CN-121991877-A - Preparation method and application of jujube extracellular vesicles

Abstract

The invention relates to the technical field of biological medicines, in particular to a preparation method and application of jujube extracellular vesicles. The preparation method comprises the steps of preparing fresh jujube into homogenate, adding PBS, and carrying out multistage centrifugation to obtain a crude extract of the jujube extracellular vesicles, incubating the crude extract of the jujube extracellular vesicles with paraformaldehyde and dithiothreitol in PBS, and then carrying out centrifugal filtration to obtain the jujube extracellular vesicles. The preparation method has the characteristics of stable process, low cost, good biological safety and the like, and has wide application prospect in the field of medicines. The prepared jujube extracellular vesicles can be applied to anti-inflammatory drugs and immunoregulatory drugs, and provide technical support for developing novel natural source drugs.

Inventors

• LI MUXIA
• CHEN JIANPING
• WU JIAZHEN
• HUANG SHIYING
• ZENG HUIHUAN

Assignees

• 深圳市中医院

Dates

Publication Date

20260508

Application Date

20251225

Claims (10)

1. 1. The preparation method of the jujube extracellular vesicles is characterized by comprising the following steps: Preparing fresh jujube into homogenate, adding PBS (phosphate buffer solution) for multistage centrifugation to obtain crude extract of the jujube extracellular vesicles, incubating the crude extract of the jujube extracellular vesicles together with paraformaldehyde and dithiothreitol in the PBS, and centrifugally filtering to obtain the jujube extracellular vesicles.
2. 2. The method for preparing the jujube extracellular vesicles according to claim 1, comprising the following steps: S1, removing cores of fresh Chinese dates, crushing the fresh Chinese dates into homogenate, adding PBS, filtering and centrifuging, and collecting supernatant to obtain a Chinese date crude extract; S2, centrifuging and concentrating the crude jujube extract to obtain a concentrated solution, centrifuging the concentrated solution, collecting supernatant, and centrifuging again to obtain a jujube extracellular vesicle precipitate, wherein the jujube extracellular vesicle precipitate is added with PBS buffer solution for blowing and filtering to obtain a crude jujube extracellular vesicle extract; s3, carrying out centrifugation on the crude extract of the jujube extracellular vesicles, then carrying out precipitation and resuspension, then incubating the crude extract of the jujube extracellular vesicles with paraformaldehyde and dithiothreitol in PBS, and carrying out centrifugal filtration after incubation is completed to obtain the jujube extracellular vesicles.
3. 3. The method for preparing the jujube extracellular vesicles according to claim 2, wherein the centrifugation in S1 is performed for 20 to 30min under 8000 to 12000 g.
4. 4. The method for preparing the jujube extracellular vesicles according to claim 2, wherein the centrifugal temperatures of the S1 and the S2 are 3-5 ℃.
5. 5. The preparation method of the jujube extracellular vesicles according to claim 2 is characterized in that S2 specifically comprises the steps of centrifuging 2300-2700 g of crude jujube extract to obtain concentrated solution, centrifuging 8000-12000 g of concentrated solution for 8-12 min, collecting supernatant, centrifuging 48000-5200 g of supernatant for 80-100 min to obtain jujube extracellular vesicle sediment, adding PBS buffer into the jujube extracellular vesicle sediment to blow, and filtering to obtain the crude jujube extracellular vesicle extract.
6. 6. The preparation method of the jujube extracellular vesicles according to claim 2 is characterized in that S3 specifically comprises the steps of centrifuging 200-240 g of crude extract of the jujube extracellular vesicles for 4-6 min to obtain precipitate, re-suspending the precipitate, incubating the precipitate with paraformaldehyde and dithiothreitol in PBS, centrifuging 200-240 g of the supernatant for 4.5-5.5 min after incubation, centrifuging 9000-10000 g of the supernatant for 13-17 min, collecting the supernatant again, centrifuging 3300-3700 g of the supernatant for 12-18 min, and washing the supernatant with PBS to obtain the jujube extracellular vesicles.
7. 7. The method for preparing jujube extracellular vesicles according to claim 2, wherein the centrifugation temperature of S3 is room temperature.
8. 8. The method for preparing the jujube extracellular vesicles according to claim 2, wherein the conditions of the incubation in S3 are that the incubation is performed for 2.5 to 3.5 hours under the environment of 35 to 39 ℃ and 4.5 to 5.5% co 2 .
9. 9. Application of fructus Jujubae extracellular vesicles in preparing antiinflammatory agent is provided.
10. 10. Application of fructus Jujubae extracellular vesicles in preparation of immunomodulating drugs is provided.

Description

Preparation method and application of jujube extracellular vesicles Technical Field The invention relates to the technical field of biological medicines, in particular to a preparation method and application of jujube extracellular vesicles. Background Extracellular vesicles (Extracellular Vesicles, EVs) are nanoscale membranous vesicles secreted by cells and have the ability to carry bioactive molecules such as proteins, nucleic acids, lipids and the like, and play an important role in intercellular communication. In recent years, plant-derived extracellular vesicles have been attracting attention because of their wide sources, high safety, and various biological activities. The efficacy of the plant extracellular vesicles is closely related to the source of the plant extracellular vesicles, the existing research discovers that the plant extracellular vesicles from ginseng have an immunoregulatory effect, the plant extracellular vesicles from ginger have an anti-inflammatory effect, the plant extracellular vesicles from platycodon root have an anti-tumor effect, and no report exists at present that the plant extracellular vesicles from Chinese date have the anti-inflammatory and immunoregulatory effects. The Chinese date (Ziziphus jujuba Mill.) is used as a traditional Chinese medicinal material for both medicine and food, and has the effects of invigorating spleen and replenishing qi, nourishing blood and tranquillizing. In Ben Jing (book of meridian) is recorded "the heart and abdomen pathogenic factors are dominant, the spleen is nourished in the middle warmer, and the twelve meridians are helped. Stomach qi calming, nine orifices dredging, qi deficiency tonifying, body fluid deficiency tonifying, convulsion, limb weight and general drug; modern researches have shown that the Chinese date contains rich polysaccharide, flavone and triterpene compounds, and has various pharmacological activities such as antioxidation, immunoregulation, anti-inflammatory and the like. In the prior art, the extraction method of the plant extracellular vesicles is various, the jujube yield is high, and the effective extraction method and application research of the extracellular vesicles have important values. Disclosure of Invention The invention aims to solve the technical problems of a preparation method of jujube extracellular vesicles and application of the jujube extracellular vesicles in anti-inflammatory drugs and immunoregulatory drugs. In order to solve the technical problems, the technical scheme adopted by the invention is that the preparation method of the jujube extracellular vesicles comprises the following steps: homogenizing fresh jujube, adding PBS (phosphate buffered saline) for multistage centrifugation to obtain crude extract of the jujube extracellular vesicles, incubating the crude extract of the jujube extracellular vesicles with paraformaldehyde and dithiothreitol in PBS, and centrifugally filtering to obtain the jujube extracellular vesicles. The invention adopts another technical scheme that the jujube extracellular vesicles are applied to the preparation of anti-inflammatory drugs. The invention adopts a further technical scheme that the jujube extracellular vesicles are applied to the preparation of immunoregulatory medicines. The preparation method of the jujube extracellular vesicles has the advantages of being stable in process, low in cost, good in biological safety and the like, and has wide application prospects in the field of medicines. The prepared jujube extracellular vesicles can be applied to anti-inflammatory drugs and immunoregulatory drugs, and provide technical support for developing novel natural source drugs. Drawings FIG. 1 is a diagram showing the morphology of the jujube extracellular vesicles under transmission electron microscopy in an embodiment of the invention; FIG. 2 shows the particle size distribution of extracellular vesicles of date detected by DLS (dynamic light Scattering) in the example of the present invention; FIG. 3 is a graph showing Zeta potential (electrokinetic potential) detection of extracellular vesicles of jujube in an embodiment of the invention; FIG. 4 is a bar graph showing the effect of jujube extracellular vesicles on inhibiting macrophage inflammatory factor release in accordance with an embodiment of the present invention; FIG. 5 is a statistical chart of the flow cytometry detection of macrophage M2 phenotype transformation results in an embodiment of the present invention. Detailed Description In order to describe the technical contents, the achieved objects and effects of the present invention in detail, the following description will be made with reference to the embodiments in conjunction with the accompanying drawings. A preparation method of jujube extracellular vesicles comprises the following steps: Preparing fresh jujube into homogenate, adding PBS (phosphate buffer solution) for multistage centrifugation to obtain crude extract of the