Preparation method and application of jujube extracellular vesicles
Extracellular vesicles of jujube were extracted from fresh jujubes by multi-stage centrifugation and treatment with paraformaldehyde and dithiothreitol. This solved the problem of the application of extracellular vesicles of jujube in anti-inflammatory and immunomodulatory drugs, and achieved highly efficient anti-inflammatory and immunomodulatory effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN TRADITIONAL CHINESE MEDICINE HOSPITAL
- Filing Date
- 2025-12-25
- Publication Date
- 2026-05-08
AI Technical Summary
There is no existing research on the application of extracellular vesicles derived from jujube in anti-inflammatory and immunomodulatory aspects, and there is a lack of effective preparation methods.
Extracellular vesicles of jujube were extracted from fresh jujube homogenate using a multi-stage centrifugation method combined with paraformaldehyde and dithiothreitol. Impurities were removed by centrifugation filtration and incubation to obtain purified extracellular vesicles.
The prepared jujube cell extracellular vesicles have anti-inflammatory and immunomodulatory activities, can significantly inhibit the release of inflammatory factors, and promote the transformation of macrophages to M2 type, and can be applied to anti-inflammatory and immunomodulatory drugs.
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Figure CN121991877A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing extracellular vesicles of jujube cells and their application. Background Technology
[0002] Extracellular vesicles (EVs) are nanoscale membrane-bound vesicles secreted by cells, capable of carrying bioactive molecules such as proteins, nucleic acids, and lipids, playing a crucial role in intercellular communication. In recent years, plant-derived EVs have attracted widespread attention due to their wide availability, high safety profile, and diverse biological activities. The efficacy of plant EVs is closely related to their source. Existing research has found that ginseng-derived EVs have immunomodulatory effects, ginger-derived EVs have anti-inflammatory effects, and bellflower-derived EVs have anti-tumor effects. Currently, there are no reports of anti-inflammatory and immunomodulatory effects from jujube-derived EVs.
[0003] Jujube (Ziziphus jujuba Mill.) is a traditional Chinese medicine used for both food and medicine, possessing the effects of tonifying the middle energizer, replenishing qi, nourishing blood, and calming the mind. The *Shennong Bencao Jing* records that it "treats evil qi in the heart and abdomen, soothes the middle energizer and nourishes the spleen, assists the twelve meridians, regulates stomach qi, opens the nine orifices, replenishes qi and body fluids, treats deficiencies in the body, fright, heaviness in the limbs, and harmonizes with all medicines." Modern research shows that jujube contains abundant polysaccharides, flavonoids, and triterpenoids, exhibiting various pharmacological activities such as antioxidant, immunomodulatory, and anti-inflammatory effects. Currently, there are diverse methods for extracting plant extracellular vesicles. Given the high yield of jujube, the effective extraction methods and applications of its extracellular vesicles are of significant value. Summary of the Invention
[0004] The technical problem to be solved by this invention is: a method for preparing extracellular vesicles of jujube cells and their application in anti-inflammatory drugs and immunomodulatory drugs.
[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing extracellular vesicles of jujube cells, comprising the following steps: Fresh jujubes were homogenized and then added to PBS (phosphate-buffered saline) for multi-stage centrifugation to obtain crude extract of jujube extracellular vesicles. The crude extract of jujube extracellular vesicles was incubated with paraformaldehyde and dithiothreitol in PBS, and then centrifuged and filtered to obtain jujube extracellular vesicles.
[0006] Another technical solution adopted in this invention is the application of jujube extracellular vesicles in the preparation of anti-inflammatory drugs.
[0007] Another technical solution adopted in this invention is the application of jujube extracellular vesicles in the preparation of immunomodulatory drugs.
[0008] The beneficial effects of this invention are as follows: the method for preparing jujube extracellular vesicles of this invention has the characteristics of stable process, low cost, and good biosafety, and has broad application prospects in the pharmaceutical field. The prepared jujube extracellular vesicles can be used in anti-inflammatory drugs and immunomodulatory drugs, providing technical support for the development of novel natural-derived drugs. Attached Figure Description
[0009] Figure 1 This is a morphological diagram of extracellular vesicles in jujube cells under transmission electron microscopy in an embodiment of the present invention. Figure 2 This invention provides an example of using DLS (Dynamic Light Scattering) to detect the extracellular vesicle size distribution in jujube cells. Figure 3 This is a diagram showing the surface potential of extracellular vesicles in jujube cells detected by zeta potential (electrokinetic potential) in an embodiment of the present invention. Figure 4 This is a bar graph illustrating the effect of extracellular vesicles of jujube cells inhibiting the release of inflammatory factors from macrophages in an embodiment of the present invention. Figure 5 This is a statistical graph showing the results of flow cytometry detection of macrophage M2 phenotype transformation in an embodiment of the present invention. Detailed Implementation
[0010] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.
[0011] A method for preparing extracellular vesicles of jujube cells, comprising the following steps: Fresh jujubes were homogenized and then added to PBS for multi-stage centrifugation to obtain crude extract of jujube extracellular vesicles. The crude extract of jujube extracellular vesicles was incubated with paraformaldehyde and dithiothreitol in PBS, and then centrifuged and filtered to obtain jujube extracellular vesicles.
[0012] As can be seen from the above description, the beneficial effects of this invention are as follows: The method for preparing jujube extracellular vesicles involves homogenizing fresh jujubes, centrifuging to obtain a crude extract, then removing impurities through multi-stage centrifugation combined with incubation with paraformaldehyde and dithiothreitol, and finally purifying the jujube extracellular vesicles by centrifugation and filtration. The preparation method of this invention is simple and feasible, and the obtained product exhibits clear activity, providing a new technical approach for the high-value utilization of jujube resources.
[0013] Furthermore, this includes the following steps: S1: Remove the pits from fresh jujubes, crush them into a homogenate, add PBS, filter and centrifuge, collect the supernatant to obtain crude jujube extract; S2: The crude jujube extract is centrifuged and concentrated to obtain a concentrated solution. After centrifugation of the concentrated solution, the supernatant is collected and centrifuged again to obtain jujube extracellular vesicle precipitate. The jujube extracellular vesicle precipitate is added to PBS buffer and subjected to impingement and filtration to obtain crude jujube extracellular vesicle extract. S3: After centrifuging the crude extract of jujube extracellular vesicles, the precipitate was resuspended and then incubated with paraformaldehyde and dithiothreitol in PBS. After incubation, the extract was centrifuged and filtered to obtain jujube extracellular vesicles.
[0014] Furthermore, the centrifugation conditions in S1 are: centrifugation at 8000~12000g for 20~30min.
[0015] Furthermore, the crude extract of jujube should be stored at -25 to -15°C.
[0016] As described above, vesicles are composed of a lipid bilayer and are highly sensitive to temperature, freeze-thaw cycles, and repeated agitation. Storing them at -25°C to -15°C can maximize their structural integrity and functional activity, preventing vesicle rupture, aggregation, or leakage of contents caused by repeated freeze-thaw cycles or prolonged high temperatures.
[0017] Furthermore, the centrifugation temperature for both S1 and S2 is 3~5℃.
[0018] As can be seen from the above description, centrifugation at low temperatures can prevent excessively high temperatures from damaging the integrity of the vesicles.
[0019] Further, S2 specifically involves: concentrating the crude jujube extract by centrifugation at 2300-2700g for 12-18 minutes to obtain a concentrated solution; collecting the supernatant after centrifuging the concentrated solution at 8000-12000g for 8-12 minutes; centrifuging the supernatant at 48000-52000g for 80-100 minutes to obtain the jujube extracellular vesicle precipitate; adding PBS buffer to the jujube extracellular vesicle precipitate and agitating it, then filtering to obtain the crude jujube extracellular vesicle extract.
[0020] Further, S2 specifically involves: concentrating the crude jujube extract at 2300-2700g for 12-18 minutes at 3-5℃ to obtain a concentrated solution; collecting the supernatant after centrifuging the concentrated solution at 8000-12000g for 8-12 minutes at 3-5℃; centrifuging the supernatant at 48000-52000g for 80-100 minutes at 3-5℃ to obtain jujube extracellular vesicle precipitate; adding PBS buffer to the jujube extracellular vesicle precipitate, refluxing, and then filtering to obtain the crude jujube extracellular vesicle extract.
[0021] As described above, the crude jujube extract of the present invention undergoes low-speed pre-centrifugation to precipitate and concentrate larger impurities such as vesicles and cell debris, reducing the volume requiring ultracentrifugation and increasing the vesicle recovery rate. Then, it is centrifuged at a lower speed to remove impurities. Next, it is centrifuged at a higher speed to achieve solid-liquid separation, obtaining jujube extracellular vesicle precipitate. The jujube extracellular vesicle precipitate is added to PBS buffer and agitated, then filtered to remove bacteria, thus obtaining the crude jujube extracellular vesicle extract.
[0022] S2 uses differential centrifugation to separate extracellular vesicles from cells, cell debris, and other impurities.
[0023] Further, S3 specifically involves: centrifuging the crude extract of jujube extracellular vesicles at 200-240g for 4-6 min to obtain a precipitate; resuspending the precipitate in PBS and incubating it with paraformaldehyde and dithiothreitol in PBS; collecting the supernatant by centrifugation at 200-240g for 4.5-5.5 min after incubation; collecting the supernatant by centrifugation at 9000-10000g for 13-17 min and collecting the supernatant again; concentrating the supernatant by centrifugation at 3300-3700g for 12-18 min; and washing with PBS to obtain jujube extracellular vesicles.
[0024] As described above, after centrifuging and resuspending the crude jujube extract, S3 was incubated with paraformaldehyde and dithiothreitol in PBS. After incubation, the extract was first centrifuged at a lower centrifugal force and then centrifuged at a higher centrifugal force to screen out the supernatant containing plant extracellular vesicles. Then, the extract was centrifuged, concentrated, and washed to remove impurities and obtain jujube extracellular vesicles.
[0025] Furthermore, the centrifugation temperature for S3 is room temperature, which is 20~25℃.
[0026] As can be seen from the above description, paraformaldehyde and dithiothreitol are added to S3 to maintain the integrity and stability of the vesicles, and they can be centrifuged at room temperature.
[0027] Further, S3 specifically involves: centrifuging the crude extract of jujube extracellular vesicles at 200-240g for 4-6 min to obtain a precipitate; resuspending the precipitate and incubating it with paraformaldehyde and dithiothreitol in PBS; after incubation, centrifuging at 200-240g for 4.5-5.5 min at room temperature to collect the supernatant; centrifuging the supernatant at 9000-10000g for 13-17 min at room temperature and collecting the supernatant again; then concentrating the supernatant by centrifugation at 3300-3700g for 12-18 min at room temperature, and finally washing with PBS to obtain jujube extracellular vesicles.
[0028] Furthermore, the concentration of paraformaldehyde was 1% (w / v) and the molar concentration of dithiothreitol was 3 mM.
[0029] Furthermore, the incubation conditions in S3 are as follows: incubation at 35~39℃ and 4.5~5.5% CO2 for 2.5~3.5h.
[0030] Furthermore, the particle size of extracellular vesicles in jujube cells ranges from 50 nm to 200 nm.
[0031] Another technical solution adopted in this invention is the application of jujube extracellular vesicles in the preparation of anti-inflammatory drugs.
[0032] Another technical solution adopted in this invention is the application of jujube extracellular vesicles in the preparation of immunomodulatory drugs.
[0033] As can be seen from the above description, the extracellular vesicles of jujube prepared by the present invention have typical vesicle morphological characteristics. Electron microscopy shows that they have a typical cup-shaped morphology, can significantly inhibit the release of inflammatory factors, promote the transformation of macrophages to M2 type, and have good anti-inflammatory and immunomodulatory activities.
[0034] Another technical solution adopted in this invention is: a pharmaceutical composition comprising jujube extracellular vesicles and a pharmaceutically acceptable carrier.
[0035] Furthermore, the dosage form of the pharmaceutical composition is an injection, an oral solution, or a topical preparation.
[0036] Example 1 of the present invention is: a method for preparing extracellular vesicles of jujube cells, the specific steps of which are as follows: S1: Remove the pits from 50g of fresh jujubes, crush them into a homogenate, add 200mL of PBS, filter and centrifuge at 10000g for 25min at 4℃, collect the supernatant, obtain the crude jujube extract and store it at -20℃. S2: The crude extract of jujube was concentrated by centrifugation at 2500g for 15 min at 4℃ to obtain a concentrate; the concentrate was then centrifuged at 10000g for 10 min at 4℃ and the supernatant was collected; the supernatant was then centrifuged at 50000g for 90 min at 4℃ to obtain the jujube extracellular vesicle precipitate; 10 mL of PBS buffer was added to the jujube extracellular vesicle precipitate and the mixture was pipetted, then filtered through a 0.22 μm filter membrane to obtain the crude extract of jujube extracellular vesicles. S3: Centrifuge the crude extract of jujube extracellular vesicles at 220g for 5min to obtain a precipitate; add 5mL PBS to the precipitate to resuspend it and obtain a suspension; add 1% (w / v) paraformaldehyde and 3mM dithiothreitol and incubate at 37℃ and 5% CO2 for 3h; after incubation, centrifuge at 220g for 5min at room temperature to collect the supernatant; centrifuge the supernatant at 9500g for 15min at room temperature and collect the supernatant again; then concentrate it at 3500g for 15min using a 100kDa centrifuge filter at room temperature; wash with 2mL PBS; and repeat the washing twice with an equal volume of PBS to obtain jujube extracellular vesicles.
[0037] Example 2 of the present invention is a method for preparing extracellular vesicles of jujube cells, the specific steps of which are as follows: S1: Remove the pits from 50g of fresh jujubes, crush them into a homogenate, add 200mL of PBS, filter and centrifuge at 8000g for 30min at 3℃, collect the supernatant, obtain the crude jujube extract and store it in an environment of -25℃. S2: The crude extract of jujube was concentrated by centrifugation at 2300g for 18 min at 3℃ to obtain a concentrate; the concentrate was then centrifuged at 8000g for 12 min at 3℃ and the supernatant was collected; the supernatant was then centrifuged at 48000g for 100 min at 3℃ to obtain the jujube extracellular vesicle precipitate; 10 mL of PBS buffer was added to the jujube extracellular vesicle precipitate and the mixture was pipetted, then filtered through a 0.22 μm filter membrane to obtain the crude extract of jujube extracellular vesicles. S3: Centrifuge the crude extract of jujube extracellular vesicles at 200g for 6min to obtain a precipitate; ... Add 5mL PBS to the precipitate to resuspend it and obtain a suspension. Add 1% (w / v) paraformaldehyde and 3mM dithiothreitol and incubate at 35℃ and 4.5% CO2 for 3.5h; After incubation, centrifuge at 200g for 5.5min at room temperature to collect the supernatant; Centrifuge the supernatant at 9000g for 17min at room temperature and collect the supernatant again. Then, concentrate the supernatant at 3300g for 18min using a 100kDa centrifuge filter at room temperature, wash with 2mL PBS, and repeat the washing once with an equal volume of PBS to obtain jujube extracellular vesicles.
[0038] Example 3 of the present invention is: a method for preparing extracellular vesicles of jujube cells, the specific steps of which are as follows: S1: Remove the pits from 50g of fresh jujubes, crush them into a homogenate, add 200mL of PBS, filter and centrifuge at 12000g for 20min at 5℃, collect the supernatant, obtain the crude jujube extract and store it at -15℃. S2: The crude extract of jujube was concentrated by centrifugation at 2700g for 12 min at 5℃ to obtain a concentrated solution; the concentrated solution was centrifuged at 12000g for 8 min at 5℃ and the supernatant was collected; the supernatant was centrifuged at 52000g for 80 min at 5℃ to obtain the jujube extracellular vesicle precipitate; 10 mL of PBS buffer was added to the jujube extracellular vesicle precipitate and the mixture was pipetted, and then filtered through a 0.22 μm filter membrane to obtain the crude extract of jujube extracellular vesicles. S3: Centrifuge the crude extract of jujube extracellular vesicles at 240g for 4min to obtain a precipitate; ... Add 5mL PBS to the precipitate to resuspend it and obtain a suspension. Add 1% (w / v) paraformaldehyde and 3mM dithiothreitol and incubate at 39℃ and 5.5% CO2 for 2.5h; After incubation, centrifuge at 240g for 4.5min at room temperature to collect the supernatant; Centrifuge the supernatant at 10000g for 13min at room temperature and collect the supernatant again. Then, concentrate the supernatant at 3700g for 12min using a 100kDa centrifuge filter at room temperature, wash with 2mL PBS, and repeat the washing with an equal amount of PBS 3 times to obtain jujube extracellular vesicles.
[0039] Example 4 of the present invention is to verify the extracellular vesicles of jujube cells prepared in Example 1.
[0040] 1. Observe the morphology of extracellular vesicles in jujube cells using transmission electron microscopy, such as... Figure 1 ; 2. The size of extracellular vesicles in jujube cells was detected using DLS, such as... Figure 2 The particle size of extracellular vesicles in jujube cells ranges from 50 nm to 400 nm. 3. The surface potential of extracellular vesicles in jujube cells was detected using zeta potential, such as... Figure 3 .
[0041] Example 5 of the present invention is: evaluating the anti-inflammatory activity of the jujube extracellular vesicles prepared in Example 1. RAW264.7 macrophages in the logarithmic growth phase were used for cell counting, and the cell density was adjusted to 5 × 10⁻⁶. 5 Cell suspension was plated into 12-well plates at a density of 1 mL / well, with 100 ng / mL lipopolysaccharide (LPS) added to each well for cell modeling. Jujube extracellular vesicles (EVVs) were then added, and the plates were incubated for 48 hours. Cell supernatants were collected and analyzed by ELISA (enzyme-linked immunosorbent assay). ELISA results showed that jujube extracellular vesicles significantly inhibited the release of inflammatory factors such as TNF-α and IL-6. Figure 4 .
[0042] Example 6 of the present invention is: evaluating the immunomodulatory activity of the jujube extracellular vesicles prepared in Example 1. RAW264.7 macrophages in the logarithmic growth phase were used for cell counting, and the cell density was adjusted to 5 × 10⁻⁶. 5 Cell suspension was plated into 12-well plates at a density of 1 mL / well, with 40 ng / mL of STI (interleukin-4 + 40 ng / mL interleukin-13) added to induce cell modeling. 100 μg / mL of jujube cell extracellular vesicles were also added. Cells were cultured in a cell culture incubator for 48 h, and cells were collected. Flow cytometry fixative was added to the cells, and the cells were incubated at room temperature in the dark for 20 min, followed by washing once with 1 mL of PBS. After washing, flow cytometry permeabilization buffer was added, and the cells were incubated at room temperature in the dark for 10 min, followed by washing once with 1 mL of PBS. Flow cytometry antibody (CD206, 5 μL / sample) was added, and the cells were incubated at room temperature in the dark for 30 min, followed by washing twice with 1 mL of PBS. The cells were resuspended in 500 μL of PBS and analyzed by flow cytometry. Results are shown in [Figure number missing]. Figure 5 The results showed that extracellular vesicles of jujube cells can promote the transformation of macrophages into the M2 type.
[0043] In summary, the method for preparing extracellular vesicles of jujube provided by this invention and its application have the following advantages: 1. The preparation method is simple and feasible, and the activity of the obtained product is well-defined.
[0044] 2. Jujube extracellular vesicles can significantly inhibit the release of inflammatory factors and promote the transformation of macrophages into M2 type, exhibiting good anti-inflammatory and immunomodulatory activities.
[0045] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.
Claims
1. A method for preparing extracellular vesicles of jujube cells, characterized in that, Includes the following steps: Fresh jujubes were homogenized and then added to PBS for multi-stage centrifugation to obtain crude extract of jujube extracellular vesicles. The crude extract of jujube extracellular vesicles was incubated with paraformaldehyde and dithiothreitol in PBS, and then centrifuged and filtered to obtain jujube extracellular vesicles.
2. The method for preparing extracellular vesicles of jujube cells according to claim 1, characterized in that, Includes the following steps: S1: Remove the pits from fresh jujubes, crush them into a homogenate, add PBS, filter and centrifuge, collect the supernatant to obtain crude jujube extract; S2: The crude jujube extract is centrifuged and concentrated to obtain a concentrated solution. After centrifugation of the concentrated solution, the supernatant is collected and centrifuged again to obtain jujube extracellular vesicle precipitate. The jujube extracellular vesicle precipitate is added to PBS buffer and subjected to impingement and filtration to obtain crude jujube extracellular vesicle extract. S3: After centrifuging the crude extract of jujube extracellular vesicles, the precipitate was resuspended and then incubated with paraformaldehyde and dithiothreitol in PBS. After incubation, the extract was centrifuged and filtered to obtain jujube extracellular vesicles.
3. The method for preparing extracellular vesicles of jujube cells according to claim 2, characterized in that, The centrifugation conditions in S1 are: centrifugation at 8000~12000g for 20~30min.
4. The method for preparing extracellular vesicles of jujube cells according to claim 2, characterized in that, The centrifugation temperature of both S1 and S2 is 3~5℃.
5. The method for preparing extracellular vesicles of jujube cells according to claim 2, characterized in that, S2 specifically involves: concentrating the crude jujube extract by centrifugation at 2300-2700g to obtain a concentrated solution; collecting the supernatant after centrifuging the concentrated solution at 8000-12000g for 8-12 minutes; centrifuging the supernatant at 48000-52000g for 80-100 minutes to obtain jujube extracellular vesicle precipitate; adding PBS buffer to the jujube extracellular vesicle precipitate and agitating it, then filtering to obtain the crude jujube extracellular vesicle extract.
6. The method for preparing extracellular vesicles of jujube cells according to claim 2, characterized in that, S3 specifically involves: centrifuging the crude extract of jujube extracellular vesicles at 200-240g for 4-6 min to obtain a precipitate; resuspending the precipitate and incubating it with paraformaldehyde and dithiothreitol in PBS; collecting the supernatant by centrifugation at 200-240g for 4.5-5.5 min after incubation; centrifuging the supernatant at 9000-10000g for 13-17 min and collecting the supernatant again; then concentrating the supernatant by centrifugation at 3300-3700g for 12-18 min; and finally washing with PBS to obtain jujube extracellular vesicles.
7. The method for preparing extracellular vesicles of jujube cells according to claim 2, characterized in that, The centrifugation temperature of S3 is room temperature.
8. The method for preparing extracellular vesicles of jujube cells according to claim 2, characterized in that, The incubation conditions in S3 are as follows: incubation at 35~39℃ and 4.5~5.5% CO2 for 2.5~3.5h.
9. Application of jujube extracellular vesicles in the preparation of anti-inflammatory drugs.
10. Application of jujube cell extracellular vesicles in the preparation of immunomodulatory drugs.