Extraction method of plant exosome with anti-inflammatory and anti-oxidation effects

Abstract

The invention relates to a method for extracting plant exosomes with anti-inflammatory and anti-oxidation effects, and belongs to the technical field of exosome separation.The method comprises the steps that low-speed centrifugation and 0.4-0.45-micron filter membrane filtration operation are firstly used for improving the purity of the plant exosomes, then two kinds of polyethylene glycol with different molecular weights are used for precipitating the plant exosomes, and the plant exosomes are separated; and after the obtained precipitate is resuspended, the precipitate is filtered by a 0.22-0.25 mu m filter membrane, and the plant exosome is precipitated by using PEG1000, so that the extraction efficiency and the purity of the plant exosome are remarkably improved. The extracted plant exosome is high in biological activity and has excellent anti-inflammatory and anti-oxidation capability.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome separation, and specifically relates to a method for extracting plant exosomes with anti-inflammatory and antioxidant effects. Background Art

[0002] Exosomes are tiny vesicles released into the cell fluid by living cells. They have important functions such as intercellular information flow and material transport. Almost all human cells and plant cells can release exosomes, and plant exosomes have the pharmacological effects of the source plants. At present, ultracentrifugation is one of the most commonly used methods for separating exosomes in plant exosome separation methods, but it will cause agglomeration and destruction of exosomes, and there are problems of time-consuming and high cost. The polymer precipitation method usually uses polyethylene glycol (PEG) as a medium, and harvests exosomes under centrifugal conditions by reducing the solubility of exosomes. The polymer precipitation method is simple to operate and has a short analysis time, but the purity of the exosomes obtained is low. Therefore, it is necessary to provide a plant exosome extraction method that can solve the above problems. Summary of the invention

[0003] The purpose of the present invention is to provide a method for extracting plant exosomes with anti-inflammatory and antioxidant effects. First, low-speed centrifugation and 0.4-0.45μm filter membrane filtration are used to improve the purity of plant exosomes, and then two polyethylene glycols with different molecular weights are used to precipitate plant exosomes, and the obtained precipitate is resuspended and first filtered through a 0.22-0.25μm filter membrane, and then PEG1000 is used to precipitate plant exosomes, which significantly improves the extraction efficiency and purity of plant exosomes. The extracted plant exosomes have high biological activity and excellent anti-inflammatory and antioxidant abilities.

[0004] Technical problem to be solved by the present invention: Ultracentrifugation is one of the most commonly used methods for isolating exosomes, but it can cause aggregation and destruction of exosomes, and is time-consuming and costly. The polymer precipitation method usually uses PEG as a medium to reduce the solubility of exosomes and harvest exosomes under centrifugal conditions. The polymer precipitation method is simple to operate and takes a short analysis time, but the purity of the exosomes obtained is low.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A method for extracting plant exosomes with anti-inflammatory and antioxidant effects comprises the following steps:

[0007] A1. After cleaning the plant tissue, add it into PBS solution, break the wall, filter and collect the filtrate;

[0008] Furthermore, in step A1, the plant is Polygonatum sibiricum.

[0009] Furthermore, in step A1, filtration is performed using a 100-200 mesh nylon filter.

[0010] Furthermore, in step A1, the volume ratio of the plant tissue and the PBS solution is (1-2): (1-2).

[0011] A2, centrifuging the filtrate at 1000-1300g for 10-15min, and then at 5000-6000g for 30-40min. After the centrifugation, filtering the supernatant filtrate through a 0.4-0.45μm filter membrane to obtain a filtrate;

[0012] Furthermore, in step A2, the centrifugation temperature is 0-4°C.

[0013] A3, dissolving PEG600 and PEG6000 in PBS solution, then adding the filtrate obtained in step A2 to obtain a mixed solution, placing the mixed solution at 2-8°C for 12-24h, centrifuging to obtain a precipitate a;

[0014] Furthermore, in step A3, the mass ratio of PEG600 to PEG6000 is (0.5-1):(1.5-2.5).

[0015] Furthermore, in step A3, the final mass concentration of PEG600 in the mixed solution is 1-3%; the final mass concentration of PEG6000 in the mixed solution is 4-8%.

[0016] Furthermore, in step A3, the centrifugation conditions are: centrifugation at a centrifugal force of 10000-15000 g at 2-8° C. for 25-40 min.

[0017] A4, resuspending the precipitate a with a PBS solution, filtering the obtained solution through a 0.22-0.25 μm filter membrane, and then adding the filtered solution to a PBS solution containing PEG1000, mixing them thoroughly to obtain a mixed solution, and centrifuging to obtain a precipitate b;

[0018] Furthermore, in step A4, the centrifugation conditions are: centrifugation at a centrifugal force of 10000-15000 g at 0-4° C. for 10-15 min.

[0019] Furthermore, in step A4, the mass concentration of PEG1000 in the PBS solution containing PEG1000 is 20-40%, and the mass concentration of PEG1000 in the mixed solution is 2-4%.

[0020] A5. Resuspend the precipitate b with PBS solution to obtain a plant exosome suspension.

[0021] Beneficial effects of the present invention:

[0022] In the technical scheme of the present invention, low-speed centrifugation and 0.4-0.45 μm filter membrane filtration are first used to improve the purity of plant exosomes, and then two polyethylene glycols with different molecular weights are used to precipitate plant exosomes. The obtained precipitate is resuspended and first filtered through a 0.22-0.25 μm filter membrane, and then PEG1000 is used to precipitate plant exosomes. The above operations significantly improve the extraction efficiency and purity of plant exosomes. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] A method for extracting plant exosomes with anti-inflammatory and antioxidant effects comprises the following steps:

[0026] A1. Wash fresh Polygonatum sibiricum and add it into PBS solution, break the wall, filter it with a 100-mesh nylon filter, and collect the filtrate, wherein the volume ratio of plant tissue to PBS solution is 1:1;

[0027] A2, centrifuging the filtrate at 0°C and 1000 g centrifugal force for 10 min, and then centrifuging at 0°C and 5000 g centrifugal force for 30 min. After the centrifugation, filtering the supernatant filtrate through a 0.4 μm filter membrane to obtain a filtrate;

[0028] A3, dissolving PEG600 and PEG6000 in PBS solution, the mass ratio of PEG600 to PEG6000 is 0.5:1.5, then adding the filtrate obtained in step A2 to obtain a mixed solution, the final mass concentration of PEG600 in the mixed solution is 1%, and the final mass concentration of PEG600 in the mixed solution is 8%, then placing the mixed solution at 2°C for 12h, centrifuging at 10000g centrifugal force at 2°C for 25min, to obtain precipitate a;

[0029] A4. Resuspend the precipitate a with a PBS solution, filter the resulting solution through a 0.22 μm filter membrane, and then add the filtered solution into a PBS solution containing PEG1000 with a mass concentration of 20%, mix thoroughly to obtain a mixed solution, wherein the mass concentration of PEG1000 in the mixed solution is 2%, and then centrifuge at 10000 g for 10 min at 0° C. to obtain a precipitate b;

[0030] A5. Resuspend the precipitate b with PBS solution to obtain a plant exosome suspension.

[0031] Example 2

[0032] A method for extracting plant exosomes with anti-inflammatory and antioxidant effects comprises the following steps:

[0033] A1. Wash fresh Polygonatum sibiricum and add it into PBS solution, break the wall, filter it with a 150-mesh nylon filter, and collect the filtrate, where the volume ratio of plant tissue to PBS solution is 1:1.5;

[0034] A2, centrifuge the filtrate at 4°C and 1100 g centrifugal force for 12 min, then centrifuge at 4°C and 5000 g centrifugal force for 35 min. After the centrifugation, filter the supernatant filtrate through a 0.42 μm filter membrane to obtain a filtrate;

[0035] A3, dissolving PEG600 and PEG6000 in PBS solution, the mass ratio of PEG600 to PEG6000 is 0.5:2, then adding the filtrate obtained in step A2 to obtain a mixed solution, the final mass concentration of PEG600 in the mixed solution is 1.5%, the final mass concentration of PEG600 in the mixed solution is 7%, then placing the mixed solution at 2°C for 18h, centrifuging at 12000g centrifugal force at 4°C for 30min, to obtain precipitate a;

[0036] A4. Resuspend the precipitate a with a PBS solution, filter the resulting solution through a 0.22 μm filter membrane, and then add the filtered solution into a PBS solution containing PEG1000 with a mass concentration of 25%, mix thoroughly to obtain a mixed solution, wherein the mass concentration of PEG1000 in the mixed solution is 2.8%, and then centrifuge at a centrifugal force of 12000 g at 4° C. for 12 min to obtain a precipitate b;

[0037] A5. Resuspend the precipitate b with PBS solution to obtain a plant exosome suspension.

[0038] Example 3

[0039] A method for extracting plant exosomes with anti-inflammatory and antioxidant effects comprises the following steps:

[0040] A1. Wash fresh Polygonatum sibiricum and add it into PBS solution, break the wall, filter it with a 180-mesh nylon filter, and collect the filtrate, wherein the volume ratio of plant tissue to PBS solution is 2:1;

[0041] A2, centrifuge the filtrate at 4°C and 1200 g centrifugal force for 15 min, then centrifuge at 4°C and 5500 g centrifugal force for 35 min. After the centrifugation, filter the supernatant filtrate through a 0.43 μm filter membrane to obtain a filtrate;

[0042] A3, dissolving PEG600 and PEG6000 in PBS solution, the mass ratio of PEG600 to PEG6000 is 1:1.5, then adding the filtrate obtained in step A2 to obtain a mixed solution, the final mass concentration of PEG600 in the mixed solution is 2.8%, the final mass concentration of PEG600 in the mixed solution is 6.5%, then placing the mixed solution at 4°C for 24 hours, centrifuging at 13000g centrifugal force at 4°C for 35 minutes, to obtain precipitate a;

[0043] A4. Resuspend the precipitate a with a PBS solution, filter the resulting solution through a 0.22 μm filter membrane, and then add the filtered solution into a PBS solution containing PEG1000 with a mass concentration of 30%, mix thoroughly to obtain a mixed solution, wherein the mass concentration of PEG1000 in the mixed solution is 3%, and then centrifuge at a centrifugal force of 13000 g at 4° C. for 13 min to obtain a precipitate b;

[0044] A5. Resuspend the precipitate b with PBS solution to obtain a plant exosome suspension.

[0045] Example 4

[0046] A method for extracting plant exosomes with anti-inflammatory and antioxidant effects comprises the following steps:

[0047] A1. Wash fresh Polygonatum sibiricum and add it into PBS solution, break the wall, filter it with a 200-mesh nylon filter, and collect the filtrate, wherein the volume ratio of plant tissue to PBS solution is 1.5:1;

[0048] A2, centrifuge the filtrate at 4°C and 1300g for 15 min, then centrifuge at 4°C and 5000-6000g for 30 min. After the centrifugation, filter the supernatant filtrate through a 0.45 μm filter membrane to obtain a filtrate;

[0049] A3, dissolving PEG600 and PEG6000 in PBS solution, the mass ratio of PEG600 to PEG6000 is 1:2, then adding the filtrate obtained in step A2 to obtain a mixed solution, the final mass concentration of PEG600 in the mixed solution is 2.5%, the final mass concentration of PEG600 in the mixed solution is 7%, then placing the mixed solution at 8°C for 24h, centrifuging at 15000g centrifugal force at 8°C for 40min, to obtain precipitate a;

[0050] A4. Resuspend the precipitate a with a PBS solution, filter the resulting solution through a 0.25 μm filter membrane, and then add the filtered solution into a PBS solution containing PEG1000 with a mass concentration of 35%, mix thoroughly to obtain a mixed solution, wherein the mass concentration of PEG1000 in the mixed solution is 3.5%, and then centrifuge at a centrifugal force of 15000 g at 4° C. for 15 min to obtain a precipitate b;

[0051] A5. Resuspend the precipitate b with PBS solution to obtain a plant exosome suspension.

[0052] Example 5

[0053] A method for extracting plant exosomes with anti-inflammatory and antioxidant effects comprises the following steps:

[0054] A1. Wash fresh Polygonatum sibiricum and add it into PBS solution, break the wall, filter it with a 200-mesh nylon filter, and collect the filtrate, where the volume ratio of plant tissue to PBS solution is 2:1.5;

[0055] A2, centrifuge the filtrate at 4°C and 1300g for 15 min, then centrifuge at 4°C and 6000g for 40 min. After the centrifugation, filter the supernatant filtrate through a 0.45 μm filter membrane to obtain a filtrate;

[0056] A3, dissolving PEG600 and PEG6000 in PBS solution, the mass ratio of PEG600 to PEG6000 is 1:2.5, then adding the filtrate obtained in step A2 to obtain a mixed solution, the final mass concentration of PEG600 in the mixed solution is 3%, the final mass concentration of PEG600 in the mixed solution is 4%, then placing the mixed solution at 4°C for 24 hours, centrifuging at 15000g centrifugal force at 4°C for 40 minutes, to obtain precipitate a;

[0057] A4. Resuspend the precipitate a with a PBS solution, filter the resulting solution through a 0.25 μm filter membrane, and then add the filtered solution into a PBS solution containing PEG1000 with a mass concentration of 40%, mix thoroughly to obtain a mixed solution, wherein the mass concentration of PEG1000 in the mixed solution is 4%, and then centrifuge at a centrifugal force of 15000 g at 4° C. for 15 min to obtain a precipitate b;

[0058] A5. Resuspend the precipitate b with PBS solution to obtain a plant exosome suspension.

[0059] Comparative Example 1

[0060] Compared with Example 3, the supernatant filtrate in step A2 of Comparative Example 1 was not filtered with a 0.4-0.45 μm filter membrane, and the other steps and raw materials were the same as those in Example 3.

[0061] Comparative Example 2

[0062] Compared with Example 3, only PEG600 was used in Step A3 of Comparative Example 2, and the other steps and raw materials were the same as those of Example 3.

[0063] Comparative Example 3

[0064] Compared with Example 3, only PEG6000 was used in Step A3 of Comparative Example 3, and the other steps and raw materials were the same as those of Example 3.

[0065] Comparative Example 4

[0066] Compared with Example 3, the solution obtained in step A4 of Comparative Example 4 was not filtered through a 0.22-0.25 μm filter membrane, and the other steps and raw materials were the same as those of Example 3.

[0067] Performance Testing

[0068] (1) Particle size analysis: The particle size of plant exosomes was measured using the MalvernNano ZS instrument;

[0069] (2) Protein concentration: The total protein concentration was determined using the BCA protein concentration assay kit;

[0070] (3) Antioxidant capacity: The plant exosomes extracted from Examples 1-5 and Comparative Examples 1-4 were mixed with a 0.1 mmol / L DPPH anhydrous ethanol solution at a volume ratio of 1:1, and placed in a dark place at room temperature for 60 min. The absorbance value was measured at 517 nm and recorded as A 1 ; Anhydrous ethanol and DPPH anhydrous ethanol solution were mixed in a volume ratio of 1:1 as a control, placed in the dark at room temperature for 60 minutes, and the absorbance value was measured at 517nm and recorded as A 0 The plant exosomes prepared in Examples 1-5 and Comparative Examples 1-4 were mixed with anhydrous ethanol at a volume ratio of 1:1 as a control, placed in a dark place at room temperature for 60 minutes, and the absorbance value was measured at 517 nm and recorded as A 2 Each group was run three times in parallel. The calculation formula for DPPH free radical scavenging rate is as follows: DPPH free radical scavenging rate (%) = (A 0 -(A 1 -A 2 )) / A 0 ×100%;

[0071] (4) Anti-inflammatory ability: The COX-2 inhibition test was tested in the kit.

[0072] Table 1

[0073]

[0074]

[0075] It can be seen from the data in Table 1 that the particle size of the plant exosomes extracted by the present invention belongs to the exosome particle size range, and the total protein concentration of the plant exosomes extracted by the embodiment is higher than that of the comparative example, indicating that the plant exosome yield obtained by the extraction method provided by the present invention is higher. At the same time, the plant exosomes extracted by the present invention have excellent antioxidant and anti-inflammatory effects. It can be seen from the data of Example 3 and Comparative Example 1 that in step A2, the supernatant filtrate is not filtered by a 0.4-0.45 μm filter membrane, so that the obtained plant exosomes have increased macromolecular impurities, reduced purity, and reduced antioxidant and anti-inflammatory effects. It can be seen from the data of Example 3 and Comparative Example 2 and Comparative Example 3 that in step A3, only PEG600 or PEG6000 is used as a precipitant to precipitate plant exosomes, so that the obtained plant exosome protein concentration is reduced and the yield is reduced. It can be seen from the data of Example 3 and Comparative Example 4 that when the obtained solution is not filtered by a 0.22-0.25 μm filter membrane in step A4, the obtained plant exosomes have increased impurities, miscellaneous peaks appear, and the antioxidant and anti-inflammatory effects are reduced.

[0076] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0077] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for extracting plant exosomes with anti-inflammatory and antioxidant effects, characterized in that: The following steps are involved: A1. After cleaning the plant tissue, add it into PBS solution, break the wall, filter and collect the filtrate; A2, centrifuging the filtrate at 1000-1300g for 10-15min, and then at 5000-6000g for 30-40min. After the centrifugation, filtering the supernatant filtrate through a 0.4-0.45μm filter membrane to obtain a filtrate; A3, dissolving PEG600 and PEG6000 in PBS solution, then adding the filtrate obtained in step A2 to obtain a mixed solution, placing the mixed solution at 2-8°C for 12-24h, centrifuging to obtain a precipitate a; A4, resuspending the precipitate a with a PBS solution, filtering the obtained solution through a 0.22-0.25 μm filter membrane, and then adding the filtered solution to a PBS solution containing PEG1000, mixing them thoroughly to obtain a mixed solution, and centrifuging to obtain a precipitate b; A5. Resuspend the precipitate b with PBS solution to obtain a plant exosome suspension.

2. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In the step A1, the plant is Polygonatum sibiricum.

3. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In the step A1, the filtration is performed using a 100-200 mesh nylon filter.

4. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In the step A1, the volume ratio of the plant tissue to the PBS solution is (1-2): (1-2).

5. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In step A2, the centrifugation temperature is 0-4°C.

6. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In step A3, the mass ratio of PEG600 to PEG6000 is (0.5-1):(1.5-2.5).

7. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In step A3, the final mass concentration of PEG600 in the mixed solution is 1-3%; the final mass concentration of PEG6000 in the mixed solution is 4-8%.

8. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In step A3, the centrifugation condition is: centrifugation at a centrifugal force of 10000-15000 g at 2-8° C. for 25-40 min.

9. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In step A4, the centrifugation condition is: centrifugation at a centrifugal force of 10000-15000 g at 0-4° C. for 10-15 min.

10. The method for extracting plant exosomes with anti-inflammatory and antioxidant effects according to claim 1, characterized in that: In the step A4, the mass concentration of PEG1000 in the PBS solution containing PEG1000 is 20-40%, and the mass concentration of PEG1000 in the mixed solution is 2-4%.

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