Extraction method and application of angelica dahurica exosome-like nano vesicles

Abstract

The invention belongs to the technical field of plant extraction, and particularly relates to an extraction method and application of angelica dahurica exosome-like nano vesicles. Compared with the traditional extraction method, the method provided by the invention has the advantages that the equipment requirement is low and the cost is low by combining the differential centrifugation method with the sodium carboxymethyl cellulose; the method has the characteristics of simple operation, low cost, high treatment capacity and high flexibility, the contents of protein and RNA in the extracted angelica dahurica exosome-like nano-vesicles can respectively reach 47.9% and 3.12%, which are respectively improved by 15.9% and 16.34% compared with the traditional method, and the sneezing number and nose scratching frequency generated by mice stimulated by ovalbumin (OVA) can be effectively reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and in particular relates to an extraction method of angelica dahurica exosome-like nanovesicles and application thereof. Background Art

[0002] Allergic rhinitis, also known as allergic rhinitis (AR), is a chronic non-infectious inflammation of the Th2 type nasal mucosa mediated by specific immunoglobulin E (IgE) caused by human contact with allergens. It is prone to cause 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 Umbelliferae family. In addition to its analgesic and antioxidant pharmacological effects, it also has anti-inflammatory effects and is often used to treat allergic rhinitis. The traditional Angelica dahurica preparations required for the treatment or relief of allergic rhinitis are generally extracted by boiling water or ethanol extraction. The active ingredients are generally Angelica dahurica flavonoids, phenols and terpenes. However, whether it is water extraction or ethanol extraction, the extraction efficiency is limited; and the proteins, fats and miRNAs in Angelica dahurica cannot be extracted, resulting in a certain waste of resources; and there is little literature on the extraction of Angelica dahurica exosome-like nanovesicles and their specific effects on the human body.

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

[0005] In order to solve the above technical problems, the present invention provides an extraction method of Angelica dahurica exosome-like nanovesicles and its application. Angelica dahurica exosome-like nanovesicles are obtained by using differential centrifugation, flocculant-assisted and gradient sucrose centrifugation. 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: S1, ultrafinely grinding the raw material of Angelica dahurica and then adding phosphate buffer solution for homogenization to obtain Angelica dahurica homogenate; S2, subjecting the homogenate obtained in S1 to differential centrifugation to obtain a differential centrifugation pretreatment solution of Angelica dahurica exosome-like nanovesicles; S3, adding sodium carboxymethyl cellulose to the Angelica dahurica exosome-like nanovesicle differential centrifugation pretreatment solution, standing at 4°C overnight, centrifuging, collecting the precipitate, and obtaining a crude extract of Angelica dahurica exosome-like nanovesicles; S4, resuspending 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; S5, sucrose density gradient centrifugation purification: add 15-60% sucrose solution into a centrifuge tube, add the resuspension obtained in S4 onto the sucrose solution for 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 the sucrose, collect the precipitate, and obtain the Angelica dahurica exosome-like nanovesicle extract purified by sucrose density gradient centrifugation.

[0007] Preferably, in S1, the Angelica dahurica raw material is dried Angelica dahurica slices, which are crushed to 120-200 meshes using an ultrafine grinder; after crushing, 8-10 times the mass volume of phosphate buffer solution is added and mixed to obtain an Angelica dahurica homogenate; the pH of the phosphate buffer solution is 7.2, and the concentration is 0.01M.

[0008] Preferably, in S2, the differential centrifugation is: first centrifugation at 2000-5000g for 0.5-1 hour to collect the supernatant, then the supernatant is centrifuged at 6000-15000g for 1-2 hours to collect the supernatant again, and the supernatant is filtered to obtain the Angelica dahurica exosome-like nanovesicle differential centrifugation pretreatment liquid.

[0009] Preferably, the filtration is to filter the supernatant through a 0.45 μm water filter membrane.

[0010] Preferably, in S3, the amount of sodium carboxymethyl cellulose added is 0.05-2% of the mass of the system; and the centrifugation is performed at 10,000 rpm for 60 minutes.

[0011] Preferably, in 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.

[0012] Preferably, in S5, the sucrose concentrations of 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 band between the 30%-45% sucrose solution absorbed; and the mixture is centrifuged at 100,000-150,000 g for 1 to 2 hours.

[0013] In a second aspect of the present invention, there is provided a use of the Angelica dahurica exosome-like nanovesicles prepared by the above method in alleviating allergic rhinitis.

[0014] Preferably, the Angelica dahurica exosome-like nanovesicles can reduce the sneezing and nose scratching symptoms of allergic rhinitis caused by ovalbumin (OVA) stimulation.

[0015] 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).

[0016] The beneficial effects of the present invention are: (1) The method for extracting Angelica dahurica exosome-like nanovesicles provided by the present invention can achieve protein and RNA contents of 47.9% and 3.12% respectively in the extracted Angelica dahurica exosome-like nanovesicles, which are 15.9% and 16.34% higher than those of the traditional method, greatly improving the protein and RNA contents; (2) The present invention uses differential centrifugation combined with sodium carboxymethyl cellulose. Compared with the traditional extraction method, the method of the present invention has the advantages of low equipment requirements and low cost; large processing volume and high flexibility; it can avoid mechanical damage, protect sample activity, improve precipitation specificity, and is easy to separate; (3) The exosome-like nanovesicles extracted from Angelica dahurica in the present invention can effectively reduce the number of sneezes and nose scratching caused by ovalbumin (OVA) stimulation in the nose of mice, and can effectively relieve the symptoms of allergic rhinitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of transmission electron microscopy and particle size detection of Angelica dahurica exosome-like nanovesicles; Figure 2 The effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on cellular NO levels; Figure 3 The effect of Angelica dahurica exosome-like nanovesicles and boiling water extract on the level of inflammatory factor IL-6; Figure 4 This is the effect of Angelica dahurica exosome-like nanovesicles and boiling water extract on the level of cellular inflammatory factor IL-1β. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0019] Example 1 A method for extracting exosome-like nanovesicles of Angelica dahurica comprises the following steps: S1, using an ultrafine grinder to grind the dried Angelica dahurica slices into about 160 mesh; after grinding, add 9 times the mass volume of Angelica dahurica 0.01M phosphate buffer solution (pH=7.2) and mix well to obtain Angelica dahurica homogenate; S2, subjecting the homogenate obtained in S1 to differential centrifugation, centrifuging at 5000 g for 1 hour to collect the supernatant, and centrifuging the supernatant at 15000 g for 2 hours to collect the supernatant again, to obtain the Angelica dahurica exosome-like nanovesicle differential centrifugation pretreatment solution; S3, adding 0.5% of the system weight of sodium carboxymethyl cellulose to the Angelica dahurica exosome-like nanovesicle differential centrifugation pretreatment solution, standing at 4°C overnight, centrifuging at 10000 rpm for 60 minutes, collecting the precipitate, and obtaining a crude sample of Angelica dahurica exosome-like nanovesicles; S4, resuspending the crude extract of Angelica dahurica exosome-like nanovesicles with 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 to the centrifuge tube in sequence, the resuspension obtained in S4 was added to the top of the sucrose solution, centrifuged at 150,000 g for 1 hour, the bands between 30% and 45% sucrose solutions were aspirated and collected in a bottle, 20 times the volume of the aspirated bands was added with phosphate buffer solution, and centrifuged again at 150,000 g for 1 hour to wash with sucrose, and the precipitate was collected to obtain the Angelica dahurica exosome-like nanovesicle extract purified by sucrose density gradient centrifugation.

[0020] The transmission electron microscopy results and particle size detection results of the obtained Angelica dahurica exosome-like nanovesicle extract are shown in the figure. Figure 1 It can be seen that the structure of the detected Angelica dahurica exosome-like nanovesicles is "teacup-shaped", with an average particle size of 258.4nm, which is consistent with the characteristics of plant exosomes.

[0021] Example 2 The difference from Example 1 is that in S3, the amount of sodium carboxymethyl cellulose added is 1% of the body mass, and the other steps are the same as Example 1.

[0022] Example 3 The difference from Example 1 is that in S3, the amount of sodium carboxymethyl cellulose added is 2% of the body mass, and the other steps are the same as Example 1.

[0023] Example 4 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 the supernatant is collected again after centrifugation at 6000g for 2 hours. The other steps are the same as in Example 1.

[0024] Comparative Example 1 The difference from Example 1 is that step S3 is not performed, and step S4 is performed directly, and the other steps are the same as those in Example 1.

[0025] Comparative Example 2 The difference from Example 1 is that in S3, polyvinyl alcohol is used to replace sodium carboxymethyl cellulose, and the other steps are the same as those in Example 1.

[0026] Comparative Example 3 The difference from Example 1 is that S3 does not use sodium carboxymethyl cellulose, but uses ultracentrifugation, specifically: the Angelica dahurica exosome-like nanovesicle differential centrifugation pretreatment liquid is ultracentrifuged at 100000g for 60 minutes, and the precipitate is collected.

[0027] Comparative Example 4 The difference from Comparative Example 3 is that sodium carboxymethyl cellulose is added after ultracentrifugation in S3, and the added amount is the same as that in Example 1, and the other steps are the same as those in Comparative Example 3.

[0028] Test Example 1 The BCA protein concentration determination method was used to determine the protein content in the Angelica dahurica exosome-like nanovesicles obtained in Examples 1-4 and Comparative Examples 1-4. The results are shown in Table 1.

[0029] Table 1 Protein content in the exosome-like nanovesicles of Angelica dahurica extracted from various examples and comparative examples Group Protein content in extracted exosome-like nanovesicles (%) RNA content in extracted 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

[0030] According to Table 1, the protein content in the Angelica dahurica exosome-like nanovesicles extracted in Example 1 is the highest, reaching 47.9%, and the RNA content is 3.12%. Compared with the Angelica dahurica exosome-like nanovesicles extracted by differential centrifugation + ultracentrifugation method, the protein content is increased by 15.9%, and the RNA content is increased by 16.34%.

[0031] Test Example 2 Cell experiments Mouse mononuclear macrophage leukemia cells (RAW264.7) were cultured in plastic culture dishes in DMEM high glucose (Genview) supplemented with 10% fetal calf serum (Applied Biosystems) and 1% double antibody.

[0032] The 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 the exosome-like nanovesicle sample obtained in Example 1), and a water extract group (the group treated with the boiling water extraction sample). RAW264.7 cells with good growth and a fusion rate of 80% were digested and made into a cell suspension and inoculated into a 24-well cell culture plate (Fisher). After the cells were completely attached to the wall, a mixture of 250 ng / ml LPS and the exosome sample of Angelica dahurica or the water extract sample of Angelica dahurica was added for 24 hours, and the cell culture supernatants were collected respectively, and the levels of NO, IL-6, and IL-1β were determined according to the ELISA instructions.

[0033] NO detection kit: 1. Take out Griess Reagent Ⅰ and Ⅱ and return them to room temperature.

[0034] 2. Dilute the standard (1-100µM) in the same solution as the samples to be tested.

[0035] 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 can usually be 0, 1, 2, 5, 10, 20, 40, 60, 100µM.

[0036] 3. Add standards and samples to the 96-well plate at 50 µl / well.

[0037] If the sample is the culture supernatant, it can be sampled directly. If there is a precipitate, it needs to be centrifuged and the supernatant needs to be taken. If the sample is a cell or tissue, it can be quickly frozen and thawed, and then centrifuged to precipitate and take the supernatant. If the volume is less than 50µl, it can be diluted with redistilled water or 0.9% NaCl (correspondingly, the standard should also be diluted with redistilled water or 0.9% NaCl). Cells or tissues can also be used for Western or IP lysis buffer (no inhibitors need to be added) lysis, and the standard should also be diluted accordingly.

[0038] 4. Add 50 µl / well of room temperature Griess Reagent I to each well.

[0039] 5. Add 50 µl / well of room temperature Griess Reagent II to each well.

[0040] 6. Measure absorbance at 540 nm.

[0041] 7. Calculate the concentration of nitric oxide in the sample based on the standard curve.

[0042] ELISA kit method: (DAKEWEI) 1. Before use, mix all reagents thoroughly to avoid foaming. Reagents should be stored according to the instructions on the bottle label and equilibrated at room temperature for 20-30 minutes before use.

[0043] 2. Determine the number of strips required based on the number of experimental wells (blanks and standards).

[0044] 3. Sample addition: Add 100 μL / well of diluted Cytokine standard to the standard well, add 100 μL / well of sample to the sample well, set up a blank well, and replace the sample and standard with Dilution buffer R (1×).

[0045] 4. Add detection antibody: Add diluted Biotinylated antibody at 50 μL / well. After mixing, cover with sealing film and incubate at room temperature (18-25°C) (IL-6, IL-1β incubate for 1 h).

[0046] 5. Wash the plate: remove the liquid K in the wells, add 300μL / well 1× washing buffer; leave for 1 minute and then discard the liquid in the wells. Repeat 3 times, and dry on filter paper each time.

[0047] 6. Add enzyme: 100 μL / well of diluted Streptavidin-HRP working solution. Cover with sealing film and incubate at room temperature (18-25°C) for 20 minutes.

[0048] 7. Wash the plate: Repeat step 5.

[0049] 8. Color development: Add TMB at 100 μL / well, incubate at room temperature (18-25°C) in the dark for 5-30 minutes, and terminate the reaction based on the depth of the color in the well (dark blue). Usually, a color development of 10-20 minutes can achieve good results.

[0050] 9. Stop the reaction: Quickly add 100 μL / well Stop solution to stop the reaction.

[0051] 10. Plate reading: Read the value at a detection wavelength of 450nm within 10 minutes after termination. It is recommended to use dual wavelengths, i.e., a detection wavelength of 450nm and a reference wavelength or calibration wavelength of 610-630nm, to read the plate simultaneously, so that the measurement results will be more accurate. (Microplate reader: SpectraMaxABS).

[0052] Effects of different treatments on cells Figure 2-Figure 4 As shown in Tables 2-4, Figure 2 As shown in Table 2, LPS treatment of RAW264.7 cells induced a significant increase in 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 the IL-6 level in RAW264.7 cells ( p <0.0001), after treatment with different concentrations of Angelica dahurica exosome-like nanovesicles, the level of IL-6 was significantly lower than that of the LPS group ( p<0.0001), the concentration of Angelica dahurica water-extracted group was only 0.01μg / ml significantly lower than that of LPS group, but its effect was still not as good as that of exosome group. The results showed that Angelica dahurica exosome-like nanovesicles were more effective than water-extracted Angelica dahurica. Figure 4 As shown in Table 4, LPS induced a significant increase in the level of IL-1β in RAW264.7 cells ( p <0.0001), after treatment with different concentrations of Angelica dahurica exosome-like nanovesicles, the level of IL-1β was significantly reduced compared with the LPS group (0.01-1μg / ml, p <0.001), the water extraction effect was not as significant as that of exosomes, indicating that the Angelica dahurica exosome-like nanovesicle group was better than the Angelica dahurica water extraction group as a whole.

[0053] Table 2 Effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on cellular NO levels Group NO(μM) Group NO(μM) normal 0.4917 LPS 9.9145 Angelica dahurica exosomes 0.01μg / ml 4.9216 Angelica dahurica water extract 0.01 μg / m 10.0343 Angelica dahurica exosomes 0.1μg / ml 5.0452 Angelica dahurica water extract 0.1 μg / m 7.4315 Angelica dahurica exosomes 1μg / ml 6.0832 Angelica dahurica water extract 1 μg / m 8.6365 Angelica dahurica exosomes 10μg / ml 6.4541 Angelica dahurica water extract 10 μg / m 8.5156 Angelica dahurica exosomes 100μg / ml 6.9516 Angelica dahurica water extract 100 μg / m 9.5531

[0054] Table 3 Effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on the level of inflammatory factor IL-6 Group IL-6 (pg / ml) Group IL-6 (pg / ml) normal 7.3543 LPS 122.2166 Angelica dahurica exosomes 0.01μg / ml 41.2686 Angelica dahurica water extract 0.01 μg / m 94.8384 Angelica dahurica exosomes 0.1μg / ml 52.3598 Angelica dahurica water extract 0.1 μg / m 108.4768 Angelica dahurica exosomes 1μg / ml 64.3923 Angelica dahurica water extract 1 μg / m 131.7134 Angelica dahurica exosomes 10μg / ml 71.4723 Angelica dahurica water extract 10 μg / m 130.8643 Angelica dahurica exosomes 100μg / ml 79.7366 Angelica dahurica water extract 100 μg / m 119.7415

[0055] Table 4 Effects of Angelica dahurica exosome-like nanovesicles and boiling water extract on the level of inflammatory factor IL-6 Group IL-1β (pg / ml) Group IL-1β (pg / ml) normal 3.4532 LPS 18.7286 Angelica dahurica exosomes 0.01μg / ml 6.1648 Angelica dahurica water extract 0.01 μg / m 9.9853 Angelica dahurica exosomes 0.1μg / ml 7.0328 Angelica dahurica water extract 0.1 μg / m 12.0961 Angelica dahurica exosomes 1μg / ml 9.6752 Angelica dahurica water extract 1 μg / m 15.9127 Angelica dahurica exosomes 10μg / ml 13.2096 Angelica dahurica water extract 10 μg / m 16.6535 Angelica dahurica exosomes 100μg / ml 13.8625 Angelica dahurica water extract 100 μg / m 18.0862

[0056] Test Example 3 Animal Experimentation: 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 samples obtained in Example 1), and a water extract group (low (3.2 μg / ml), medium (16 μg / ml), and high (80 μg / ml) dose treatment groups of boiling water extraction samples).

[0057] Modeling steps: 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 samples 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 boiling water extract samples) were intraperitoneally injected with 200 μl of sensitization solution for basic sensitization. The normal control group was injected with an equal dose of normal saline.

[0058] 2. From day 21 to day 27, 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 samples 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 samples) were stimulated intranasally (10 μl / side) daily to maintain a continuous inflammatory state. The normal control group was replaced with an equal amount of normal saline instead of OVA. All treatment groups were given drugs 30 minutes before nasal drops.

[0059] 3. On day 27, after the last OVA intranasal challenge, nasal symptoms were recorded for 15 minutes to calculate the frequency of nasal friction and nasal sneezing.

[0060] On day 4.28, the mice were killed and samples were collected.

[0061] The effects of different treatments on sneezing and nose scratching symptoms caused by ovalbumin (OVA) stimulation in the nose of mice are shown in Table 5.

[0062] Table 5 Effects of each group on sneezing and nose scratching symptoms in mice stimulated with ovalbumin (OVA) Group Average number of sneezes Average number of nose scratching normal 1.6 4.8 Modeling 15.5 46.7 Low dose of exosomes (3.2 μg / ml) 6.5 29.5 Exosome medium dose (16μg / ml) 5.5 23.3 Exosome high dose (80μg / ml) 7 30.6 Low dose of water extraction (3.2μg / ml) 8.4 30.2 Medium dose of water extraction (16μg / ml) 10.3 36 Water increased dose (80μg / ml) 11.8 41.2

[0063] As shown in Table 5, the Angelica dahurica exosome-like nanovesicles extracted by the present invention can effectively reduce the number of sneezes and nose scratching caused by ovalbumin (OVA) stimulation in the nose of mice.

Claims

1. A method for extracting exosome-like nanovesicles from Angelica dahurica, characterized in that: The following steps are involved: S1, ultrafinely grinding the raw material of Angelica dahurica and then adding phosphate buffer solution for homogenization to obtain Angelica dahurica homogenate; S2, subjecting the homogenate obtained in S1 to differential centrifugation to obtain a differential centrifugation pretreatment solution of Angelica dahurica exosome-like nanovesicles; S3, adding sodium carboxymethyl cellulose to the Angelica dahurica exosome-like nanovesicle differential centrifugation pretreatment solution, standing at 4°C overnight, centrifuging, collecting the precipitate, and obtaining a crude extract of Angelica dahurica exosome-like nanovesicles; S4, resuspending 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; S5, sucrose density gradient centrifugation purification: add 15-60% sucrose solution into a centrifuge tube, add the resuspension obtained in S4 onto the sucrose solution for 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 the sucrose, collect the precipitate, and obtain the Angelica dahurica exosome-like nanovesicle extract purified by sucrose density gradient centrifugation.

2. The method according to claim 1, characterized in that In S1, the Angelica dahurica raw material is dried Angelica dahurica slices, which are crushed to 120-200 meshes by an ultrafine grinder; the volume of the phosphate buffer solution added is 8-10 times the mass of the Angelica dahurica powder; the pH of the phosphate buffer solution is 7.2, and the concentration is 0.01M.

3. The method according to claim 1, characterized in that In S2, the differential centrifugation is as follows: the supernatant is collected by first centrifugation at 2000-5000g for 0.5-1 hour, and then the supernatant is collected again by centrifugation at 6000-15000g for 1-2 hours, and the supernatant is filtered to obtain the Angelica dahurica exosome-like nanovesicle differential centrifugation pretreatment liquid.

4. The method according to claim 3, characterized in that The filtration is performed by passing the supernatant through a 0.45 μm water filter membrane.

5. The method according to claim 1, characterized in that In S3, the amount of sodium carboxymethyl cellulose added is 0.05-2% of the system mass.

6. The method according to claim 1, characterized in that In S3, centrifugation was performed at 10,000 rpm for 60 minutes.

7. The method according to claim 1, characterized in that In S4, the volume of the phosphate buffer solution was 4 to 6 times the weight of the crude extract of Angelica dahurica exosome-like nanovesicles.

8. The method according to claim 1, characterized in that In S5, the ultra-high speed centrifugation is performed at 100,000-150,000 g for 1-2 hours.

9. The method according to claim 1, characterized in that In S5, the sucrose concentrations of 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 for washing the sucrose is 15 to 20 times the volume of the band between the 30% and 45% sucrose solution absorbed.

10. Use of the Angelica dahurica exosome-like nanovesicles prepared by the method according to any one of claims 1 to 9 in alleviating allergic rhinitis.

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