Extraction method and application of extracellular vesicles of dry coptis chinensis

Abstract

The invention discloses a method for extracting extracellular vesicles of dried coptis chinensis, which comprises the following steps: step 1, taking out the dried coptis chinensis, washing sandy soil with tap water, and immersing the dried coptis chinensis with a phosphate buffer solution for 24 hours; 2, peeling the coptis chinensis in the step 1, juicing with a juicer, and filtering; 3, treating the filtrate in the step 2 by using a differential centrifugation method to obtain a crude extract of the dried coptis chinensis; step 4, carrying out primary purification on the crude extract in the step 3 by using sephadex G25; step 5, carrying out purification treatment of sucrose density gradient centrifugation on the crude extract primarily purified in the step 4 by using an ultra-speed centrifuge to obtain the dried coptis chinensis extracellular vesicles; according to the preparation method, the dry coptis chinensis extracellular vesicles with higher protein content can be successfully prepared, and the tea-support-shaped extracellular vesicles are good in stability and dispersity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a method for extracting dry Coptis chinensis extracellular vesicles. BACKGROUND

[0002] Extracellular vesicles are membrane-bound nanovesicles (50-1000 nm in diameter) released by cells through the endosomal pathway. Extracellular vesicles (EVs) contain various components such as nucleic acids, proteins and metabolites, and usually have potential therapeutic functions similar to their parent cells, making them suitable for cell-free therapy; however, the use of expensive cytokines and growth factors is required to induce cell differentiation in vitro for animal and human cells with specific functions; in addition, for certain functional cells such as stem cells, there is a lack of commercial cell lines, and progenitor cells need to be gradually isolated and induced from tissues, which makes the process more complicated.

[0003] In recent years, plant-derived exosome-like nanovesicles have received increasing attention due to their wide availability and low cost; in the research of traditional Chinese medicine, Coptis chinensis as an important heat-clearing and detoxifying medicinal material, its core active ingredient berberine has been proven to have broad-spectrum antibacterial, anti-inflammatory and intestinal flora regulation effects; however, the single berberine component has technical bottlenecks such as low bioavailability, insufficient targeting and easy metabolic degradation in practical application; and the extracellular vesicles derived from Coptis chinensis contain active ingredients such as berberine and miRNA, which have therapeutic effects, but there is still a blank in the extraction method of Coptis chinensis.

[0004] Secondly, most of the current plant extracellular vesicles are extracted by selecting fresh plants as raw materials, but due to the different harvesting times of different plants, it is impossible to meet the needs of using fresh plants. For example, if the plant is harvested in spring, there will be no fresh plants as raw materials in other seasons, but in order to be stored for a long time, the plant will be dried and treated. Therefore, it is particularly important to explore whether the long-term stored plants can still be used to extract plant extracellular vesicles, which lays a foundation for realizing large-scale production. In addition, the existing technology still has technical defects in the development of EVs from traditional Chinese medicine, such as the difficulty of traditional ultracentrifugation method to realize high-purity separation of EVs in complex matrix of traditional Chinese medicine. SUMMARY

[0005] The purpose of the present application is to provide a method for extracting dry Coptis chinensis extracellular vesicles, which can effectively solve the technical problems mentioned in the background.

[0006] To achieve the above purpose, the present application provides the following technical scheme: A method for extracting dry Coptis chinensis extracellular vesicles, comprising the following steps, Step 1: Take out the dry Coptis deltoidea and wash the sand with tap water, immerse the dry Coptis deltoidea in phosphate buffer solution (PBS) for 24 hours; Step 2: Peel the Coptis deltoidea in step 1, then juice it with a juicer and filter it; Step 3: Treat the filtrate in step 2 with differential centrifugation to obtain the crude extract of dry Coptis deltoidea; Step 4: Preliminarily purify the crude extract in step 3 with dextran gel G25; Step 5: Purify the crude extract preliminarily purified in step 4 with sucrose density gradient centrifugation by using an ultracentrifuge to obtain the extracellular vesicles of dry Coptis deltoidea; Step 6: Determine the toxicity of the extracellular vesicles of dry Coptis deltoidea in step 5 by using RAW264.7 macrophages and HUVEC cells.

[0007] Preferably, when the dry Coptis deltoidea is immersed in the phosphate buffer solution, the amount of the phosphate buffer solution and the dry Coptis deltoidea satisfies that 3 mL of the phosphate buffer solution is added to each gram of the dry Coptis deltoidea. Preferably, 120 g of the dry Coptis deltoidea is immersed in 360 mL of the phosphate buffer solution.

[0008] Preferably, in step 2, the filtration is performed by using gauze or a filter screen.

[0009] Preferably, in step 3, the filtrate in step 2 is first subjected to centrifugation twice in sequence, and the centrifugation is performed at a relative centrifugal force of 2200xg for 25 min and at a relative centrifugal force of 10000xg for 60 min, respectively, and the supernatant is taken after each centrifugation; and then the centrifugation is performed at a relative centrifugal force of 150,000xg for 90 min, and the precipitate is taken.

[0010] Preferably, in step 4, 2 mL of the crude extract in step 3 is treated each time by using dextran gel G25 (Smartdex G-25), and large-particle substances, which are generally macromolecular impurities including proteins, polysaccharides or others, are screened out.

[0011] Preferably, in step 5, the sucrose concentrations used are 8%, 30%, 45% and 60%, respectively, and the dry Coptis deltoidea extracellular vesicles are collected from the sucrose solution with a sucrose concentration of 30%-45% under the condition of a relative centrifugal force of 150,000xg for 90 min.

[0012] Preferably, in step 3, the treatment of the filtrate by using differential centrifugation is completed within 2-3 days after the filtrate in step 2 is obtained.

[0013] Preferably, in the above-mentioned steps 3 and 5, the centrifugation temperature is controlled at 4℃, and the dry Coptis chinensis extracellular vesicles (EVs) are stored at 4℃ for 3-6 months.

[0014] The dry Coptis chinensis extracellular vesicles prepared by the above-mentioned preparation method are applied to promote tissue regeneration.

[0015] Compared with the prior art, the present application has the following beneficial effects: The present application constructs an extraction method of Coptis chinensis extracellular vesicles, proves that dry Coptis chinensis can also separate extracellular vesicles, and the extracted extracellular vesicles have higher purity and content. Compared with the prior art patent CN118834817A, the present application adopts enzymolysis, differential centrifugation and sucrose gradient centrifugation, while the present application adopts differential centrifugation, dextran gel G25 and sucrose gradient centrifugation; the present application selects the fleshy part of dry Coptis chinensis instead of powder, retains the medicinal value of the original Coptis chinensis, and makes the obtained extracellular vesicles have more NTA; in addition, by filtering with dextran gel G25 before sucrose gradient centrifugation, the prepared dry Coptis chinensis extracellular vesicles have higher protein content, are basically characterized by tea tray-shaped extracellular vesicles with good stability (potential less than -10 mV, which represents stable nanoparticles) and good dispersibility (dispersibility coefficient is low, and a dispersibility coefficient less than 0.3 represents good dispersibility); and have better biocompatibility; under the action of the Coptis chinensis extracellular vesicles, there is a trend to promote the proliferation of HUVEC cells, which indicates that the Coptis chinensis extracellular vesicles have therapeutic potential to promote the growth of endothelial cells. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The figure is a particle size diagram of the dry Coptis chinensis extracellular vesicles of the present application; Figure 2 The figure is a polydispersity index diagram (PDI) of the dry Coptis chinensis extracellular vesicles of the present application; Figure 3 The figure is a potential diagram of the dry Coptis chinensis extracellular vesicles of the present application; Figure 4 The figure is a protein concentration detection diagram of the dry Coptis chinensis extracellular vesicles of the present application; Figure 5 The figure is a diagram of the change of the particle concentration of the dry Coptis chinensis extracellular vesicles with the particle size in the present application; Figure 6 The figure is an electron microscope diagram of the dry Coptis chinensis extracellular vesicles in the present application, and the scale is 100 nm; Figure 7 The figure is a cell toxicity (RAW 264.7) experimental result diagram of the dry Coptis chinensis extracellular vesicles of the present application; Figure 8The figure in the middle shows the cytotoxicity (HUVEC) experiment results of extracellular vesicles of dried Coptis chinensis in this application embodiment. Detailed Implementation

[0017] The technical solution of the present invention will be described in detail below with reference to the embodiments, but the scope of protection is not limited thereto.

[0018] Example 1: A method for extracting extracellular vesicles from dried Coptis chinensis includes the following steps: Step 1: Take out the dried Coptis chinensis, wash off the sand with tap water, and soak the dried Coptis chinensis in phosphate buffered solution (PBS) for 24 hours; When soaking dried Coptis chinensis in phosphate buffer solution, the amount of phosphate buffer solution and dried Coptis chinensis should be such that 3 mL of phosphate buffer solution is added for every gram of dried Coptis chinensis. Weigh out 60 g of dried Coptis chinensis and immerse it in 180 mL of phosphate buffer solution. Step 2: Peel the Coptis chinensis from Step 1, juice it using a juicer, and then filter it. Step 3: The filtrate from Step 2 was processed by differential centrifugation to obtain a crude extract of dried Coptis chinensis. The filtrate from step 2 was centrifuged twice, once at a relative centrifugal force of 2200×g for 25 min and once at a relative centrifugal force of 10000×g for 60 min, and the supernatant was collected from both centrifugations. Then, the precipitate was collected after centrifugation at a relative centrifugal force of 150,000×g for 90 min. Step 4: Perform preliminary purification of the crude extract from Step 3 using dextran gel G25; Use Smartdex G-25 dextran gel to process 2 mL of the crude extract in step 3 each time to remove large particles. Large particles are generally high molecular weight impurities, including proteins, polysaccharides or other impurities. Step 5: The crude extract that has undergone preliminary purification in Step 4 is purified by sucrose density gradient centrifugation using an ultracentrifuge to obtain dried Coptis chinensis extracellular vesicles. Centrifugation temperature was controlled at 4℃. Sucrose mixed with dried Coptis chinensis extracellular vesicles (EVs) was selected and stored at 4℃ for 3-6 months. The sucrose concentrations used were 8%, 30%, 45% and 60%, respectively. The sucrose concentrations were centrifuged for 90 min under a relative centrifugal force of 150,000×g, and the extracellular vesicles of dried Coptis chinensis cells were collected from sucrose solutions with sucrose concentrations of 30%-45%. Step 6: Determine the toxicity of the extracellular vesicles of Coptis chinensis obtained in Step 5 using RAW264.7 macrophages and HUVEC cells.

[0019] Particle size and PDI determination: The dried Coptis chinensis extracellular vesicles (EVs) prepared in Example 1 were diluted with ultrapure water, and the particle size, polydispersity index (PDI) and potential of the EVs were detected using a Malvern particle size and potential analyzer. Experimental results: The particle size detected by the laser diffractometer (NANO ZS90) was 318.56±3.33 nm, the PDI was 0.15±0.03, and the potential was -36.63±0.27 mV. Figures 1-3 The above basic characterization indicates that extracellular vesicles were successfully prepared and extracted in Example 1 of the present invention. Protein concentration determination: The extracellular vesicles of dried Coptis chinensis extracted in Example 1 were diluted 1-fold and added to 96-well plates, 20 μl per well, with three replicates per group. 200 μl of a protein quantification kit (BCA kit) was added to each well, and the plates were incubated at 37°C for 30 min. Finally, the absorbance of the samples at 562 nm was measured using a microplate reader. A standard curve was plotted with the standard concentration as the x-axis and the OD value of the standard wells as the y-axis. The formula for the standard curve was derived, and the protein content of the extracellular vesicles of dried Coptis chinensis was calculated based on the formula. Experimental results: Protein concentration was 0.65 ± 0.05 mg / mL ( Figure 4 The high protein concentration measured in this invention indicates that the extracted extracellular vesicles of Coptis chinensis contain a large number of protein particles, and the yield of extracellular vesicles of Coptis chinensis in this invention is high. Transmission electron microscope (TEM) imaging: Place a copper grid on adhesive paper, fix the extracellular vesicles of dried Coptis chinensis from Example 1 with glutaraldehyde, then add 5-10 μl of the dried Coptis chinensis extracellular vesicle solution to the copper grid and let it stand for 10 min (waiting for adsorption), then remove excess sample from the copper grid with filter paper, then add 10 μl of 2% phosphotungstic acid staining solution to the copper grid and stain for 2 min, carefully remove excess staining solution with filter paper; dry the copper grid under an infrared lamp, and finally observe the morphology of EVs using a transmission electron microscope. Experimental results: Figure 6 As can be seen from the electron microscopy morphology, the extracellular vesicles (EVs) of dried Coptis chinensis conform to the saucer-like structure unique to exosomes; this basic characterization proves that the present invention has successfully completed the extraction of extracellular vesicles. Nanoparticle Tracking Analysis (NTA): Take 10 μl of the dried Coptis chinensis extracellular vesicles (EVs) prepared in Example 1 above and analyze them using Zetaview software to obtain the particle concentration within a specific particle size range. Experimental results: From Figure 5 The results showed that the extracellular vesicle (EV) size of the diluted dried Coptis chinensis was 171 nm, and the total concentration before dilution was 2.1 × 10⁻⁶. 11 particles / mL, which is consistent with the basic characteristics of plant exosome-like nanovesicles; Cytotoxicity assay: The toxic effects of different concentrations of dried Coptis chinensis extracellular vesicles (EVs) prepared in Example 1 on RAW264.7 and HUVECs were studied using the Methylthiazolyldiphenyl-tetrazolium bromide (MTT) method. HUVECs and RAW264.7 cells in logarithmic growth phase were digested and then subjected to 8 × 10⁻⁶ ions / mL of cytotoxicity assay. 3 Cells were seeded at a density of 100 μl of cell suspension in each well of a 96-well plate. After cell attachment and reaching 80% confluence, 100 μL of extracellular vesicle solutions containing protein concentrations of 0, 4, 8, 16, 32, and 64 μg / mL of Coptis chinensis were added to each well. After incubation with the extracellular vesicles for 48 h, the extracellular vesicles were removed, and 100 μL of 1 mg / mL MTT solution was added. After incubation for 4 h, the MTT solution was removed. Then, 100 μL of DMSO was added to each well, and the plate was shaken at room temperature for 15 min until the formazan was completely dissolved. The absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated. The principle of the MTT assay relies on the activity of intracellular succinate dehydrogenase; this enzyme catalyzes the reduction of MTT molecules within the cell, forming water-insoluble blue-violet formazan crystals, which then deposit within the cell. Living cells possess this enzyme's function and are therefore able to produce formazan crystals, while dead cells lack this ability. Dimethyl sulfoxide (DMSO) is used to dissolve the formazan crystals within the cells; the absorbance of the dissolved formazan is measured using a microplate reader at wavelengths of 490 nm or 570 nm. Experimental results: From Figure 7 and Figure 8 Extracellular vesicles of dried Coptis chinensis at protein concentrations between 0 and 64 μg / mL did not induce cytotoxicity in RAW264.7 and HUVEC cells. Figure 8The results showed that the extracellular vesicles of dried Coptis chinensis at a protein concentration of 64 μg / mL promoted the proliferation of HUVEC cells, indicating that the extracellular vesicles of dried Coptis chinensis prepared by us have certain application significance such as promoting tissue regeneration. Finally, it should be noted that the above 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 with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for extracting extracellular vesicles from dried Coptis chinensis, characterized in that, Includes the following steps: Step 1: Take out the dried Coptis chinensis, wash off the sand with tap water, and immerse the dried Coptis chinensis in phosphate buffer solution; Step 2: Peel the Coptis chinensis from Step 1, juice it using a juicer, and then filter it. Step 3: The filtrate from Step 2 was processed by differential centrifugation to obtain a crude extract of dried Coptis chinensis. Step 4: Perform preliminary purification of the crude extract from Step 3 using dextran gel G25; Step 5: The crude extract that has undergone preliminary purification in Step 4 is purified by sucrose density gradient centrifugation using an ultracentrifuge to obtain dried Coptis chinensis extracellular vesicles. Step 6: Determine the toxicity of the extracellular vesicles of Coptis chinensis obtained in Step 5 using RAW264.7 macrophages and HUVEC cells.

2. The method for extracting extracellular vesicles from dried Coptis chinensis according to claim 1, characterized in that, Weigh out dried Coptis chinensis and immerse it in phosphate buffer solution for 24 hours. The amount of phosphate buffer solution and dried Coptis chinensis should be such that 3 mL of phosphate buffer solution is added for every gram of dried Coptis chinensis.

3. The method for extracting extracellular vesicles from dried Coptis chinensis according to claim 1, characterized in that, In step 2, the filter is made with gauze or a filter screen.

4. The method for extracting extracellular vesicles from dried Coptis chinensis according to claim 1, characterized in that, In step 3, the filtrate from step 2 is centrifuged twice, with a relative centrifugal force of 2200×g for 25 min and a relative centrifugal force of 10000×g for 60 min, and the supernatant is collected after each centrifugation. Then, the precipitate is collected after centrifugation at a relative centrifugal force of 150,000×g for 90 min.

5. The method for extracting extracellular vesicles of dried Coptis chinensis according to claim 1, wherein in step 4, the crude extract in step 3 is treated with dextran gel G25 to remove high molecular weight proteins, polysaccharides or other impurities.

6. The method for extracting extracellular vesicles from dried Coptis chinensis according to claim 1, characterized in that, In step 5, the sucrose concentrations used were 8%, 30%, 45% and 60%, respectively. The sucrose concentrations were centrifuged for 90 min under a relative centrifugal force of 150,000×g, and the extracellular vesicles of dried Coptis chinensis cells were collected from the sucrose solution with a sucrose concentration of 30%-45%.

7. The method for extracting extracellular vesicles from dried Coptis chinensis according to claim 1, characterized in that, In step 3, after obtaining the filtrate from step 2, the filtrate is processed using differential centrifugation within 2-3 days.

8. The method for extracting extracellular vesicles from dried Coptis chinensis according to claim 1, characterized in that, In steps 3 and 5, the centrifugation temperature is controlled at 4°C, and the sucrose-mixed dried Coptis chinensis extracellular vesicles are stored at 4°C for 3-6 months.

9. The application of the dried Coptis chinensis extracellular vesicles prepared by the preparation method according to any one of claims 1-8 in promoting tissue regeneration.

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