Sterile extraction method of Chinese herbal medicine exosome

Abstract

The invention relates to a sterile extraction method of a Chinese herbal medicine exosome. The method comprises the following steps: soaking a Chinese herbal medicine in a solution containing antibiotics, and extracting the exosome. The sterile extraction method of the Chinese herbal medicine exosome can maintain the original properties of the exosome, provides guarantee for subsequent application of the exosome, is simple and safe to operate, and is suitable for Chinese herbal medicines with various properties.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome separation, and in particular to a method for aseptically extracting exosomes from Chinese herbal medicines. Background Art

[0002] Exosomes are tiny vesicles (30-150nm) secreted by cells. They are mainly derived from multivesicular bodies formed by the invagination of intracellular lysosomal particles. After the multivesicular outer membrane fuses with the cell membrane, it is released into the extracellular matrix and can transfer DNA, RNA or protein between cells, thereby affecting the function of the recipient cells. The function of exosomes depends on the cell type from which they come, and can participate in the body's immune response, antigen presentation, cell migration, cell differentiation, tumor invasion and other aspects.

[0003] Exosomes from Chinese herbal medicines are often extracted by ultracentrifugation, but since Chinese herbal medicines themselves contain bacteria, exosomes obtained by direct pretreatment and ultracentrifugation often carry microorganisms. Traditional methods often choose to filter and sterilize to obtain sterile exosomes, but this method has a very high loss rate, is not conducive to obtaining full-sized exosomes, and is time-consuming and labor-intensive. Therefore, it is possible to consider sterilizing Chinese herbal medicines from the source.

[0004] Sterilization of Chinese herbal medicines includes moist heat sterilization, irradiation sterilization, microwave sterilization and ozone sterilization. Among them, moist heat sterilization is not suitable for the sterilization of special Chinese medicines such as those that are not heat-resistant and easily hydrolyzed. It will change the appearance color of the Chinese medicine, reduce the content of volatile oils, and increase the moisture content. In particular, it will cause the Chinese medicine powder to agglomerate, become damp, and even exceed the moisture content. Irradiation sterilization has high regulatory requirements and needs to be selected with caution. Microwave sterilization is not suitable for products with low water content. Ozone sterilization has many influencing factors and is limited to the sterilization of medicines that do not contain easily oxidizable ingredients. CN113355272A discloses a method for extracting plant exosomes, which is not sterilized, which is not conducive to practical application. CN113502256A discloses a method for extracting sterile exosomes, but this method is only for the sterile extraction method of animal cell supernatant, and the sterile extraction method of plant-derived exosomes is not described in detail. Summary of the invention

[0005] Problem that the invention aims to solve

[0006] In view of the above problems existing in the prior art, the object of the present invention is to provide a sterile extraction method for exosomes of most Chinese herbal medicines.

[0007] Solutions for solving problems

[0008] The present invention provides a method for aseptically extracting exosomes from Chinese herbal medicines. The method comprises soaking the Chinese herbal medicines in a solution containing antibiotics and then extracting the exosomes.

[0009] Preferably, the method comprises the following steps:

[0010] (1) Clean the Chinese herbal medicines and soak them in a solution containing antibiotics.

[0011] Preferably, the antibiotic in step (1) is selected from one or more of penicillin, streptomycin, amphotericin B, gentamicin, azithromycin and clindamycin.

[0012] Preferably, the final concentration of the solution containing antibiotics in step (1) is 0.1-5×.

[0013] Preferably, the method further comprises the steps of:

[0014] (2) discarding the waste water, washing the Chinese herbal medicines soaked in step (1), and squeezing out the juice after adding a solvent;

[0015] (3) collecting the squeezed juice obtained in step (2), and placing it into a sterilized centrifuge tube after pretreatment;

[0016] (4) collecting the supernatant A obtained in step (3) and centrifuging at high speed;

[0017] (5) Collecting the supernatant B obtained in step (4), performing ultracentrifugation, and retaining the precipitate, which is the Chinese herbal exosomes.

[0018] Preferably, the solvent in step (2) is sterile PBS.

[0019] Preferably, the pretreatment in step (3) comprises multiple low-speed centrifugation.

[0020] Preferably, the sterilized centrifuge tube in step (3) is sterilized by heating at high temperature and high pressure.

[0021] Preferably, the ultracentrifugation in step (5) comprises two ultracentrifugations.

[0022] Preferably, the instruments and containers used in the method are sterilized.

[0023] Effects of the Invention

[0024] The aseptic extraction method of Chinese herbal medicine exosomes of the present invention can maintain the original properties of the exosomes, provide a guarantee for the subsequent application of the exosomes, and is simple and safe to operate, and is applicable to Chinese herbal medicines of various properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Flow chart of the extraction method.

[0026] Figure 2 This is a transmission electron microscopy image of the exosomes in Example 1.

[0027] Figure 3 This is a particle size distribution curve of exosomes in Example 1.

[0028] Figure 4 This is the gel electrophoresis protein band diagram of exosomes in Example 1, where the PC group is the positive control.

[0029] Figure 5 The total colony count test results of exosomes prepared in Example 1 (left) and Comparative Example 1 (right). DETAILED DESCRIPTION

[0030] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0031] As used herein, the term "final concentration" refers to the concentration of a solute in a solution. For example, in certain embodiments, the final concentration is the concentration of the sum of all antibiotics in the solution.

[0032] The present invention provides a method for aseptically extracting exosomes from Chinese herbal medicines. The method comprises soaking the Chinese herbal medicines in a solution containing antibiotics and then extracting the exosomes.

[0033] In certain embodiments, the method comprises the steps of:

[0034] (1) Clean the Chinese herbal medicines and soak them in a solution containing antibiotics.

[0035] In certain embodiments, the antibiotic in step (1) is selected from one or more of penicillin, streptomycin, amphotericin B, gentamicin, azithromycin, and clindamycin.

[0036] In certain embodiments, the antibiotic in step (1) is selected from one or more of penicillin, streptomycin, and amphotericin B.

[0037] In certain embodiments, the antibiotics in step (1) are penicillin, streptomycin and amphotericin B.

[0038] In certain embodiments, the antibiotic in step (1) is a triple antibiotic of penicillin-streptomycin-amphotericin B 100×.

[0039] In certain embodiments, the solvent of the solution in step (1) is sterile water.

[0040] In certain embodiments, the final concentration of the solution containing the antibiotic in step (1) is 0.1-5×.

[0041] In certain embodiments, the final concentration of the solution containing the antibiotic in step (1) is 0.5-2×.

[0042] In certain embodiments, the final concentration of the solution containing the antibiotic in step (1) is 1×.

[0043] In certain embodiments, the cleaning in step (1) is performed using sterile water.

[0044] In certain embodiments, the soaking in step (1) is for 6-24 hours.

[0045] In certain embodiments, the soaking in step (1) is for 12 hours.

[0046] In certain embodiments, step (1) comprises washing the Chinese herbal medicine with sterile water for three times and then transferring the mixture into a sterile bottle, adding sterile water into the bottle on a clean bench, adding a mixed solution of penicillin-streptomycin-amphotericin B and soaking the mixture overnight.

[0047] In certain embodiments, the Chinese herbal medicine is delivered to the production workshop after being treated with air blowing.

[0048] In certain embodiments, the method further comprises the steps of:

[0049] (2) discarding the waste water, washing the Chinese herbal medicines soaked in step (1), and squeezing out the juice after adding a solvent;

[0050] (3) collecting the squeezed juice obtained in step (2), and placing it into a sterilized centrifuge tube after pretreatment;

[0051] (4) collecting the supernatant A obtained in step (3) and centrifuging at high speed;

[0052] (5) collecting the supernatant B obtained in step (4), performing ultracentrifugation, and retaining the precipitate, which is the Chinese herbal exosomes.

[0053] In certain embodiments, the cleaning in step (2) is performed using sterile water.

[0054] In certain embodiments, the solvent in step (2) is sterile PBS.

[0055] In certain embodiments, the juicing in step (2) is performed for 5-10 minutes.

[0056] In certain embodiments, step (2) includes discarding the waste water, washing the Chinese herbal medicine soaked in step (1) with sterile water 1-2 times, adding sterile PBS in a clean bench, transferring to a sterilized wall-breaking machine, sealing the opening with a sealing film, and squeezing the juice for 5-10 minutes.

[0057] In certain embodiments, the disinfection is 75% alcohol disinfection.

[0058] In certain embodiments, the sterilization is ultraviolet sterilization.

[0059] In certain embodiments, the pretreatment in step (3) comprises multiple low-speed centrifugation.

[0060] In certain embodiments, the pretreatment in step (3) comprises a first low-speed centrifugation, a second low-speed centrifugation, and a third low-speed centrifugation.

[0061] In certain embodiments, the parameters of the first low-speed centrifugation are 4°C, 500g, and 10 min.

[0062] In certain embodiments, the parameters of the second low-speed centrifugation are 4° C., 2000 g, and 20 min.

[0063] In certain embodiments, the parameters of the third low-speed centrifugation are 4°C, 4000g, and 30 min.

[0064] In certain embodiments, the sterilized centrifuge tube in step (3) is sterilized by heating at high temperature and high pressure.

[0065] In certain embodiments, the high temperature and high pressure sterilization is sterilization at 121° C. for 30 min.

[0066] In certain embodiments, step (3) includes collecting the squeezed juice obtained in step (2) in a clean bench, transferring it to a sterile centrifuge tube, sealing the opening with a sealing film after it is filled, and then performing a first low-speed centrifugation, a second low-speed centrifugation, and a third low-speed centrifugation.

[0067] In certain embodiments, the parameters of the high-speed centrifugation in step (4) are 4°C, 10,000 g, and 60 min.

[0068] In certain embodiments, step (4) comprises collecting the supernatant A obtained in step 3 in a clean bench, transferring it to a sterile centrifuge tube sterilized by high temperature and high pressure heating, and centrifuging it at high speed.

[0069] In certain embodiments, the ultracentrifugation in step (5) comprises two ultracentrifugations.

[0070] In certain embodiments, the ultracentrifugation parameters in step (5) are 4°C, 100,000 g, and 70 min.

[0071] In certain embodiments, step (5) includes collecting the supernatant B obtained in step (4), performing a first ultracentrifugation, retaining the precipitate, resuspending and performing a second ultracentrifugation, and retaining the precipitate, which is the Chinese herbal exosomes.

[0072] In certain embodiments, the resuspension in step (5) is performed using sterile PBS.

[0073] In certain embodiments, the instruments and containers used in the method are sterilized.

[0074] In certain embodiments, the sterilization treatment includes alcohol disinfection, ultraviolet sterilization, and high temperature and high pressure heating sterilization.

[0075] In certain embodiments, the method is performed completely aseptically.

[0076] In certain embodiments, the Chinese herbal medicine is a botanical medicine.

[0077] In certain embodiments, the method further comprises the steps of:

[0078] (6) The Chinese herbal exosomes obtained in step (5) are stored at -80°C.

[0079] In certain embodiments, the storage in step (6) is freeze-dried storage.

[0080] In certain embodiments, the storage in step (6) is freeze-dried storage in the presence of a freeze-protectant.

[0081] The present invention also provides sterile Chinese herbal medicine exosomes prepared according to the method.

[0082] The present invention also provides a pharmaceutical composition, which comprises the sterile Chinese herbal medicine exosomes prepared according to the method.

[0083] The present invention also provides an application of the sterile Chinese herbal medicine exosomes prepared according to the method, or the pharmaceutical composition in the preparation of daily chemical products or medicines.

[0084] Example 1: Aseptic extraction of exosomes from white peony root

[0085] S1. Weigh 100g of white peony tablets, wash them 3 times with ultrapure water to remove impurities, place them in a beaker wiped with 75% alcohol and irradiated with ultraviolet light, place them in a clean room through a transfer window, and wash them again 3 times with sterile water in the clean bench in the workshop. Then transfer them to a sterile plastic bottle, add 495mL of sterile water and 5mL of penicillin-streptomycin-amphotericin B solution (100×, penicillin content 10kU / ml, streptomycin content 10mg / ml, amphotericin B content 25μg / ml, purchased from Solebow, product number P7630-100ml), the final concentration of the mixed solution is 1×, and the Chinese herbal medicine is not covered to ensure that the three antibodies are fully penetrated and soaked overnight.

[0086] S2. Discard the overnight water, wash three times with sterile water in the clean bench, add 500mL sterile PBS, and put it in a wall-breaking machine that has been disinfected with 75% alcohol and sterilized with ultraviolet light in advance. Seal the opening with sealing film, and squeeze the juice for 5-10 minutes to ensure that the white peony slices are completely broken.

[0087] S3. Collect all the squeezed juice in the clean bench and transfer it to a sterile 50mL centrifuge tube, make sure it is full, seal the opening with a sealing film, and remove dead cells, large plant particles and vesicles by low-speed centrifugation pretreatment. The pretreatment mainly includes three centrifugations. The parameters of the first centrifugation are: 4℃, 500g, 10min, the purpose is to remove dead cells; the parameters of the second centrifugation are: 4℃, 2000g, 20min, the purpose is to remove large plant particles; the parameters of the third centrifugation are: 4℃, 4000g, 30min, the purpose is to remove large plant vesicles.

[0088] S4. Package the centrifuge tubes required for high-speed centrifugation and ultracentrifugation in sterilization bags and sterilize them in a high-temperature and high-pressure autoclave (121°C, 30 min). Dry them and set them aside for use.

[0089] S5. In the clean bench of the workshop, the supernatant collected in step S3 is placed into the centrifuge tube required for the high-speed centrifugation, and a 1 / 1000 balance is used for balancing. A 1 / 1000 analytical balance is sterilized in advance and placed in the clean bench of the workshop to facilitate sample balancing and ensure that the operation process is completely sterile. After being tightly packaged, it is placed in the rotor of the high-speed centrifuge for centrifugation, and the centrifugation parameters are: 4°C, 10000g, 60min.

[0090] S6. After centrifugation, transfer the centrifuge tube to the clean bench in the workshop, discard the precipitate, collect the supernatant, transfer it to the centrifuge tube required for ultracentrifugation, and balance it with a 1 / 1000 analytical balance. The 1 / 1000 analytical balance is sterilized in advance and placed in the clean bench in the workshop to facilitate sample balancing and ensure that the operation process is completely sterile. After tight packaging, put it into the ultracentrifuge rotor for centrifugation. The centrifugation parameters are: 4°C, 100,000g, 70min.

[0091] S7. After centrifugation, transfer the centrifuge tube to the clean bench in the workshop, discard the supernatant, keep the precipitate, add sterile PBS to resuspend, and balance it with a 1 / 1000 analytical balance. The 1 / 1000 analytical balance is sterilized in advance and placed in the clean bench in the workshop to facilitate sample balancing and ensure that the operation process is completely sterile. After tight packaging, put it into the ultracentrifuge rotor and centrifuge it again. The centrifugation parameters are: 4°C, 100,000g, 70min.

[0092] S8. After centrifugation, transfer the centrifuge tube to the clean bench in the workshop, discard the supernatant, and retain the precipitate to obtain sterile white peony exosomes. Transfer the obtained sterile exosomes to a sterile EP tube, seal it tightly with a clean sealing film, and store it at -80°C for a short term. For long-term storage, add a lyophilization protective agent and freeze-dry.

[0093] Example 2: Aseptic extraction of white ginseng exosomes

[0094] S1. Weigh 100g of white ginseng slices, wash them 3 times with ultrapure water to remove impurities, place them in a beaker wiped with 75% alcohol and irradiated with ultraviolet light, place them in a clean room through a transfer window, and wash them again 3 times with sterile water in the clean bench in the workshop. Then transfer them to a sterile plastic bottle, add 495mL of sterile water and 5mL of penicillin-streptomycin-amphotericin B solution (100×), and the final concentration of the mixed solution is 1×, which covers the Chinese herbal medicine to ensure that the three antibodies are fully penetrated and soaked overnight.

[0095] S2. Discard the overnight water, wash with sterile water in the clean bench for 3 times, add 800mL sterile PBS, and put it in a wall-breaking machine that has been disinfected with 75% alcohol and UV sterilized in advance. Seal the opening with sealing film, and squeeze the juice for 5-10 minutes to ensure that the white ginseng slices are completely broken.

[0096] S3. Collect all the squeezed juice in the clean bench and transfer it to a sterile 50mL centrifuge tube, make sure it is full, seal the opening with a sealing film, and remove dead cells, large plant particles and vesicles by low-speed centrifugation pretreatment. The pretreatment mainly includes three centrifugations. The parameters of the first centrifugation are: 4℃, 500g, 10min, the purpose is to remove dead cells; the parameters of the second centrifugation are: 4℃, 2000g, 20min, the purpose is to remove large plant particles; the parameters of the third centrifugation are: 4℃, 4000g, 30min, the purpose is to remove large plant vesicles.

[0097] S4. Package the centrifuge tubes required for high-speed centrifugation and ultracentrifugation in sterilization bags and sterilize them in a high-temperature and high-pressure autoclave (121°C, 30 min). Dry them and set them aside for use.

[0098] S5. In the clean bench of the workshop, the supernatant collected in step S3 is placed into the centrifuge tube required for the high-speed centrifugation, and a 1 / 1000 balance is used for balancing. A 1 / 1000 analytical balance is sterilized in advance and placed in the clean bench of the workshop to facilitate sample balancing and ensure that the operation process is completely sterile. After being tightly packaged, it is placed in the rotor of the high-speed centrifuge for centrifugation, and the centrifugation parameters are: 4°C, 10000g, 60min.

[0099] S6. After centrifugation, transfer the centrifuge tube to the clean bench in the workshop, discard the precipitate, collect the supernatant, transfer it to the centrifuge tube required for ultracentrifugation, and balance it with a 1 / 1000 analytical balance. The 1 / 1000 analytical balance is sterilized in advance and placed in the clean bench in the workshop to facilitate sample balancing and ensure that the operation process is completely sterile. After tight packaging, put it into the ultracentrifuge rotor for centrifugation. The centrifugation parameters are: 4°C, 100,000g, 70min.

[0100] S7. After centrifugation, transfer the centrifuge tube to the clean bench in the workshop, discard the supernatant, keep the precipitate, add sterile PBS to resuspend, and balance it with a 1 / 1000 analytical balance. The 1 / 1000 analytical balance is sterilized in advance and placed in the clean bench in the workshop to facilitate sample balancing and ensure that the operation process is completely sterile. After tight packaging, put it into the ultracentrifuge rotor and centrifuge it again. The centrifugation parameters are: 4°C, 100,000g, 70min.

[0101] S8. After centrifugation, transfer the centrifuge tube to the clean bench in the workshop, discard the supernatant, and retain the precipitate to obtain sterile white ginseng exosomes. Transfer the obtained sterile exosomes to a sterile EP tube, seal it tightly with a clean sealing film and store it at -80°C for short-term storage. For long-term storage, add a lyophilization protective agent and freeze-dry.

[0102] Example 3: Identification of sterile exosomes

[0103] The sterile exosomes prepared in Example 1 were identified, and the identification results were as follows: Figures 2 to 4 And as shown in Table 1.

[0104] Figure 2 Transmission electron microscopy was used to directly observe the morphology of exosomes. The results showed that the exosomes were successfully extracted, with a complete exosome structure and a cup-shaped morphology.

[0105] Figure 3 To analyze the diameter and concentration of sterile white peony exosome particles by microfluidic resistive pulse sensing (MRPS), the results showed that the exosome particle size distribution was widely distributed, mainly in the range of 65-250nm, and the particle concentration was 1.14E+14particles / ml.

[0106] Figure 4By detecting the expression of sterile white peony exosome protein by SDS-PAGE, it can be seen that the proteins expressed in the exosomes extracted in the present invention are mainly distributed in 15-25 kDa, 25-35 kDa, 45-kDa and 55-70 kDa. In addition, compared with the white peony exosomes obtained without triple antibody treatment (untreated group), there is no significant change in protein distribution, proving that this method does not affect the distribution of active proteins, thereby proving the effectiveness of the method of the present invention.

[0107] Table 1: Exosome microbial detection results prepared in Example 1

[0108] Serial number Test items unit Test Results 1 Total colony count CFU / mL ＜10 2 Total mold and yeast count CFU / mL ＜10 3 Thermotolerant coliform bacteria / mL Not detected 4 Staphylococcus aureus / mL Not detected 5 Pseudomonas aeruginosa / mL Not detected

[0109] The above results indicate that the white peony exosomes obtained by this method are sterile exosomes.

[0110] Comparative Example 1: Conventional extraction of exosomes from white peony root

[0111] S1. Weigh 100g of white peony root slices, wash them three times with ultrapure water to remove impurities, place them in a plastic bottle, add 500mL of ultrapure water to cover the Chinese herbal medicine, ensure full penetration and soak overnight.

[0112] S2. Discard the overnight water, add 500 mL PBS, transfer to a wall-breaking machine, and squeeze the juice for 5-10 minutes to ensure that the white peony slices are completely broken.

[0113] S3. Collect all the squeezed juice and transfer it to a 50mL centrifuge tube, making sure it is full, and remove dead cells, large plant particles and vesicles by low-speed centrifugation pretreatment. The pretreatment mainly includes three centrifugations. The first centrifugation parameters are: 4℃, 500g, 10min, the purpose is to remove dead cells; the second centrifugation parameters are: 4℃, 2000g, 20min, the purpose is to remove large plant particles; the third centrifugation parameters are: 4℃, 4000g, 30min, the purpose is to remove large plant vesicles.

[0114] S4, the supernatant collected in step S3 is placed into the centrifuge tube required by the high-speed centrifuge, and the tube is balanced with a 1 / 1000 scale. After being tightly packaged, the tube is placed into the rotor of the high-speed centrifuge for centrifugation, and the centrifugation parameters are: 4°C, 10000g, 60min.

[0115] S5. After centrifugation, discard the precipitate, collect the supernatant, transfer it to the centrifuge tube required by the ultracentrifuge, and balance it with a 1 / 1000 analytical balance. After sealing tightly, load it into the ultracentrifuge rotor for centrifugation, and the centrifugation parameters are: 4°C, 100000g, 70min.

[0116] S6. After centrifugation, discard the supernatant, keep the precipitate, add PBS to resuspend, and balance it with a 1 / 1000 analytical balance. After sealing tightly, put it into the ultracentrifuge rotor and centrifuge it again. The centrifugation parameters are: 4℃, 100000g, 70min.

[0117] S7. After centrifugation, discard the supernatant and keep the precipitate to obtain white peony exosomes. Transfer the exosomes into an EP tube, seal it tightly with a clean sealing film and store it at -80℃ for short-term storage. For long-term storage, add a lyophilization protective agent and freeze-dry.

[0118] Example 4: Detection of total microbial colony count in exosomes

[0119] The total number of microbial colonies was detected for the exosomes prepared in Example 1 and Comparative Example 1. The detection method was performed in accordance with Chapter 5.2 of the "Technical Specifications for Safety of Cosmetics" (2015 Edition). The results are as follows: Figure 5 As shown, the left side is the result of Example 1, and no colony growth was observed in the plate; the right side is the result of Comparative Example 1, and no colonies were counted in the plate, which proves that the extraction method of the embodiment can significantly reduce the microorganisms carried by exosomes compared with the conventional extraction method.

[0120] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A method for aseptic extraction of exosomes from Chinese herbal medicine, characterized in that: The method comprises soaking the Chinese herbal medicine in a solution containing antibiotics and then extracting exosomes.

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) Clean the Chinese herbal medicines and soak them in a solution containing antibiotics.

3. The method according to claim 2, characterized in that The antibiotic in step (1) is selected from one or more of penicillin, streptomycin, amphotericin B, gentamicin, azithromycin and clindamycin.

4. The method according to claim 2, characterized in that: The final concentration of the solution containing antibiotics in step (1) is 0.1-5×.

5. The method according to claim 2, characterized in that: The method further comprises the steps of: (2) discarding the waste water, washing the Chinese herbal medicines soaked in step (1), and squeezing out the juice after adding a solvent; (3) collecting the squeezed juice obtained in step (2), and placing it into a sterilized centrifuge tube after pretreatment; (4) collecting the supernatant A obtained in step (3) and centrifuging at high speed; (5) collecting the supernatant B obtained in step (4), performing ultracentrifugation, and retaining the precipitate, which is the Chinese herbal exosomes.

6. The method according to claim 5, characterized in that The solvent in step (2) is sterile PBS.

7. The method according to claim 5, characterized in that The pretreatment in step (3) includes multiple low-speed centrifugation.

8. The method according to claim 5, characterized in that The sterilized centrifuge tube in step (3) is sterilized by heating at high temperature and high pressure.

9. The method according to claim 5, characterized in that The ultracentrifugation in step (5) includes two ultracentrifugations.

10. The method according to any one of claims 1 to 9, characterized in that The instruments and containers used in the method are sterilized.

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