from Example 1 is that in S3, the amount of sodium carboxymethyl cellulose added is 2% of the body mass, while the other steps are the same as in Example 1.

[0042] Example 4

[0043] The difference from Example 1 is that in S2, during differential centrifugation, the supernatant is collected after centrifugation at 5000g for 0.5 hours, and then collected again after centrifugation at 6000g for 2 hours. All other steps are the same as in Example 1.

[0044] Comparative Example 1

[0045] The difference from Example 1 is that step S3 is omitted, and step S4 is performed directly. All other steps are the same as in Example 1.

[0046] Comparative Example 2

[0047] The difference from Example 1 is that in S3, polyvinyl alcohol is used instead of sodium carboxymethyl cellulose, while the other steps are the same as in Example 1.

[0048] Comparative Example 3

[0049] The difference from Example 1 is that S3 does not use sodium carboxymethyl cellulose, but instead uses ultracentrifugation. Specifically, the angelica exosome-like nanovesicle pretreatment solution is subjected to ultracentrifugation at 100,000g for 60 minutes, and the precipitate is collected.

[0050] Comparative Example 4

[0051] The difference from Comparative Example 3 is that sodium carboxymethyl cellulose was added after ultracentrifugation in S3, and the amount added was the same as in Example 1. All other steps were the same as in Comparative Example 3.

[0052] Experimental Example 1

[0053] The protein content in the Angelica dahurica exosome-like nanovesicles obtained in Examples 1-4 and Comparative Examples 1-4 was determined using the BCA protein concentration assay, and the results are shown in Table 1.

[0054] Table 1. Protein content in Angelica dahurica exosome-like nanovesicles extracted from each example and comparative example.

[0055] Group Protein content (%) extracted from exosome-like nanovesicles RNA content (%) extracted from exosome-like nanovesicles Example 1 47.9 3.12 Example 2 43.1 2.81 Example 3 40.6 2.67 Example 4 37.9 2.38 Comparative Example 1 36.5 2.26 Comparative Example 2 37.4 2.34 Comparative Example 3 40.3 2.61 Comparative Example 4 38.9 2.49

[0056] As shown in Table 1, the protein content of the Angelica dahurica exosome-like nanovesicles extracted in Example 1 was the highest, reaching 47.9%, and the RNA content was 3.12%. Compared with the Angelica dahurica exosome-like nanovesicles extracted by differential centrifugation + ultracentrifugation, the protein content increased by 15.9% and the RNA content increased by 16.34%.

[0057] Experimental Example 2

[0058] Cell experiments

[0059] Mouse mononuclear macrophage leukemia cells (RAW264.7) were cultured in plastic culture dishes in DMEM high glucose (Genview) medium supplemented with 10% fetal bovine serum (Applied Biosystems) and 1% penicillin-dextrose antibody.

[0060] Cells were divided into a normal group (normal cell culture group), a modeling group (LPS group), an exosome-like nanovesicle group (the group treated with Angelica dahurica exosome-like nanovesicle samples obtained in Example 1), and an aqueous extract group (the group treated with boiling water extract samples). RAW264.7 cells with good growth and 80% confluence were digested and prepared into a cell suspension, which was then seeded into 24-well Fisher cell culture plates. After complete cell adhesion, a mixture of 250 ng / ml LPS and Angelica dahurica exosome sample or Angelica dahurica aqueous extract was added and treated for 24 h. The cell culture supernatant was collected, and the levels of NO, IL-6, and IL-1β were measured according to the ELISA instructions.

[0061] NO detection kit:

[0062] 1. Remove Griess Reagent I and II and allow them to return to room temperature.

[0063] 2. Dilute the standard (1-100µM) with the solution used for the sample to be tested.

[0064] For example, if the sample is cell culture supernatant and the cell culture medium is DMEM + 10% FBS, then the standard is diluted with DMEM + 10% FBS. The concentration of the standard is typically 0, 1, 2, 5, 10, 20, 40, 60, or 100 µM.

[0065] 3. Add the standard and sample to the 96-well plate at a rate of 50 µl / well.

[0066] The sample is the culture supernatant, which can be taken directly. If there is precipitable material, centrifugation is required to collect the supernatant. If the sample is cells or tissue, rapid freeze-thaw lysis can be performed, followed by centrifugation to collect the supernatant. If the volume is less than 50 µl, it can be diluted with redistilled water or 0.9% NaCl (correspondingly, the standards also need to be diluted with redistilled water or 0.9% NaCl). Cells or tissues can also be used for lysis with Western lysis or IP lysis buffer (without adding inhibitors), and the standards also need to be diluted accordingly.

[0067] 4. Add room temperature Griess Reagent I to each well at a rate of 50 µl / well.

[0068] 5. Add room temperature Griess Reagent II to each well at a rate of 50 µl / well.

[0069] 6. Absorbance was measured at 540 nm.

[0070] 7. Calculate the concentration of nitric oxide in the sample based on the standard curve.

[0071] ELISA kit method: (DAKEWEI)

[0072] 1. Before use, mix all reagents thoroughly to avoid foaming. Reagents should be stored according to the instructions on the label and allowed to equilibrate at room temperature for 20-30 minutes before use.

[0073] 2. Determine the required number of strips based on the number of experimental wells (blank and standard).

[0074] 3. Sample addition: Add 100 μL / well of diluted Cytokine standard to the standard wells, add 100 μL / well of sample to the sample wells, set up blank wells, and replace the sample and standard with Dilution buffer R (1×).

[0075] 4. Add detection antibody: Add 50 μL / well of diluted biotinylated antibody. After mixing, cover with sealing film and incubate at room temperature (18-25℃) (IL-6, IL-1β incubation for 1 h).

[0076] 5. Washing the plate: Remove the liquid k from the wells, add 1×washing buffer at 300 μL / well; let stand for 1 minute, then discard the liquid in the wells. Repeat 3 times, patting dry on filter paper each time.

[0077] 6. Add enzyme: Add 100 μL / well of diluted Streptavidin-HRP working solution. Cover with sealing film and incubate at room temperature (18-25℃) for 20 minutes.

[0078] 7. Wash the plate: Repeat step 5.

[0079] 8. Color development: Add 100 μL / well of TMB and incubate at room temperature (18-25℃) in the dark for 5-30 minutes. Terminate the reaction based on the intensity of the color (deep blue) in the wells. Generally, 10-20 minutes of color development is sufficient for good results.

[0080] 9. Termination of reaction: Quickly add 100 μL / well of stop solution to terminate the reaction.

[0081] 10. Plate Reading: Within 10 minutes of termination, read the value using the measurement wavelength of 450nm. It is recommended to use dual wavelengths simultaneously, i.e., the measurement wavelength of 450nm and the reference wavelength or calibration wavelength of 610-630nm, for more accurate results. (ELISA reader: SpectraMaxABS).

[0082] The effects of different treatments on cells, such as Figures 2-4 As shown in Tables 2-4, by Figure 2 As shown in Table 2, LPS treatment of RAW264.7 cells significantly increased NO levels. p <0.0001), after treatment with different concentrations of Angelica dahurica exosome-like nanovesicles, the NO level was restored to varying degrees compared with the LPS group, and the overall effect was better than that of water extraction; Figure 3 As shown in Table 3, LPS induced a significant increase in IL-6 levels in RAW264.7 cells. p <0.0001, after treatment with different concentrations of Angelica dahurica exosome-like nanovesicles, the IL-6 level was significantly lower than that in the LPS group. p <0.0001), the concentration of Angelica dahurica water extract group was only 0.01 μg / ml, which was significantly lower than that of LPS group, but its effect was still not as good as that of exosome group. The results show that Angelica dahurica exosome-like nanovesicles are more effective than water-extracted Angelica dahurica. Figure 4 As shown in Table 4, LPS induced a significant increase in IL-1β levels in RAW264.7 cells. p <0.0001), after treatment with different concentrations of Angelica dahurica exosome-like nanovesicles, the IL-1β level was significantly reduced compared with the LPS group (0.01-1 μg / ml). p <0.001), the effect of water extraction was not as significant as that of exosomes, indicating that the overall effect of the Angelica dahurica exosome-like nanovesicle group was better than that of the Angelica dahurica water extraction group.

[0083] Table 2. Effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on cellular NO levels.

[0084] Group NO(μM) Group NO(μM) normal 0.4917 LPS 9.9145 Angelica dahurica exosomes 0.01 μg / ml 4.9216 Angelica dahurica water extract 0.01μg / m 10.0343 Angelica dahurica exosomes 0.1 μg / ml 5.0452 Angelica dahurica water extract 0.1μg / m 7.4315 Angelica dahurica exosomes 1μg / ml 6.0832 Angelica dahurica water extract 1μg / m 8.6365 Angelica dahurica exosomes 10μg / ml 6.4541 Angelica dahurica water extract 10μg / m 8.5156 Angelica dahurica exosomes 100μg / ml 6.9516 Angelica dahurica water extract 100 μg / m 9.5531

[0085] Table 3. Effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on the level of the cytoinflammatory factor IL-6.

[0086] Group IL-6 (pg / ml) Group IL-6 (pg / ml) normal 7.3543 LPS 122.2166 Angelica dahurica exosomes 0.01 μg / ml 41.2686 Angelica dahurica water extract 0.01μg / m 94.8384 Angelica dahurica exosomes 0.1 μg / ml 52.3598 Angelica dahurica water extract 0.1μg / m 108.4768 Angelica dahurica exosomes 1μg / ml 64.3923 Angelica dahurica water extract 1μg / m 131.7134 Angelica dahurica exosomes 10μg / ml 71.4723 Angelica dahurica water extract 10μg / m 130.8643 Angelica dahurica exosomes 100μg / ml 79.7366 Angelica dahurica water extract 100 μg / m 119.7415

[0087] Table 4. Effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on the level of the cytoinflammatory factor IL-6.

[0088] Group IL-1β (pg / ml) Group IL-1β (pg / ml) normal 3.4532 LPS 18.7286 Angelica dahurica exosomes 0.01 μg / ml 6.1648 Angelica dahurica water extract 0.01μg / m 9.9853 Angelica dahurica exosomes 0.1 μg / ml 7.0328 Angelica dahurica water extract 0.1μg / m 12.0961 Angelica dahurica exosomes 1μg / ml 9.6752 Angelica dahurica water extract 1μg / m 15.9127 Angelica dahurica exosomes 10μg / ml 13.2096 Angelica dahurica water extract 10μg / m 16.6535 Angelica dahurica exosomes 100μg / ml 13.8625 Angelica dahurica water extract 100 μg / m 18.0862

[0089] Experimental Example 3

[0090] Animal experiments:

[0091] C57bl / 6J mice were divided into a normal group (normal mouse culture group), a modeling group (AR group), an exosome-like nanovesicle group (low (3.2 μg / ml), medium (16 μg / ml), and high (80 μg / ml) dose treatment groups of the Angelica dahurica exosome-like nanovesicle sample obtained in Example 1), and an aqueous extract group (low (3.2 μg / ml), medium (16 μg / ml), and high (80 μg / ml) dose treatment groups of the boiled water extract sample).

[0092] Modeling steps:

[0093] 1. On days 0, 7, and 14, mice in the modeling group (AR group), exosome-like nanovesicle group (low (3.2 μg / ml), medium (16 μg / ml), and high (80 μg / ml) dose treatment groups of the Angelica dahurica exosome-like nanovesicle sample obtained in Example 1), and water extract group (low (3.2 μg / ml), medium (16 μg / ml), and high (80 μg / ml) dose treatment groups of the boiling water extract sample) were intraperitoneally injected with 200 μl of sensitizing solution for basal sensitization. The normal control group was injected with an equal volume of physiological saline.

[0094] 2.21–27 days, mice in the modeling group (AR group), exosome-like nanovesicle group (low (3.2 μg / ml), medium (16 μg / ml), and high (80 μg / ml) dose treatment groups of the Angelica dahurica exosome-like nanovesicle sample obtained in Example 1), and water extract group (low (3.2 μg / ml), medium (16 μg / ml), and high (80 μg / ml) dose treatment groups of the boiling water extract sample) were subjected to daily intranasal stimulation (10 μl / side) to maintain a continuous inflammatory state. The normal control group received an equal volume of physiological saline instead of OVA. All treatment groups received the medication 30 minutes before nasal instillation.

[0095] 3. On day 27, nasal symptoms were recorded for 15 minutes after the last intranasal OVA provocation to calculate the frequency of nasal friction and nasal sneezing.

[0096] 4.28 days later, the mice were euthanized and samples were collected.

[0097] The effects of different treatments on sneezing and nose-scratching symptoms induced by ovalbumin (OVA) stimulation in mice are shown in Table 5.

[0098] Table 5. Effects of each group on sneezing and nose-scratching symptoms induced by ovalbumin (OVA) stimulation in mice.

[0099] Group Average number of sneezes Average number of times to scratch your nose normal 1.6 4.8 Modeling 15.5 46.7 Low dose of exosomes (3.2 μg / ml) 6.5 29.5 Medium dose of exosomes (16 μg / ml) 5.5 23.3 High dose of exosomes (80 μg / ml) 7 30.6 Low-dose water extraction (3.2 μg / ml) 8.4 30.2 Medium dose of water extract (16 μg / ml) 10.3 36 Increase the dosage with water (80 μg / ml) 11.8 41.2

[0100] As shown in Table 5, the Angelica dahurica exosome-like nanovesicles extracted in this invention can effectively reduce the number of sneezes and nose scratchings produced by ovalbumin (OVA) stimulation in mice.

Claims

1. A method for extracting exosome-like nanovesicles from Angelica dahurica, comprising the following steps: S1. Grind the dried Angelica dahurica slices to 160 mesh, then add 9 times the mass volume of 0.01M phosphate buffer solution to adjust the pH to 7.2, mix well, and obtain Angelica dahurica homogenate. S2, the homogenate obtained in S1 is subjected to differential centrifugation. The supernatant is collected after centrifugation at 5000g for 1 hour. The supernatant is then centrifuged at 15000g for 2 hours, and the supernatant is collected again to obtain the differential centrifugation pretreatment solution of Angelica dahurica exosome-like nanovesicles. S3, add 0.5% sodium carboxymethyl cellulose by mass to the differential centrifugation pretreatment solution of Angelica dahurica exosome-like nanovesicles, let stand at 4℃ overnight, then centrifuge at 10000rpm for 60min, collect the precipitate, and obtain crude extract of Angelica dahurica exosome-like nanovesicles. S4. The crude extract of Angelica dahurica exosome-like nanovesicles obtained in S3 was resuspended in 5 times the volume of phosphate buffer solution to obtain a resuspension of the crude extract of Angelica dahurica exosome-like nanovesicles. S5, Sucrose density gradient centrifugation purification: 15%, 30%, 45%, and 60% sucrose solutions were added sequentially to centrifuge tubes. The resuspension obtained in S4 was added above the sucrose solution. Centrifugation was performed at 150,000g for 1 hour. The band between 30% and 45% sucrose solution was aspirated and collected in a bottle. Phosphate buffer solution with a volume of 20 times that of the aspirated band was added to the bottle. The mixture was centrifuged again at 150,000g for 1 hour to wash with sucrose. The precipitate was collected, and the angelica exosome-like nanovesicle extract purified by sucrose density gradient centrifugation was obtained.

2. The application of the Angelica dahurica exosome-like nanovesicles prepared by the method of claim 1 in the preparation of drugs for relieving allergic rhinitis.

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