An extracellular vesicle of a Polygonatum plant and a preparation method thereof

Abstract

The present invention belongs to the technical field of plant extracellular vesicles, and specifically relates to an extracellular vesicle of the Polygonatum plant and a preparation method thereof. By performing sucrose density gradient centrifugation, the activity of extracellular vesicles of Polygonatum is greatly improved. In addition, due to the simple operation and strong reproducibility of this method, the extraction rate of extracellular vesicles of Polygonatum is greatly increased. The in vitro antioxidant activity assay shows that, compared with the traditional ultracentrifugation method, the extracellular vesicles of Polygonatum obtained by the preparation method provided by the present invention have 2.01 times and 4.55 times higher antioxidant capacities against DPPH and ABTS respectively, and are more suitable for large-scale extraction of highly active extracellular vesicles of Polygonatum.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extracellular vesicles, and particularly relates to an extracellular vesicle of the genus Polygonatum and a preparation method thereof. Background Art

[0002] Rhizoma polygonati is a perennial herb of the genus Polygonatum Mill. in the family Liliaceae, and contains various beneficial chemical components, such as polysaccharides, steroidal saponins, anthraquinones, alkaloids, cardiac glycosides, lignans, vitamins, and amino acids, etc. It is a traditional Chinese medicinal and edible homologous plant in China.

[0003] Extracellular vesicles (EVs) are membranous small vesicles released from cells into the extracellular matrix, with a diameter of 50 - 500 nm, and can carry components such as proteins, lipids, and nucleic acids from the parental source. In addition, as natural nanoparticles, extracellular vesicles have immune tolerance, circulatory system stability, and the ability to cross biological barriers to reach distant organs. Therefore, extracellular vesicles can be used as carriers for transporting cell bioactive molecules.

[0004] However, the current extraction method of Polygonatum extracellular vesicles is mainly the traditional ultracentrifugation method, but this method has high experimental requirements, poor repeatability, and at the same time negatively affects the structure of Polygonatum extracellular vesicles, thus being not conducive to the large-scale and high-activity extraction and production of Polygonatum extracellular vesicles. Summary of the Invention

[0005] The purpose of the present invention is to provide an extracellular vesicle of the genus Polygonatum and a preparation method thereof, which have the characteristics of simple operation, good reproducibility, and high activity of Polygonatum extracellular vesicles.

[0006] The present invention provides a preparation method of an extracellular vesicle of the genus Polygonatum, comprising the following steps: homogenizing and performing solid-liquid separation on a sample of the genus Polygonatum to obtain a supernatant; separating the supernatant through a microporous membrane filter, and then concentrating the obtained separation liquid to obtain a concentrate; resuspending the concentrate and performing sucrose density gradient centrifugation to obtain extracellular vesicles.

[0007] Preferably, the sample of the genus Polygonatum includes one or more of the roots, stems, and leaves of the genus Polygonatum.

[0008] Preferably, the homogenization treatment includes: mixing water and the sample of the genus Polygonatum according to a mass ratio of (1 - 2):1, and then performing stirring and standing.

[0009] Preferably, the solid-liquid separation includes sequentially performing filtration and centrifugation, and the centrifugation includes sequentially performing first centrifugation, second centrifugation, and third centrifugation;

[0010] The speed of the first centrifugation is 4500 - 5000g, and the time is 25 - 40 min;

[0011] The speed of the second centrifugation is 7500 - 8000g, and the time is 30 - 35 min;

[0012] The speed of the third centrifugation is 12000 - 13000g, and the time is 55 - 70 min.

[0013] Preferably, the concentration includes ultra - centrifugation; the speed of the ultra - centrifugation is 90000 - 95000g, and the time is 120 - 130 min.

[0014] Preferably, the method of sucrose density gradient centrifugation includes: transferring the resuspended suspension to the upper layer of the discontinuous dispersed sucrose gradient solution for the first ultra - centrifugation, and then re - suspending the sample layer between 30wt.% - 45wt.% sucrose solution and performing the second ultra - centrifugation.

[0015] Preferably, the speeds of the first ultra - centrifugation and the second ultra - centrifugation are 90000 - 95000g respectively, and the times are 60 - 70 min respectively.

[0016] Preferably, in the discontinuous dispersed sucrose gradient solution, the sucrose solutions of 8wt.%, 30wt.%, 45wt.% and 60wt.% are included in sequence from top to bottom.

[0017] Preferably, the volume ratio of the sucrose solutions of 8wt.%, 30wt.%, 45wt.% and 60wt.% in the discontinuous dispersed sucrose gradient solution is 2:3:3:3.

[0018] The present invention also provides the polygonatum sibiricum exosomes prepared by the above - mentioned preparation method, which is characterized in that the DPPH value of the polygonatum sibiricum exosomes is 48.18 - 53.73 μmol / g, and the ATBS value is 110.30 - 179.52 μmol / g.

[0019] Beneficial effects:

[0020] The preparation method of polygonatum sibiricum extracellular vesicles provided by the present invention promotes the release of polygonatum sibiricum extracellular vesicles by first subjecting the sample to homogenization treatment and solid-liquid separation, and then separating and concentrating through a microporous membrane, which is more conducive to the concentration of polygonatum sibiricum extracellular vesicles. Finally, extraction is carried out by sucrose density gradient centrifugation. Since the vesicle particles will not be extruded and deformed during the extraction process, the activity of extracellular vesicles is greatly improved. At the same time, this method is simple to operate and has strong reproducibility, improving the extraction rate of polygonatum sibiricum extracellular vesicles. The in vitro antioxidant activity assay shows that compared with the traditional ultracentrifugation method, the polygonatum sibiricum extracellular vesicles obtained by the preparation method provided by the present invention have 2.01 times and 4.55 times higher antioxidant capacities against DPPH and ABTS respectively, and are more suitable for large-scale extraction to obtain highly active polygonatum sibiricum extracellular vesicles. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0022] Figure 1 It is the electron microscope detection diagram of the polygonatum sibiricum extracellular vesicles prepared in Example 1 and Comparative Example 1;

[0023] Figure 2 It is the NTA particle size analysis diagram of the polygonatum sibiricum extracellular vesicles prepared in Comparative Example 1;

[0024] Figure 3 It is the NTA particle size analysis diagram of the polygonatum sibiricum extracellular vesicles prepared in Example 1;

[0025] Figure 4 It is the antioxidant activity diagram of the polygonatum sibiricum extracellular vesicles prepared in Example 1 and Comparative Example 1. Detailed Embodiments

[0026] The present invention provides a method for preparing extracellular vesicles of polygonatum plants, including the following steps: subjecting a polygonatum plant sample to homogenization treatment and solid-liquid separation to obtain a supernatant; after separating the supernatant through a microporous membrane, concentrating the obtained separation liquid to obtain a concentrate; resuspending the concentrate and performing sucrose density gradient centrifugation to obtain extracellular vesicles.

[0027] In the present invention, unless otherwise specified, the required preparation raw materials and equipment are commercially available products well-known to those skilled in the art.

[0028] The present invention subjects the polygonatum plant sample to homogenization treatment. In the present invention, the species, processing type, growth environment, etc. of the polygonatum plant are not limited. Preferably, it includes wild polygonatum, cultivated polygonatum or polygonatum tissue culture, and more preferably includes polygonatum kingianum, polygonatum cyrtonema or polygonatum delavayi. The polygonatum plant sample includes one or more of the roots, stems and leaves of the polygonatum plant.

[0029] The homogenization treatment described in the present invention preferably includes: after mixing water with the Polygonatum plant sample, stirring and standing are carried out in sequence. The mass ratio of the water to the Polygonatum plant sample in the present invention is preferably (1-2):1; more preferably 1:1; the stirring time is preferably 0.5-2 min, more preferably 1 min, the stirring temperature is preferably 2-8 °C, more preferably 4 °C; the stirring speed is preferably 17000-18000 rpm, more preferably 18000 rpm; the standing time is preferably 0.5-2 min, more preferably 1 min. In the present invention, one time of the above stirring and standing is one time of homogenization treatment. The number of times of the homogenization treatment in the present invention is preferably 5-15 times, more preferably 10 times. In a specific embodiment of the present application, the process of the homogenization treatment is: using a high-speed mixer to stir for 1 min and then stop to stand for 1 min as one cycle, and the homogenization treatment is carried out for 10 cycles in total.

[0030] After the homogenization treatment, the present invention preferably filters the mixture after the homogenization treatment. In the present invention, the filtering material is preferably gauze; the mesh number of the gauze is preferably 300-500 meshes, more preferably 300 meshes. Through filtration, the residues in the mixture can be screened to collect the filtered Polygonatum plant sample juice.

[0031] After the filtration, the present invention preferably centrifuges the filtered Polygonatum plant sample juice. In the present invention, the centrifugation preferably includes: centrifuging the filtered Polygonatum plant sample juice for the first time to obtain a first supernatant, then centrifuging the first supernatant for the second time, and then centrifuging the obtained second supernatant for the third time to obtain a third supernatant. The speed of the first centrifugation in the present invention is preferably 4500-5000 g, more preferably 5000 g, and the time is preferably 25-40 min, more preferably 30 min; the speed of the second centrifugation is preferably 7500-8000 g, more preferably 8000 g, and the time is preferably 30-35 min, more preferably 30 min; the speed of the third centrifugation is preferably 12000-13000 g, more preferably 12000 g, and the time is preferably 55-70 min, more preferably 60 min. After three times of centrifugation, it is beneficial to obtain a supernatant containing more extracellular vesicles.

[0032] After the centrifugation, the present invention preferably separates the third supernatant by a microporous membrane. In the present invention, the microporous membrane separation preferably includes: filtering the supernatant with a 0.8 μM microporous membrane by suction filtration, and then filtering it with a 0.45 μM microporous membrane by suction filtration. Through two-stage microporous membrane separation, the active ingredients in the obtained separation liquid are further purified.

[0033] After the separation by the microporous membrane, the present invention preferably concentrates the separated liquid. In the present invention, the concentration preferably includes ultracentrifugation; the speed of the ultracentrifugation is preferably 90,000 - 95,000 g, more preferably 95,000 g, and the time is preferably 120 - 130 min, more preferably 120 min. After removing the supernatant by ultracentrifugation, the effective components of extracellular vesicles in the obtained concentrate are more concentrated.

[0034] After the concentration, the present invention preferably resuspends the obtained concentrate. In the present invention, the resuspension preferably includes: dissolving and mixing the concentrate with a buffer solution. The buffer solution in the present invention preferably includes a PBS solution, and the pH value of the PBS solution is preferably 7.2 - 7.4, more preferably 7.2. When performing the dissolution, the usage amount of the PBS solution is preferably 2 - 5 mL, more preferably 4 mL; the PBS solution preferably includes a protease inhibitor, and the volume ratio of the protease inhibitor to the PBS solution is preferably 1:(100 - 150), more preferably 1:100. In the present invention, the protease inhibitor preferably uses DMSO as a solvent and further preferably includes components with the following concentrations: 200 mM of AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride), 10 mM of Bestatin, 3 mM of E64 (E64 protein inhibitor), 2 mM of Leupeptin, 2 mM of Pepstatin A, and 500 mM of Phenanthroline.

[0035] After the resuspension, the present invention preferably performs sucrose density gradient centrifugation on the suspension. In the present invention, the sucrose density gradient centrifugation preferably includes: transferring the resuspended suspension to the upper layer of a discontinuous dispersed sucrose gradient solution for the first ultracentrifugation, and then re - suspending and performing the second ultracentrifugation on the sample layer between 30 wt.% - 45 wt.% sucrose solutions.

[0036] In the present invention, the discontinuous dispersed sucrose gradient solution preferably includes 8 wt.%, 30 wt.%, 45 wt.%, and 60 wt.% sucrose solutions; the volume ratio of the 8 wt.%, 30 wt.%, 45 wt.%, and 60 wt.% sucrose solutions in the discontinuous dispersed sucrose gradient solution is 2:3:3:3. Most of the main impurities in the vesicle extraction process are proteins, and by reasonably setting the concentration and addition ratio of sucrose in the discontinuous dispersed sucrose gradient solution, it is beneficial for the vesicles to be fully separated from the protein layer.

[0037] The speed of the first ultracentrifugation in the present invention is preferably 90,000 - 95,000 g, more preferably 95,000 g, and the time is preferably 60 - 70 min, more preferably 60 min. After the first ultracentrifugation, the centrifuged suspension is dispersed in different sucrose solution gradient layers. Through sucrose density gradient centrifugation treatment, it is beneficial to ensure that the vesicle particles will not be squeezed and deformed during the extraction process, thereby greatly improving the activity of extracellular vesicles.

[0038] After the first ultracentrifugation, the present invention preferably aspirates the sample layer between 30 wt.% - 45 wt.% sucrose solution for re - suspension and the second ultracentrifugation. By selecting the sample layer of 30 wt.% - 45 wt.% sucrose solution, it is beneficial to prepare extracellular vesicles with higher activity.

[0039] In the present invention, the re - suspension preferably includes: dissolving and mixing the sample layer between 30 wt.% - 45 wt.% sucrose solution with a buffer solution to obtain a suspension again. The composition of the buffer solution in the present invention has been described and recorded in detail above, and will not be elaborated here. The dosage of the buffer solution is preferably 3 - 4 times the volume of the sample layer between 30 wt.% - 45 wt.% sucrose solution.

[0040] The present invention preferably performs the second ultracentrifugation on the suspension obtained again to obtain extracellular vesicles. In the present invention, the speed of the second ultracentrifugation is preferably 90,000 - 95,000 g, more preferably 95,000 g, and the time is preferably 60 - 70 min, more preferably 60 min. Through the second ultracentrifugation, it is beneficial to wash the sucrose and obtain extracellular vesicles with higher purity.

[0041] After obtaining the extracellular vesicles, the present invention preferably preserves the extracellular vesicles. In the present invention, the preservation method of the extracellular vesicles preferably includes: resuspending the precipitate of the polygonatum sibiricum extracellular vesicles in PBS solution and then freezing it in an - 80°C refrigerator.

[0042] After obtaining the extracellular vesicles, the present invention also preferably identifies the extracellular vesicles. In the present invention, the identification method preferably includes electron microscopy detection and / or NTA particle size analysis and identification detection. By identifying the extracellular vesicles, it is beneficial to further detect the activity of extracellular vesicles.

[0043] The extracellular vesicles obtained by the above preparation method have high activity; in addition, due to the simple operation and strong reproducibility of this method, the extraction rate of Polygonatum sibiricum extracellular vesicles has been greatly improved. The in vitro antioxidant activity assay shows that, compared with the traditional ultracentrifugation method, the antioxidant capacities of the Polygonatum sibiricum extracellular vesicles obtained by the preparation method provided by the present invention against DPPH and ABTS are increased by 2.01 times and 4.55 times respectively, and it is more suitable for large-scale extraction of highly active Polygonatum sibiricum extracellular vesicles. Furthermore, the extracellular vesicles of Polygonatum plants obtained by the above preparation method also fall within the protection scope of the present invention.

[0044] To further illustrate the present invention, a Polygonatum plant extracellular vesicle and its preparation method provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] A preparation method of a Polygonatum plant extracellular vesicle comprises the following steps:

[0047] Take the tubers of Polygonatum cyrtonema Hua produced in Quzhou, Zhejiang to prepare extracellular vesicles.

[0048] 1) Collect juice: After mixing Polygonatum sibiricum with water at a ratio of 150 g: 150 g, perform homogenization treatment in a high-speed blender, that is, turn on the high-speed blender for homogenization for 1 min and then stop and let it stand for 1 min as one cycle, and the homogenization treatment is carried out for a total of 10 cycles, totaling 20 min. Among them, the speed in the high-speed blender is 18,000 rpm and the temperature is 4°C. After the Polygonatum sibiricum is broken and homogenized, filter it with a 300-mesh gauze, discard the residue, and collect the initially filtered Polygonatum sibiricum juice.

[0049] 2) Centrifugal extraction: Centrifuge the initially filtered Polygonatum sibiricum juice obtained in step 1) at 5000 g for 30 min, then gently take the supernatant, and then centrifuge the obtained supernatant at 8000 g for 30 min and gently take the supernatant, and then place the supernatant at 12,000 g for 60 min to obtain the Polygonatum sibiricum juice supernatant.

[0050] 3) Filtration: Filter the Polygonatum sibiricum juice supernatant in step 2) with a 0.8 μM microporous filter membrane and then with a 0.45 μM microporous filter membrane. After filtration through two-stage microporous filter membranes, obtain the second-filtered Polygonatum sibiricum juice after filtration.

[0051] 4) Concentration: Ultracentrifuge the second-filtered Polygonatum sibiricum juice obtained in step 3) at 95,000 g for 120 min, discard the supernatant, and the obtained precipitate is the concentrated Polygonatum sibiricum juice.

[0052] 5) Resuspension: Pipette 5 mL of PBS buffer (wherein, protease inhibitor is added to the PBS buffer, and the volume ratio of the protease inhibitor to the PBS solution is 1:100. The protease inhibitor uses DMSO as a solvent and is composed of components with the following concentrations: 200 mM of AEBSF, 10 mM of Bestatin, 3 mM of E64, 2 mM of Leupeptin, 2 mM of Pepstatin A, and 500 mM of Phenanthroline), and dissolve and mix it with the precipitate in step 4) to obtain a suspension.

[0053] 6) Purification of Polygonatum sibiricum extracellular vesicles by sucrose gradient density centrifugation: Prepare a discontinuous dispersed sucrose gradient solution (wherein, in the discontinuous dispersed sucrose gradient solution, sucrose solutions with 8 wt.%, 30 wt.%, 45 wt.%, and 60 wt.% are included in sequence from top to bottom, and the volume ratio of each concentration of sucrose solution is 2:3:3:3).

[0054] Transfer the suspension in step 5) to the upper layer of the discontinuous dispersed sucrose gradient solution, that is, the upper layer of the 8 wt.% sucrose solution layer, and perform sucrose gradient ultracentrifugation at 95000 g for 60 min.

[0055] After that, pipette the sample layer between the 30 wt.% - 45 wt.% sucrose solutions, and simultaneously add a PBS solution with a volume 3 times that of the sample layer between the 30 wt.% - 45 wt.% sucrose solutions for resuspension (wherein, protease inhibitor is added to the PBS buffer, and the volume ratio of the protease inhibitor to the PBS solution is 1:100. The protease inhibitor uses DMSO as a solvent and is composed of components with the following concentrations: 200 mM of AEBSF, 10 mM of Bestatin, 3 mM of E64, 2 mM of Leupeptin, 2 mM of Pepstatin A, and 500 mM of Phenanthroline) to obtain a re - resuspended suspension. Place the re - resuspended suspension under the condition of 95000 g for ultracentrifugation for 60 min. After thoroughly washing the sucrose solution in this way, the obtained precipitate is the Polygonatum sibiricum extracellular vesicles.

[0056] 7) Identification of Polygonatum sibiricum extracellular vesicles: Identify the Polygonatum sibiricum extracellular vesicles in step 6) by electron microscopy and NTA particle size analysis.

[0057] Comparative Example 1

[0058] A traditional method for extracting Polygonatum sibiricum extracellular vesicles consists of the following steps:

[0059] Take the rhizome of Polygonatum cyrtonema Hua produced in Quzhou, Zhejiang to prepare extracellular vesicles.

[0060] 1) Collect juice: The same as step 1) in Example 1.

[0061] 2) Centrifugal extraction: The same as step 2) in Example 1.

[0062] 3) Filtration: The same as step 3) in Example 1.

[0063] 4) Concentration: The same as step 4) in Example 1.

[0064] 5) Collection of polygonatum sibiricum extracellular vesicles: Pipette 5 mL of PBS buffer (wherein, protease inhibitor is added to the PBS buffer, and the volume ratio of the protease inhibitor to the PBS solution is 1:100. The protease inhibitor is dissolved in DMSO and consists of the following components in concentrations: 200 mM of AEBSF, 10 mM of Bestatin, 3 mM of E64, 2 mM of Leupeptin, 2 mM of Pepstatin A, and 500 mM of Phenanthroline), and dissolve and mix it with the precipitate in step 4) to obtain a suspension, which is the polygonatum sibiricum extracellular vesicles.

[0065] 6) Identification of polygonatum sibiricum extracellular vesicles: The polygonatum sibiricum extracellular vesicles extracted in step 5) are identified by electron microscopy and NTA particle size analysis.

[0066] Results and analysis:

[0067] 1) Analysis of electron microscopy detection results: Electron microscopy detection was respectively performed on the polygonatum sibiricum extracellular vesicles prepared in Example 1 and Comparative Example 1, and the results are as Figure 1 shown (wherein, the left picture is the electron microscopy detection image of the polygonatum sibiricum extracellular vesicles prepared in Comparative Example 1; the right picture is the electron microscopy detection image of the polygonatum sibiricum extracellular vesicles prepared in Example 1, and the scale bar in the picture is 200 nm).

[0068] It can be seen from Figure 1 that the polygonatum sibiricum extracellular vesicles obtained by both preparation methods have obvious vesicle structures, and there are fewer signs of damage to the extracted extracellular vesicles. However, the background of the polygonatum sibiricum extracellular vesicles prepared by the method provided in Example 1 is clean and has better uniformity.

[0069] 2) NTA analysis detection results: NTA analysis was respectively performed on the polygonatum sibiricum extracellular vesicles prepared in Comparative Example 1 and Example 1, and the results are as Figure 2 and Figure 3 shown.

[0070] It can be seen from Figure 2 and Figure 3It can be seen that the particle sizes of the extracellular vesicles of Polygonatum sibiricum obtained by the two preparation methods are both around 140 nm, without obvious differences, and the particle size of the extracellular vesicles is similar to the particle size of Polygonatum sibiricum in its natural state. It can be seen that the preparation method provided in this application does not change the particle size of the extracellular vesicles of Polygonatum sibiricum.

[0071] 3) Determination of the antioxidant activity of the extracellular vesicles of Polygonatum sibiricum, and the results are as Figure 4 shown (wherein the marked ultracentrifugation represents Comparative Example 1, and the marked sucrose gradient density centrifugation represents Example 1).

[0072] Take 20 μL of the extracellular vesicles of Polygonatum sibiricum obtained by different preparation methods in Example 1 and Comparative Example 1 respectively for BCA protein quantification.

[0073] After measurement, the concentration of BCA of the extracellular vesicles of Polygonatum sibiricum prepared in Example 1 is: 0.3 mg / mL; the concentration of BCA of the extracellular vesicles of Polygonatum sibiricum prepared in Comparative Example 1 is: 1.6 mg / mL.

[0074] Take 400 μL of the extracellular vesicles of Polygonatum sibiricum obtained by different preparation methods in Example 1 and Comparative Example 1 respectively and put them into 1.5 mL EP tubes, add 600 μL of 0.2 mmol / L DPPH solution, shake well, and let stand for 30 min under dark conditions at room temperature. Measure the absorbance Aa at a wavelength of 517 nm. For the extracellular vesicles of Polygonatum sibiricum obtained by each preparation method, three groups are taken, that is, three replicates, and the average value is calculated. The scavenging rate is calculated as shown in formula (1):

[0075]

[0076] In the formula: Ab is the absorbance control of the sample measured by replacing DPPH with 80% methanol solution, and Ac is the absorbance control of the sample measured by replacing the extracellular vesicles of Polygonatum sibiricum with PBS.

[0077] Take 10 μL of the extracellular vesicles of Polygonatum sibiricum obtained by different preparation methods in Example 1 and Comparative Example 1 respectively, add them to a 96-well enzyme-linked immunosorbent assay plate, and add 20 μL of 10% peroxidase and 170 μL of 0.1 mmol / L ABTS working solution respectively. For the extracellular vesicles of Polygonatum sibiricum obtained by each preparation method, three groups are taken, that is, three replicates, and the average value is calculated. The scavenging rate is calculated as shown in formula (1).

[0078] The antioxidant capacity of the extracellular vesicles of Polygonatum sibiricum, combined with the protein concentration of different extracellular vesicle samples, is normalized by Trolox Equivalent Anyioxidant Capacity (TEAC), and finally expressed in μmol / g. The results are shown in Table 1, Table 2 and Figure 4 .

[0079] Table 1 DPPH values of extracellular vesicles of Polygonatum sibiricum in different treatments (unit: μmol / g)

[0080]

[0081] Table 2 ATBS values of extracellular vesicles of Polygonatum sibiricum in different treatments (unit: μmol / g)

[0082]

[0083] It can be seen from Table 1 and Table 2 that although the extracellular vesicles of Polygonatum sibiricum prepared by different methods all show the antioxidant ability to scavenge free radicals, the extracellular vesicles of Polygonatum sibiricum obtained by the preparation method provided by the present invention have DPPH values of 48.18 - 53.73 μmol / g; and ATBS values of 110.30 - 179.52 μmol / g. Combining Figure 4 it can be seen that the average antioxidant ability of the extracellular vesicles of Polygonatum sibiricum prepared by traditional ultracentrifugation in Comparative Example 1 is only 16.63 μmol / g (DPPH) and 25.2 μmol / g (ATBS), while the average antioxidant ability of the extracellular vesicles of Polygonatum sibiricum prepared by the sucrose density gradient centrifugation method provided in Example 1 is as high as 50.04 μmol / g (DPPH) and 139.78 μmol / g (ATBS).

[0084] In summary, the preparation method of the extracellular vesicles of Polygonatum sibiricum provided by the present invention can greatly improve the antioxidant ability of the extracellular vesicles of Polygonatum sibiricum, with DPPH and ATBS increased by 2.01 times and 4.55 times respectively, and the effect is remarkable. Therefore, the preparation method provided by the present invention is more suitable for large-scale extraction of extracellular vesicles of Polygonatum sibiricum with high activity.

[0085] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A method for preparing extracellular vesicles of plants of the genus Polygonatum, characterized in that, it consists of the following steps: Homogenize the plant sample of the genus Polygonatum and perform solid-liquid separation to obtain a supernatant; After separating the supernatant through a microporous membrane filter, concentrate the obtained separated liquid to obtain a concentrate; Resuspend the concentrate and perform sucrose density gradient centrifugation to obtain extracellular vesicles; The solid-liquid separation includes filtration and centrifugation in sequence, and the centrifugation is performed by first centrifugation, second centrifugation and third centrifugation in sequence; The speed of the first centrifugation is 4500-5000g, and the time is 25-40 min; The speed of the second centrifugation is 7500-8000g, and the time is 30-35 min; The speed of the third centrifugation is 12000-13000g, and the time is 55-70 min; The method of the sucrose density gradient centrifugation includes: Transfer the resuspended suspension to the upper layer of a discontinuous dispersed sucrose gradient solution for the first ultracentrifugation, and then resuspend the sample layer between 30 wt.% and 45 wt.% sucrose solutions and perform the second ultracentrifugation; The speeds of the first ultracentrifugation and the second ultracentrifugation are 90000-95000g respectively, and the times are 60-70 min respectively; The concentration is ultracentrifugation; the speed of the ultracentrifugation is 90000-95000g, and the time is 120-130 min.

2. The preparation method according to claim 1, characterized in that, the plant sample of the genus Polygonatum is one or more of the roots, stems and leaves of plants of the genus Polygonatum.

3. The preparation method according to claim 1, characterized in that, the homogenization treatment is: after mixing water and the plant sample of the genus Polygonatum according to a mass ratio of (1-2):1, stir and let stand.

4. The preparation method according to claim 1, characterized in that, in the discontinuous dispersed sucrose gradient solution, the sucrose solutions are 8 wt.%, 30 wt.%, 45 wt.% and 60 wt.% from top to bottom in sequence.

5. The preparation method according to claim 1 or 4, characterized in that, the volume ratio of the 8 wt.%, 30 wt.%, 45 wt.% and 60 wt.% sucrose solutions in the discontinuous dispersed sucrose gradient solution is 2:3:3:

3.

6. The extracellular vesicles of Polygonatum prepared by the preparation method according to any one of claims 1-5, characterized in that, the DPPH value of the extracellular vesicles of Polygonatum is 48.18-53.73 μmol / g, and the ATBS value is 110.30-179.52 μmol / g.

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