Method for rapidly extracting plant rhizome extracellular vesicles from disaccharide and compound enzyme

Abstract

The invention is applicable to the technical field of biology, and provides a method for rapidly extracting plant rhizome extracellular vesicles by disaccharide and compound enzyme. According to the method, an enzymolysis-disaccharide solution extraction coupling method is created for the first time to extract the plant extracellular vesicles, and the defect that an extraction method is single is overcome. According to the method, the conditions are mild, the primary property, the integrity and the biological activity of the vesicle structure are kept, the extraction purity is high, and the particle size distribution is uniform and concentrated in the range of 40-300 nm. Compared with PBS (Phosphate Buffer Solution) extraction treatment, the total protein extraction amounts of lactose, cane sugar and maltose solutions are respectively increased by 20.04% + / -4.50%, 35.00% + / -5.74% and 36.45% + / -2.68%. In addition, the'single ultracentrifugation 'purification process overcomes the problem of time consumption of traditional gradient centrifugation, and the time is shortened by more than 3h compared with that of a traditional extraction method. The method breaks through the technical bottleneck of large-scale production of the plant extracellular vesicles, and has great application and popularization values.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for rapidly extracting extracellular vesicles from plant root and stem cells using disaccharides and complex enzymes. Background Technology

[0002] Plant extracellular vesicles are nanometer-sized vesicles with a phospholipid bilayer structure, their membrane structure derived from the intracellular membrane system. These vesicles contain proteins, nucleic acids, lipids, and other bioactive substances from donor cells. Natural plant extracellular vesicles have been shown to have the potential to deliver effective substances, including nucleic acids, to other species, initiating interspecific or interkingdom communication, and have shown promising potential in the treatment of skin diseases, pulmonary fibrosis, inflammation, hair loss or hair regrowth, liver diseases, and tumors. As an emerging communication carrier, extracellular vesicles have received widespread attention in recent years.

[0003] Extracting high-quality extracellular vesicles is a primary prerequisite for studying their function and mechanism of action. With the continuous advancement of extracellular vesicle research, simple, efficient, and cost-effective extracellular vesicle isolation and purification techniques are constantly being developed and optimized. Currently, a common sample pretreatment method during extracellular vesicle isolation is grinding with intense shear force. This method inevitably damages the original state and integrity of the extracellular vesicles, and may even reduce their biological activity. Regarding purification methods, commonly used methods include ultracentrifugation, density gradient centrifugation, ultrafiltration, immunoaffinity capture, size exclusion chromatography, and polymer precipitation, but these methods all have certain limitations. Among these methods, ultracentrifugation is simple to operate, low in cost, high in yield, and suitable for large sample volumes, but it is time-consuming, easily damages vesicle structures, and can lead to the co-precipitation of contaminant particles. Density gradient centrifugation yields higher purity than ultracentrifugation, but it is complex to operate and time-consuming. Ultrafiltration is quick and simple to operate, but it has lower extraction yield and purity, higher material consumption, and centrifugation shear force can easily damage vesicle structures and cause the loss of small-diameter extracellular vesicles. Immunoaffinity capture has high specificity and high extraction purity, but it is time-consuming, has low yield, and lacks universal antibodies for plant extracellular vesicles, resulting in high costs. Size exclusion chromatography can preserve vesicle structure and biological function to the maximum extent, but it is time-consuming, requires special chromatographic columns and packing materials, is susceptible to lipoprotein contamination, is costly, and is difficult to mass-produce. Polymer precipitation is simple to operate and can process large sample volumes, but it is time-consuming, has low purity, can co-precipitate with other non-extracellular vesicle substances, and the polymer itself is difficult to remove.

[0004] Therefore, constructing an extraction and purification method that can promote the release of more extracellular vesicles from plant cells, maximize the preservation of vesicle integrity and biological activity, and facilitate industrial-scale production is of significant practical importance. Based on this, this invention proposes a method for the rapid extraction of extracellular vesicles from plant root and stem cells using disaccharides and complex enzymes. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapidly extracting extracellular vesicles from plant root and stem cells using disaccharides and complex enzymes, thereby addressing the problems mentioned in the background art.

[0006] The objective of this invention is achieved through the following technical solution: A method for rapid extraction of extracellular vesicles from plant root and stem cells using disaccharides and complex enzymes, comprising the following extraction steps: Step 1: After cleaning the fresh root and rhizome medicinal materials, cut them into small pieces for later use; Step 2: Add cellulase and pectinase to a 5-30 wt% disaccharide solution, and stir with a magnetic stirrer until fully dissolved to obtain an extract; Step 3: Add the tissue fragments to the extract at a material-to-liquid ratio of 1:1 to 1:10 and mix well. Incubate at 4-40℃ for 1 hour to obtain a tissue-extract mixture. Step 4: Sonicate the tissue-extract mixture with an ultrasonic power of 0-80W for 0-15 minutes to break down the cell walls. Step 5: Place the ultrasonically treated tissue-extract mixture into a silk bag, squeeze and collect the filtrate using a stainless steel wringer; Step 6: Centrifuge the filtrate after sonication and collect the supernatant; Step 7: Filter the supernatant through a filter membrane to obtain crude extract of extracellular vesicles; Step 8: Centrifuge the crude extract of extracellular vesicles at 120,000g for 2 hours at 4°C. Resuspend the precipitate in PBS solution and vortex to obtain an extracellular vesicle suspension.

[0007] Furthermore, in step 1, the cleaning includes: first washing with tap water 3 times and removing surface dirt and stains with a brush, and then rinsing with deionized water 3 times.

[0008] Furthermore, in step 2, the disaccharide is any one of lactose, sucrose, and maltose; the amount of cellulase added is 0-6 wt%, and the amount of pectinase added is 0-6 vol%.

[0009] Furthermore, in step 6, the supernatant is collected by centrifugation at 4°C and 12000g for 60 minutes.

[0010] Furthermore, in step 7, the filtration sequence is as follows: first through a 0.45μm filter membrane, then through a 0.22μm filter membrane.

[0011] Furthermore, in step 8, the extracellular vesicle suspension is stored in a -80°C freezer for later use.

[0012] Furthermore, the root and rhizome medicinal material tissue is dandelion root tissue, ginseng root tissue, or ginger tissue.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention pioneers a "coupling of enzymatic hydrolysis and disaccharide solution extraction" method for extracting plant extracellular vesicles, overcoming the drawbacks of single extraction methods. This method operates under mild conditions, preserving the primary structure, integrity, and biological activity of the vesicles, while achieving high extraction purity with a uniform particle size distribution concentrated in the 40-300 nm range, free from large particle contamination. Lactose, sucrose, and maltose solutions significantly increased the total protein extraction compared to PBS, by 20.04%±4.50%, 35.00%±5.74%, and 36.45%±2.68%, respectively. This indicates that utilizing osmotic pressure differences can promote the release of extracellular vesicles from plant cells when osmotic pressure is balanced, thereby improving extraction efficiency. Compared to PBSS extraction, synergistic treatment with compound enzymes and lactose, sucrose, and maltose solutions increased the total protein extraction by 282.49%±26.32%, 248.85%±11.61%, and 208.85%±10.69%, respectively; and compared to disaccharide solution extraction, the total protein extraction increased by 218.45%±13.13%, 158.80%±16.05%, and 126.31%±3.51%, respectively. This indicates that synergistic treatment with compound enzymes and disaccharide solutions can further improve extraction yield. Furthermore, the "single-time ultracentrifugation" purification process developed in this invention overcomes the time-consuming problem of traditional gradient centrifugation, shortening the extraction time by more than 3 hours compared to traditional methods. This invention breaks through the technical bottleneck of large-scale production of plant extracellular vesicles and has significant application and promotion value. Attached Figure Description

[0014] Figure 1 This is a particle size distribution diagram of extracellular vesicles from dandelion roots extracted and purified using the synergistic treatment method of compound enzyme and lactose solution in Example 1.

[0015] Figure 2 This is a transmission electron microscopy (TEM) image of the extracellular vesicles from dandelion roots extracted and purified using the synergistic treatment method of compound enzyme and lactose solution in Example 1.

[0016] Figure 3 This is a particle size distribution diagram of extracellular vesicles from ginseng roots extracted and purified using the synergistic treatment method of compound enzyme and sucrose solution in Example 2.

[0017] Figure 4 This is a transmission electron microscopy (TEM) image of extracellular vesicles from ginseng roots extracted and purified using the synergistic treatment method of compound enzyme and sucrose solution in Example 2.

[0018] Figure 5This is a particle size distribution diagram of the extracellular vesicles of ginger extracted and purified using the synergistic treatment method of compound enzyme and maltose solution in Example 3.

[0019] Figure 6 This is a transmission electron microscopy (TEM) image of the ginger extracellular vesicles extracted and purified using the synergistic treatment method of compound enzyme and maltose solution in Example 3.

[0020] Figure 7 The total amount of protein extracted from extracellular vesicles in the roots of dandelion in each group (PBS in the figure represents PBS extraction treatment; lactose represents treatment with lactose solution only; and compound enzyme represents treatment with compound enzyme and lactose solution in synergistic process).

[0021] Figure 8 The total amount of protein extracted from extracellular vesicles in ginseng root cells in each group (PBS in the figure represents PBS extraction treatment; sucrose represents treatment with lactose solution only; and compound enzyme represents treatment with a combination of compound enzyme and sucrose solution).

[0022] Figure 9 The total amount of protein extracted from the extracellular vesicles of ginger cells in each group is shown in the figure (PBS represents PBS extraction treatment; maltose represents treatment with lactose solution only; and compound enzyme represents treatment with compound enzyme and maltose solution).

[0023] Figure 10 This is a comparison of the theoretical time required for traditional extraction methods and the extraction method of this invention.

[0024] Figure 11 This is a technical roadmap for the present invention. Detailed Implementation

[0025] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0026] The invention selects plant roots and stems as raw materials, such as dandelion, ginseng, or ginger (including but not limited to the roots and stems of the above three plants). The technical roadmap of this invention is shown below. Figure 11 As shown.

[0027] The specific implementation of the present invention will be described in detail below with reference to specific embodiments. The cellulase activity used in the embodiments is 1*102 5 IU / g, pectinase activity is 1*10 4 IU / mL; cellulase and pectinase were purchased from Ningxia Heshibi Biotechnology Co., Ltd. and Nanjing Pangbo Bioengineering Co., Ltd., respectively.

[0028] Example 1: Using dandelion roots as raw material, the steps of a method for rapidly extracting extracellular vesicles from plant root and stem cells using disaccharides and complex enzymes are as follows: Step 1, Sample pretreatment: Wash 50g of fresh dandelion root tissue with tap water 3 times, remove surface dirt and stains with a brush, rinse with deionized water 3 times, and cut it into uniform fine shreds with a diameter of about 0.4cm or slices with a thickness of 0.4cm using a shredder or slicer to obtain tissue fragments for later use. Step 2, Preparation of extract: Add 2wt% (5.0g) cellulase and 6vol% (15.0mL) pectinase to 15wt% lactose solution (250mL). Place the mixture in a magnetic stirrer and stir at 400rpm / min for 3-5min until completely dissolved to obtain the extract. Step 3: Preparation of extracellular vesicle exudate: Add the tissue fragments from Step 1 to the extract at a material-to-liquid ratio of 1:5 and mix well (e.g., add 50g of tissue fragments to 250mL of extract). Incubate at 36℃ for 1h to obtain a mixture of root tissue extracellular vesicle exudate and tissue fragments (referred to as tissue-extract mixture). Step 4, Ultrasonic disruption of cell walls: The tissue-extract mixture from Step 3 is subjected to ultrasonic treatment with an ultrasonic power of 30W and a time of 15min to disrupt the cell walls; Step 5, Filtrate Collection: Place the tissue-extract mixture from Step 4 into a silk bag, squeeze and collect the filtrate using a stainless steel squeezer; Step 6, Preparation of crude extract of extracellular vesicles: Centrifuge the filtrate obtained in step 5 at 4℃ and 12000g for 60 min and collect the supernatant; Step 7: Filtration of crude extracellular vesicle extract: The supernatant obtained in step 6 is filtered through 0.45μm and 0.22μm filter membranes to remove fine debris and large particulate contaminants, thus obtaining crude extracellular vesicle extract; Step 8: Purification of extracellular vesicles: The crude extract of extracellular vesicles obtained in Step 7 was centrifuged at 120,000g for 2 hours at 4°C. The supernatant was discarded, and the precipitate was resuspended in 2 mL of PBS (0.137 M NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, 0.01 M Na2HPO4). The solution was placed on a vortex mixer and vortexed at 3000 rpm for 3-5 minutes. After mixing, the extracellular vesicle suspension was obtained and stored at -80°C for later use.

[0029] Example 2: Using ginseng root as raw material, the steps of a method for rapidly extracting extracellular vesicles from plant root and stem cells using disaccharides and complex enzymes are as follows: The difference from the steps in Example 1 is as follows: the dandelion root tissue in step 1 of Example 1 is replaced with ginseng root tissue; in step 2, 4wt% (12.0g) cellulase and 5vol% (15.0mL) pectinase are added to a 5wt% sucrose solution (300mL); in step 3, the tissue fragments from step 1 are added to the extract at a material-to-liquid ratio of 1:6 and mixed well (e.g., 50g of tissue fragments are added to 300mL of extract), and incubated at 24℃ for 1h; the ultrasonic treatment conditions in step 4 are ultrasonic power of 50W and time of 10min, and the remaining operations are the same as in the steps in Example 1.

[0030] Example 3: Using ginger as raw material, the steps of a method for rapidly extracting extracellular vesicles from plant root and stem cells using disaccharides and complex enzymes are as follows: The difference from the steps in Example 1 is as follows: the dandelion root tissue in step 1 of Example 1 is replaced with ginger tissue; in step 2, 3wt% (4.5g) cellulase and 4vol% (6.0mL) pectinase are added to 20wt% maltose solution (150mL); in step 3, the tissue fragments from step 1 are added to the extract at a material-to-liquid ratio of 1:3 and mixed well (e.g., 50g of tissue fragments are added to 150mL of extract), and incubated at 16℃ for 1h; the ultrasonic treatment conditions in step 4 are 60W ultrasonic power and 5min time, and the remaining operations are the same as in the steps in Example 1.

[0031] Example 4: Particle size and potential characterization; The samples obtained in Examples 1-3 (extracellular vesicles of dandelion roots extracted and purified by the co-processing method of compound enzyme and lactose solution, extracellular vesicles of ginseng roots extracted and purified by the co-processing method of compound enzyme and sucrose solution, and extracellular vesicles of ginger extracted and purified by the co-processing method of compound enzyme and maltose solution) were diluted with PBS solution (0.137M NaCl, 2.7mM KCl, 1.8mM KH2PO4, 0.01M Na2HPO4) and then detected using a Malvern ZS-90 nanoparticle zeta potential analyzer.

[0032] The results are as follows Figure 1 , Figure 3 and Figure 5As shown, the extracellular vesicles extracted by the method of this invention have high purity, with particle sizes concentrated in the 40-300 nm range, and no obvious large particle contamination. The average particle sizes of extracellular vesicles from different root and stem medicinal materials are 181.9 nm (dandelion), 179.8 nm (ginseng), and 180.6 nm (ginger), respectively, with Zeta potentials of -10.2 mV, -8.64 mV, and -11.0 mV, and polydispersity index (PdI) of 0.130, 0.186, and 0.085, respectively. It is noteworthy that even under the same isolation and purification conditions, the Zeta potentials of extracellular vesicles from different plant roots or stems (such as those from ginseng and ginger) show systematic differences. This suggests that species origin is an independent influencing factor leading to differences in surface charge. This difference may be related to differences in plant cell wall composition or the distribution characteristics of polar groups in membrane proteins, indicating that the charge characteristics of extracellular vesicles are mainly regulated by the plant's genetic background and are unrelated to the isolation and purification method of this invention.

[0033] Example 5: Transmission electron microscopy characterization; In this embodiment, the morphology of extracellular vesicles was characterized using transmission electron microscopy. The specific steps are as follows: 1) Take 10 μL of the samples obtained in Examples 1-3 and drop them onto a copper grid, allowing them to stand for 5 min to adsorb. 2) Use filter paper to absorb the excess sample on the copper mesh, and drop 2% phosphotungstic acid solution vertically onto the surface of the copper mesh for negative staining for 5 minutes. After that, use filter paper to absorb the excess negative staining solution again. 3) Wash the copper mesh three times with PBS solution (0.137M NaCl, 2.7mM KCl, 1.8mM KH2PO4, 0.01M Na2HPO4), 5 min each time; 4) Dry at room temperature overnight; 5) Observe the morphology of the sample using a transmission electron microscope at a voltage of 80 kV.

[0034] The results are as follows Figure 2 , Figure 4 and Figure 6 As shown in the figure, the extracellular vesicles from different rhizome-based medicinal materials exhibit typical vesicle morphologies, mostly round or cup-shaped structures with one side concave. This indicates that the extracellular vesicles extracted by the method of this invention can be clearly observed in morphology using transmission electron microscopy, verifying the effectiveness of the extraction method.

[0035] Example 6: Protein quantification (BCA method); 1) Preparation of standard protein: Before use, add 1 mL of 1xPBS to 2 mg of BSA standard protein lyophilized powder and mix well to achieve a concentration of 2 mg / mL.

[0036] 2) Preparation of working solution: Mix BCA Reagent A and BCA Reagent B in a ratio of 50:1. Prepare and use immediately according to the experimental dosage.

[0037] 3) Create a standard curve according to the table below.

[0038] Table 1. Preparation of Standard Curve for Protein Quantification Using BCA Method

[0039] 4) Experimental steps: Preheat the microplate reader for 30 min, adjust the wavelength to 562 nm, prepare 20 μL of standard, add 20 μL of sample to a 96-well plate, add 200 μL of BCA working solution to each well, mix well by pipetting, incubate at 37 °C for 30 min, and measure the OD value at a wavelength of 562 nm.

[0040] The protein concentration of extracellular vesicles from dandelion roots, ginseng roots, and ginger was determined using the BCA method described above to compare extraction efficiency.

[0041] The results were as follows regarding extracellular vesicles in dandelion roots: Figure 7 As shown, the protein content of PBS (after PBS extraction) was 0.410 mg, 0.379 mg, and 0.409 mg (results from three independent experiments); the protein content of lactose (extraction using lactose solution only) was 0.474 mg, 0.477 mg, and 0.485 mg (results from three independent experiments). Compared with PBS, lactose increased the total protein extraction by 20.04% ± 4.50%. Furthermore, the protein content of the compound enzyme (using a synergistic treatment method with compound enzyme and lactose solution) was significantly increased to 1.446 mg, 1.524 mg, and 1.606 mg (results from three independent experiments). Compared with PBS and lactose, the compound enzyme increased the total protein extraction by 282.49% ± 26.32% and 218.45% ± 13.13%, respectively.

[0042] The results were as follows regarding extracellular vesicles in ginseng roots: Figure 8As shown, the protein content of PBS (after PBS extraction) was 0.357 mg, 0.390 mg, and 0.404 mg (results from three independent experiments); the protein content of sucrose (extraction with sucrose solution only) was 0.520 mg, 0.531 mg, and 0.501 mg (results from three independent experiments). Compared with PBS, sucrose increased the total protein extraction by 35.00% ± 5.74%. Furthermore, the protein content of the compound enzyme (using a synergistic treatment method with compound enzyme and sucrose solution) was significantly increased to 1.336 mg, 1.463 mg, and 1.222 mg (results from three independent experiments). Compared with PBS and sucrose, the total protein extraction by the compound enzyme treatment was increased by 248.85% ± 11.61% and 158.80% ± 16.05%, respectively.

[0043] For ginger extracellular vesicles, the results are as follows Figure 9 As shown, the protein content of PBS (after PBS extraction) was 0.594 mg, 0.573 mg, and 0.565 mg (results from three independent experiments); the protein content of maltose (extraction with maltose solution only) was 0.755 mg, 0.825 mg, and 0.784 mg (results from three independent experiments). Compared with PBS, maltose increased the total protein extraction by 36.45% ± 2.68%. Furthermore, the protein content of the compound enzyme (using a combined treatment method with maltose solution) was significantly increased to 1.678 mg, 1.785 mg, and 1.888 mg (results from three independent experiments). Compared with PBS and maltose treatment, the total protein extraction by the compound enzyme treatment was increased by 208.85% ± 10.69% and 126.31% ± 3.51%, respectively.

[0044] The above results indicate that extraction with disaccharide solution and synergistic extraction with compound enzyme and disaccharide solution, compared with PBS extraction, can effectively promote the release of extracellular vesicles in ginger cells and increase yield.

[0045] Example 7: Comparison of theoretical time; The theoretical time required to compare traditional extraction methods with that of the extraction method of this invention ( Figure 10 It was found that the theoretical time required for the extraction method of the present invention (265 min, about 4.4 h) is much lower than the theoretical time of the traditional extraction method (480 min, i.e. 8 h), which shortens the time by more than 3 h, demonstrating the significant advantage of the extraction method of the present invention in terms of time efficiency.

[0046] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for rapidly extracting extracellular vesicles from plant root and stem cells using disaccharides and complex enzymes, characterized in that, The extraction process includes the following steps: Step 1: After cleaning the fresh root and rhizome medicinal materials, cut them into small pieces for later use; Step 2: Add cellulase and pectinase to a 5-30 wt% disaccharide solution, and stir with a magnetic stirrer until fully dissolved to obtain an extract; Step 3: Add the tissue fragments to the extract at a material-to-liquid ratio of 1:1 to 1:10 and mix well. Incubate at 4-40℃ for 1 hour to obtain a tissue-extract mixture. Step 4: Sonicate the tissue-extract mixture with an ultrasonic power of 0-80W for 0-15 minutes to break down the cell walls. Step 5: Place the ultrasonically treated tissue-extract mixture into a silk bag, squeeze and collect the filtrate using a stainless steel wringer; Step 6: Centrifuge the filtrate after sonication and collect the supernatant; Step 7: Filter the supernatant through a filter membrane to obtain crude extract of extracellular vesicles; Step 8: Centrifuge the crude extract of extracellular vesicles at 120,000g for 2 hours at 4°C. Resuspend the precipitate in PBS solution and vortex to obtain an extracellular vesicle suspension.

2. The method according to claim 1, characterized in that, In step 1, the cleaning process includes: first rinsing with tap water 3 times and removing surface dirt and stains with a brush, then rinsing with deionized water 3 times.

3. The method according to claim 1, characterized in that, In step 2, the disaccharide is any one of lactose, sucrose, and maltose; the amount of cellulase added is 0-6 wt%, and the amount of pectinase added is 0-6 vol%.

4. The method according to claim 1, characterized in that, In step 6, the mixture is centrifuged at 4°C and 12000g for 60 minutes, and the supernatant is collected.

5. The method according to claim 1, characterized in that, In step 7, the filtration sequence is as follows: first through a 0.45μm filter membrane, then through a 0.22μm filter membrane.

6. The method according to claim 1, characterized in that, In step 8, the extracellular vesicle suspension is stored in a -80°C freezer for later use.

Images