Preparation method of peony exosome

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of peony exosomes. The preparation method of the peony exosome comprises the following steps: crushing peony plants, filtering, and collecting supernatant to obtain first supernatant; carrying out anion exchange column chromatography on the first supernatant, taking a PBS (Phosphate Buffer Solution) as an elution buffer solution, and collecting a fourth cut-off branch effluent; performing freeze drying on the fourth cut-off branch effluent to obtain freeze-dried powder; dissolving the freeze-dried powder with a PBS buffer solution; obtaining a freeze-dried solution; purifying the freeze-dried solution through sephadex column chromatography, eluting and collecting to obtain a second supernatant; and filtering the second supernate through a microporous filter membrane to obtain the peony exosome. The preparation method of the peony exosome provided by the invention not only obviously improves the extraction efficiency of the peony exosome, but also has the advantages of simplicity and convenience in operation and cost saving, and lays a solid foundation for wide application of the peony exosome in industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to peony exosomes and a preparation method thereof. Background Art

[0002] Recent studies have found that plant exosomes may play an important role in plant material transport, signal transmission, immune defense, and interaction with other organisms. They can also be well integrated with skin lipids to diffuse, absorb, and take effect. In addition, plant exosomes can carry a variety of functional ingredients deep into the skin to exert their effects. Their unique structure and functional characteristics provide new ideas and solutions for skin health, repair, and beauty.

[0003] Peony is a traditional famous flower in my country with a long history of cultivation and a profound cultural foundation. Peony has ornamental value, medicinal and edible value, and cultural value. At present, the peony industry is more focused on cultivation technology, seedling breeding, potted flower and cut flower production, etc. Peony exosomes as raw materials have great potential and development space for deep processing and derivative products of peony.

[0004] At present, the extraction method of peony exosomes adopts conventional plant-derived exosome extraction technology. In exosome extraction technology, differential centrifugation and density gradient centrifugation are usually used to extract plant-derived exosomes. However, the extraction efficiency of peony exosomes extracted by these two methods is low. Therefore, it is necessary to provide a method for efficiently extracting peony exosomes. Summary of the invention

[0005] In order to solve the problem of low extraction efficiency of peony exosomes extracted by conventional plant-derived exosome extraction technology in the prior art, the present invention provides a method for preparing peony exosomes. To achieve the above purpose, the present invention adopts the following technical solution.

[0006] The present invention provides a method for preparing peony exosomes, comprising the following steps:

[0007] The peony plant is mixed with a buffer solution, crushed, filtered to remove the peony plant cell residues, and the filtrate is collected; the buffer solution is a separation buffer solution, which is used to maintain the stability of the peony exosome structure and separate the peony plant cell residues and the filtrate at the same time.

[0008] The filtrate was chromatographed through anion exchange column, eluted with PBS buffer as elution buffer to remove impurities such as foreign proteins and endotoxins, and 1900 mL to 2100 mL was used as a cut-off branch, and the effluent from the 4th cut-off branch was collected. The collected effluent from the 4th cut-off branch was the first batch of branch effluent containing peony exosomes.

[0009] The effluent from the fourth intercepting branch is freeze-dried to obtain a freeze-dried powder; the freeze-dried powder is dissolved in PBS buffer to obtain a freeze-dried solution.

[0010] The lyophilized solution was purified by dextran gel column chromatography, eluted with PBS buffer as the eluent, and the impurities and other small molecule impurities were removed again, and 8 mL to 12 mL was used as an interception branch, and the second interception branch was collected to obtain the second supernatant. The collected second interception branch is the second batch of branches containing peony exosomes.

[0011] The second supernatant is filtered through a microporous filter membrane to collect the filtrate; the filtrate is then precipitated with gelatin to enrich peony exosomes.

[0012] The preparation method of the peony exosomes provided by the present invention uses ion exchange column chromatography and dextran gel column chromatography combined with gelatin precipitation technology for multiple enrichment, which significantly improves the concentration of peony exosomes and significantly improves the extraction efficiency of peony exosomes.

[0013] Preferably, the filling medium in the chromatographic column of the anion exchange column chromatography is Q Sepharose Fast Flow ion exchange chromatography filler. Q Sepharose FF ion exchange chromatography filler is a highly cross-linked agarose filler, which greatly enhances the mechanical properties and has a very fast flow rate. It is used for preparative protein separation and is suitable for industrial-scale production. The filler is decharged, nonspecific adsorption is very low, the recovery rate is also improved, and it has high chemical stability, and a fully verified in-situ cleaning (CIP) and disinfection scheme can be used. The present invention uses Q Sepharose FF ion exchange chromatography filler to increase the concentration of peony exosomes and improve the extraction efficiency of peony exosomes.

[0014] Preferably, the chromatographic column filling medium of the dextran gel column chromatography is Sephadex G25. Sephadex G25 is a chromatographic medium based on dextran gel, which can be used for the separation of proteins of different molecular weights. The present invention uses Sephadex G25 to increase the concentration of peony exosomes and improve the extraction efficiency of peony exosomes.

[0015] Preferably, the pH of the PBS buffer is 7.3-7.5.

[0016] Preferably, the buffer is a separation buffer; the components of the separation buffer include Na2HPO4, K2HPO4, NaCl, CaCl2 and water; the molar concentration of K2HPO4 is 38mmol / L to 42mmol / L ，The molar concentration of the Na2HPO4 is 28 mmol / L to 32 mmol / L, the molar concentration of the NaCl is 8 mmol / L to 12 mmol / L, and the molar concentration of the CaCl2 is 0.5 mmol / L to 0.7 mmol / L. The solvent of the separation buffer is water. The separation buffer can maintain the structure and biological properties of the biological protein, thereby improving the extraction efficiency of peony exosomes.

[0017] Preferably, the mass ratio of the buffer solution to the peony plant is 1:4-6.

[0018] Preferably, the freeze-drying temperature is -18°C to -22°C.

[0019] Preferably, the method of filtering with a microporous membrane is as follows: filtering the second supernatant through microporous membranes with pore sizes of 0.75 μm to 0.85 μm, 0.40 μm to 0.50 μm and 0.20 μm to 0.24 μm in sequence. More preferably, the second supernatant is filtered through microporous membranes with pore sizes of 0.8 μm, 0.45 μm and 0.22 μm in sequence. The use of different pore sizes to filter larger particles of impurities can remove bacterial microorganisms, make the sample clear and clean, and play a role in increasing the concentration of peony exosomes.

[0020] Preferably, the method of gelatin precipitation is as follows:

[0021] The filtrate and gelatin are mixed at a mass ratio of 7 to 9:1 to obtain a reaction solution; the reaction solution is then centrifuged to obtain the peony exosomes.

[0022] The centrifugal conditions are: 9000 rpm to 15000 rpm, 10 min to 50 min.

[0023] This operation step can precipitate proteins, remove other impurities, and increase the concentration of peony exosomes.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention provides a method for preparing peony exosomes. The core objective of the present invention is to create a method for preparing peony exosomes, aiming to efficiently obtain peony exosomes. The method for preparing peony exosomes provided by the present invention first crushes the peony plant, and then sequentially passes it through anion exchange column chromatography and dextran gel column chromatography, and combines gelatin precipitation technology for multiple enrichment to extract peony exosomes. The method for preparing peony exosomes provided by the present invention achieves the effect of significantly improving the extraction efficiency of peony exosomes through the cooperation of the above methods, and also has the advantages of simple operation and cost saving, which lays a solid foundation for the wide application of peony exosomes in industrial production.

[0026] 2. The present invention uses ion exchange column chromatography and dextran gel column chromatography combined with gelatin precipitation technology for multiple enrichment, which significantly improves the concentration of peony exosomes. It has great flexibility and can be customized according to the specific properties of exosomes and experimental requirements. For example, different ion exchangers, buffers, and elution conditions can be selected to optimize the enrichment effect. And it avoids the use of expensive ultra-high-speed refrigerated centrifuges and other professional equipment and high-cost commercial purification columns or kits. Only common laboratory chromatography columns and centrifuges and low-cost microporous filter membranes are required. It has strong operability, simple and easy operation methods, and reduces the cost of isolating peony exosomes.

[0027] 3. The preparation method of peony exosomes provided by the present invention has high separation efficiency. The identification and characterization confirmed that the extracted peony exosomes have a typical hemispherical or saucer-like structure and an ideal particle size, with an average particle size of 77.3 nm and a concentration of 1.21×10 11 Particles / mL, and its protein concentration reached 25.056μg / μL, with extremely high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a transmission electron micrograph of peony exosomes in Example 1 of the present invention.

[0029] Figure 2 This is the NanoFCM characterization result of peony exosomes in Example 1 of the present invention.

[0030] Figure 3 This is the NanoFCM characterization result of peony exosomes in Comparative Example 1 of the present invention.

[0031] Figure 4 This is the NanoFCM characterization result of peony exosomes in Comparative Example 2 of the present invention.

[0032] Figure 5 This is the NanoFCM characterization result of peony exosomes in Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0034] The materials and reagents used in the following examples are as follows:

[0035] The peony plant comes from Luoyang peony.

[0036] PBS buffer was abbreviated as PBS, which was purchased from Shanghai Titan Technology Co., Ltd. with a concentration of 1×, premixed granule, and a pH of 7.4.

[0037] The 0.8 μm microporous filter membrane, 0.45 μm microporous filter membrane and 0.22 μm microporous filter membrane were purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.

[0038] Example 1: Preparation of Peony Exosomes

[0039] 1. Preparation of Peony Exosomes

[0040] (1) After the peony plant is cleaned with double distilled water, it is mixed with separation buffer at a mass ratio of 1:5 and then crushed. After crushing, it is filtered with gauze to collect the supernatant to obtain the first supernatant.

[0041] Among them, the components of the separation buffer include Na2HPO4, K2HPO4, NaCl, CaCl2 and water, the molar concentration of K2HPO4 is 40mmol / L, the molar concentration of Na2HPO4 is 30mmol / L, the molar concentration of NaCl is 10mmol / L, the molar concentration of CaCl2 is 0.6mmol / L, the solvent of the separation buffer is water, and the pH value of the separation buffer is 7.3.

[0042] (2) The first supernatant is passed through an ion exchange column for chromatography, and impurities are removed by utilizing the adsorption characteristics of different ions, and the effluent is collected. The specific steps are as follows:

[0043] (a) The volume of the first supernatant loaded on the ion exchange column was 300 mL.

[0044] (b) The filling volume of the ion exchange column chromatography was 12000 mL.

[0045] (c) The eluent volume of the ion exchange column chromatography was 18000 mL.

[0046] (d) The medium filled in the chromatographic column of the ion exchange column chromatography is Q Sepharose Fast Flow ion exchange chromatography filler.

[0047] (e) Ion exchange column chromatography, with 2000 mL as one cut-off branch, and the fourth cut-off branch was collected.

[0048] (f) The elution buffer was PBS buffer.

[0049] Among them, the concentrations of the elution buffer are 0.25mmol / L, 0.5mmol / L, 1mmol / L, 5mmol / L, 20mmol / L, 50mmol / L and 100mmol / L.

[0050] (3) adding a lyophilization protective agent to the effluent of the fourth interception branch for freeze-drying to obtain a lyophilized powder; dissolving the lyophilized powder in PBS buffer to obtain a lyophilized solution.

[0051] Among them, freeze drying includes the following steps: first, cool the vacuum freeze dryer to -20°C and keep it for 8 hours; then turn on the vacuum pump, control the vacuum degree to 1Pa, gradually increase the shelf temperature to 25°C, and freeze dry for 30 hours.

[0052] The volume ratio of the lyophilized powder to PBS buffer was 1:6.

[0053] The freeze-drying protective agent includes one or more of mannitol, trehalose, glucose and dextran 20000.

[0054] The total amount of the freeze-dried protective agent is 10% of the total mass of the fourth intercepting branch in terms of mass percentage.

[0055] (4) The freeze-dried solution is further separated and purified by dextran gel column chromatography to obtain a second supernatant. The specific steps are as follows:

[0056] (a) The volume of the lyophilized solution loaded for the dextran gel column chromatography was 5 mL.

[0057] (b) The column filling volume for the dextran gel column chromatography was 50 mL.

[0058] (c) The eluent volume for ion exchange column chromatography was 50 mL.

[0059] (d) The medium filled in the chromatographic column of the dextran gel column chromatography is Sephadex G25.

[0060] (e) Sephadex column chromatography, with 10 mL as one cut-off branch, and the second cut-off branch was collected.

[0061] (f) The eluent was PBS buffer.

[0062] The concentration of the elution buffer was 0.45 mmol / L.

[0063] (5) Filter the second supernatant through a microporous filter membrane to obtain a preliminary exosome filtrate.

[0064] Among them, the microporous membrane filtration includes the following steps: after filtering through a microporous membrane with a pore size of 0.8 μm, the filtrate is collected and then filtered through a microporous membrane with a pore size of 0.45 μm, the collected filtrate is finally filtered through a microporous membrane with a pore size of 0.22 μm, and the filtrate is collected to obtain the preliminary filtrate of exosomes.

[0065] (6) The exosome preliminary filtrate and gelatin are mixed and stirred to obtain a reaction solution.

[0066] Among them, the mass ratio of exosome preliminary filtrate to gelatin was 8:1.

[0067] A magnetic stirrer was used for the stirring process. The speed of the magnetic stirrer was set to 300 rpm and the stirring time was 15 h.

[0068] (7) The reaction solution was centrifuged, the supernatant was discarded, and the precipitate was resuspended with ultrapure water to obtain a heavy suspension containing peony exosomes.

[0069] The centrifugal conditions are as follows: centrifugal speed: 12000 rpm, centrifugal time: 40 min.

[0070] (8) Centrifuging the heavy suspension containing peony exosomes, taking the supernatant, and obtaining peony exosomes.

[0071] The centrifugal conditions are as follows: centrifugal speed: 12000 rpm, centrifugal time: 15 min.

[0072] 2. Characterization and Analysis of Peony Exosomes

[0073] 10 μL of the peony exosomes obtained above was taken out and dropped on a copper mesh to precipitate for 1 min. After the floating liquid was removed by filter paper, 10 μL of uranyl acetate was dropped on the copper mesh to precipitate for 1 min. The floating liquid was removed by filter paper.

[0074] The exosome sample on the copper grid was dried at room temperature for several minutes to obtain an air-dried sample after staining.

[0075] The stained air-dried samples were imaged by electron microscopy at 80 kV. The transmission electron microscopy imaging results are shown below: Figure 1 .

[0076] NanoFCM instrument was used to detect the concentration of exosomes. The particle size and concentration of peony exosomes were shown in Figure 1 and Figure 2 .

[0077] Depend on Figure 1 It can be seen that the peony exosomes prepared in Example 1 have a typical hemispherical or saucer-like structure.

[0078] Depend on Figure 2 It can be seen that the concentration of peony exosomes prepared in Example 1 is 1.21E+11Particles / mL, that is, 1.21×10 11 Particles / mL.

[0079] Comparative Example 1

[0080] This comparative example refers to the method disclosed in the patent number CN 114306409 A, "A method for extracting maca exosomes", to extract peony exosomes. The specific steps are as follows:

[0081] Peony plants were selected and firstly washed thoroughly three times with pure water. The cleaned peony plants were then sent into a juicer for three consecutive crushing and juicing.

[0082] After the juice is squeezed, the juice is filtered through gauze and centrifuged continuously: first, centrifuged at a centrifugal force of 1000g for 10 minutes, then at a centrifugal force of 3000g for 20 minutes, and finally at a centrifugal force of 10000g for 40 minutes. After the differential centrifugation, the supernatant is taken, the precipitate is discarded, and the supernatant is further subjected to ultra-high-speed centrifugation at a centrifugal force of 150000g for 120 minutes. After the centrifugation is completed, the precipitate is collected and suspended in ice PBS buffer to prepare a precipitate suspension.

[0083] Next, 8% wt, 30% wt, 45% wt and 60% wt sucrose solutions were added to the centrifuge tube in order according to the concentration gradient, and then the above-prepared precipitate suspension was added to the top layer. After that, ultra-high-speed centrifugation was performed again at a centrifugal force of 150,000 g for multiple times, each time for 120 minutes.

[0084] After centrifugation, the liquid in each sucrose concentration layer was collected into a cleaned centrifuge tube, ice PBS buffer was added and mixed evenly, and then centrifuged again at a centrifugal force of 150,000 g for 90 min to remove the sucrose.

[0085] Finally, the precipitate was aliquoted and resuspended in 1 to 3 ml of PBS buffer, filtered through a 0.22 μL filter membrane, and then the particle size was measured.

[0086] The concentration of the obtained peony exosomes was detected by NanoFCM instrument, and the results were as follows Figure 3 shown.

[0087] Depend on Figure 3 It can be seen that the concentration of peony exosomes obtained by the method provided in Comparative Example 1 is 2.4E+10 Particles / mL, that is, 2.4×10 10 Particles / mL.

[0088] Comparative Example 2

[0089] This comparative example refers to the method disclosed in the patent number CN 110302278 B, "A method for preparing exosomes of Rehmannia glutinosa", and the specific steps are as follows:

[0090] Peony plants were selected, washed thoroughly with pure water, and then squeezed into peony juice using a juicer, and the obtained peony juice was frozen for subsequent use. In order to prepare peony exosomes, the frozen peony juice was first thawed and then subjected to a series of centrifugation treatments. First, supernatant A was obtained by centrifugation at a centrifugal force of 300g for 10 minutes; then, supernatant A was centrifuged again at a centrifugal force of 2000g for 10 minutes to obtain supernatant B; then, supernatant B was centrifuged at a centrifugal force of 10000g for 30 minutes to obtain supernatant C. Finally, supernatant C was centrifuged at a centrifugal force of 100000g for up to 80 minutes to obtain precipitate D. The precipitate D was suspended with PBS buffer to obtain the total extract of peony exosomes.

[0091] In order to obtain more refined peony exosomes, PEG (polyethylene glycol) with a molecular weight of 4000 was prepared into a 50% by volume PEG solution, and then the total extract of peony exosomes was added to the 50% by volume PEG solution, stirred and allowed to stand for 24 hours, and then centrifuged. The precipitate obtained was the peony exosomes.

[0092] The obtained peony exosomes were tested for the concentration of exosomes using NanoFCM instrument. The results are shown in Figure 4 .

[0093] Depend on Figure 4 It can be seen that the concentration of peony exosomes obtained by the method provided in Comparative Example 2 is 1.1E+9 Particles / mL, that is, 1.1×10 9 Particles / mL.

[0094] Comparative Example 3

[0095] This comparative example refers to the method disclosed in the patent number CN 114507634A, "A method for extracting ginger exosomes", to extract peony exosomes. The specific steps are as follows:

[0096] The peony plants are fully stirred to homogenize, and then filtered through a gauze to remove the residue, and preliminary peony juice is collected, which is the first peony juice. The first peony juice is centrifuged at a centrifugal force of 4000g for 15 minutes, and then the supernatant is taken; the supernatant is further centrifuged at a centrifugal force of 4000g for 90 minutes, and the supernatant is also taken; finally, the supernatant is centrifuged at a centrifugal force of 10000g for 60 minutes, and the supernatant is taken again.

[0097] The supernatant after the above treatment was filtered through 0.8 μm and 0.45 μm microporous membranes in turn to obtain a purer peony juice, which is the second peony juice. Subsequently, PEG6000 was added according to the volume of the second peony juice to a final concentration of 10% wt. After PEG6000 was completely dissolved, the solution was allowed to stand for 12 hours.

[0098] The static solution is centrifuged at a centrifugal force of 10,000 g for 30 minutes, and then the precipitate is collected to obtain peony exosomes.

[0099] The obtained peony exosomes were tested for the concentration of exosomes using NanoFCM instrument. The results are shown in Figure 5 .

[0100] Depend on Figure 5 It can be seen that the concentration of peony exosomes obtained by the method provided in Comparative Example 3 is 2.09E+10Particles / mL, that is, 2.09×10 10 Particles / mL.

[0101] The concentrations of peony exosomes obtained in Example 1 and Comparative Examples 1 to 3 are shown in Table 1.

[0102] Table 1 Peony exosome concentration table

[0103]

[0104] From the results in Table 1, it can be seen that the concentration of peony exosomes obtained by the preparation method provided by the present invention is higher, which significantly improves the extraction efficiency of peony exosomes and also has the advantages of simple operation and cost saving.

[0105] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0106] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A method for preparing peony exosomes, characterized in that: The steps include: The peony plant is mixed with a buffer solution, crushed, filtered to remove the peony plant cell residues, and the filtrate is collected; The filtrate was chromatographed on an anion exchange column using PBS buffer as the elution buffer to remove impurities such as foreign proteins and endotoxins, and the first branch outflow containing peony exosomes was collected; The first batch of tributary effluent containing peony exosomes is freeze-dried to obtain a freeze-dried powder; the freeze-dried powder is dissolved in PBS buffer; obtaining a lyophilized solution; Purifying the lyophilized solution by dextran gel column chromatography, eluting with PBS buffer as an eluent, removing protein impurities again, collecting a second batch of branches containing peony exosomes, and obtaining a second supernatant; The second supernatant is filtered through a microporous filter membrane to collect the filtrate; The filtrate was then precipitated with gelatin to enrich peony exosomes.

2. The method for preparing peony exosomes according to claim 1, characterized in that: The filling medium in the chromatographic column of the anion exchange column chromatography is Q Sepharose Fast Flow ion exchange chromatography filler.

3. The method for preparing peony exosomes according to claim 1, characterized in that: The chromatographic column filling medium of the dextran gel column chromatography is Sephadex G25.

4. The method for preparing peony exosomes according to claim 1, characterized in that: The pH of the PBS buffer is 7.3-7.

5.

5. The method for preparing peony exosomes according to claim 1, characterized in that: The buffer is a separation buffer; the components of the separation buffer include Na2HPO4, K2HPO4, NaCl, CaCl2 and water; the molar concentration of K2HPO4 is 38mmol / L to 42mmol / L ， The molar concentration of the Na2HPO4 is 28mmol / L to 32mmol / L, the molar concentration of the NaCl is 8mmol / L to 12mmol / L, and the molar concentration of the CaCl2 is 0.5mmol / L to 0.7mmol / L.

6. The method for preparing peony exosomes according to claim 1, characterized in that: When performing anion exchange column chromatography, 1900 mL to 2100 mL is used as a cut-off branch, and the collected 4th cut-off branch effluent is the first batch of branch effluent containing peony exosomes.

7. The method for preparing peony exosomes according to claim 1, characterized in that: When performing the dextran gel column chromatography purification, 8 mL to 12 mL is used as one cut-off branch, and the collected second cut-off branch is the second batch of branches containing peony exosomes.

8. The method for preparing peony exosomes according to claim 1, characterized in that: The method of filtering with microporous membrane is as follows: filtering the second supernatant through microporous membranes with pore sizes of 0.75 μm to 0.85 μm, 0.40 μm to 0.50 μm and 0.20 μm to 0.24 μm in sequence.

9. The method for preparing peony exosomes according to claim 1, characterized in that: The method of the gelatin precipitation is as follows: The filtrate and gelatin are mixed at a mass ratio of 7 to 9:1 to obtain a reaction solution; the reaction solution is then centrifuged to obtain the peony exosomes; The centrifugal conditions are: 9000 rpm to 15000 rpm, 10 min to 50 min.

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