Efficient extraction method of plant cell vesicles based on synergistic effect of three-dimensional gradient micro vortex and sound wave fluidization and application of efficient extraction method

Abstract

The invention discloses a plant cell vesicle efficient extraction method based on the synergistic effect of three-dimensional gradient micro vortex and sound wave fluidization and application of the plant cell vesicle efficient extraction method. According to the plant cell vesicle extraction method provided by the invention, high-yield, high-purity and high-activity extraction of plant cell vesicles is realized through an active enzymolysis-acoustic cracking-three-dimensional gradient separation full-chain process, and the plant cell vesicle extraction method can be used for large-scale production. The method overcomes the defects of low yield and purity, chemical pollution, low extraction efficiency, uncontrollable vesicle size, easily damaged structure, long time and the like of the traditional extraction method, and can be used for producing and preparing more cosmetics and medicines containing plant cell vesicles on a large scale. The method systematically solves the global problem of extraction and application of the plant vesicles through multidisciplinary cross innovation (acoustics, fluid mechanics and molecular bionics), is high in extraction efficiency, good in activity effect and comprehensive and advanced in technical parameters, and has important significance on extraction and development of the plant cell vesicles.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and cosmetics, and in particular to a method for efficiently extracting plant cell vesicles based on the synergistic effect of three-dimensional gradient micro-vortex and sonic fluidization and its application. Background Art

[0002] Plant-derived extracellular vesicles (PEVs) are nanoscale membrane structures secreted by plant cells. They carry a variety of bioactive molecules, such as proteins, lipids, and nucleic acids, and have broad potential for biomedical applications, including drug delivery, immunomodulation, and disease treatment. For example, Centella asiatica-derived extracellular vesicles (CA-EVs), currently under investigation, are nanoscale lipid bilayer structures (30–150 nm in diameter) secreted from the cell membrane. They are rich in triterpenoid saponins (such as asiaticoside and madecassoside), polyphenols (such as quercetin and kaempferol), and non-coding RNAs (such as miR-21 and miR-29b). Studies have shown that CA-EVs can promote collagen synthesis by activating the TGF-β / Smad pathway in skin fibroblasts and reduce the release of inflammatory factors (IL-6 and TNF-α) by inhibiting the NF-κB pathway. These CA-EVs offer unique advantages in anti-aging, scar repair, and sensitive skin care.

[0003] Currently, the industrial application of plant cell vesicles faces the following core challenges: (1) The contradiction between extraction efficiency and activity retention: Traditional ultracentrifugation requires multiple differential centrifugation cycles (e.g., 300×g→2000×g→10,000×g→>100,000×g), which takes more than 12 hours or even longer, resulting in a loss of more than 30% of vesicle membrane surface proteins due to oxidative stress. At the same time, the shear force generated by high-speed centrifugation (>100,000×g) can destroy the vesicle structure, such as the leakage rate of asiaticoside as high as 45%. (2) Chemical contamination and safety hazards: Although the polyethylene glycol (PEG) precipitation method can shorten the extraction time to 4 hours, the residual PEG can damage the skin barrier function and induce contact dermatitis (clinical reports show an allergy rate of >3%). Although the ultrafiltration method (300 kDa membrane package) does not require chemical addition, the vesicle retention rate is only 58% due to membrane pore blockage, and it is impossible to separate vesicles from apoptotic bodies (the two have overlapping densities). (3) Bottlenecks of particle size uniformity and transdermal efficiency: Plant cell vesicles obtained by existing technologies are mostly polydisperse (PDI>0.3), with particle sizes ranging from 100 to 400 nm, and poor transdermal efficiency. Therefore, the development of an efficient, low-cost, and high-purity plant cell vesicle extraction technology has important scientific significance and application value. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the problems of low yield, low purity, low activity, low efficiency, chemical pollution, poor transdermal efficiency, and inability to produce on a large scale in existing plant cell vesicle extraction methods. The present invention provides an efficient extraction method for plant cell vesicles based on the synergistic effect of three-dimensional gradient microvortex and acoustic wave fluidization and its application.

[0005] The first object of the present invention is to provide a method for efficiently extracting plant cell vesicles.

[0006] The second object of the present invention is to provide a plant cell vesicle.

[0007] A third object of the present invention is to provide a product.

[0008] The fourth object of the present invention is to provide applications of the above-mentioned plant cell vesicles and products.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides a method for extracting plant cell vesicles based on the synergistic effect of three-dimensional gradient micro-vortex and sonic fluidization, comprising the following steps: S1. Enzymatic pretreatment: The plant sample to be extracted is enzymatically hydrolyzed using cell wall degrading enzymes in a water bath at 30-38°C, 100-150 rpm, for 1-3 h to obtain a plant enzymatic hydrolyzate. S2, sonic fluidization lysis: The enzymatic hydrolyzate was transferred to a reactor and first subjected to low-frequency cavitation at a frequency of 15-25 kHz for 3-7 min, then switched to high-frequency penetration at a frequency of 0.8-1.2 MHz for 8-12 min to obtain a reaction solution; S3, gradient centrifugation: The medium solution used is a three-layer medium solution with different densities, of which the bottom solution has a density of 1.1~2 g / cm 3 The high-density barrier layer solution and the middle layer solution use a density of 1.1~1.5 g / cm 3 The isotonic capture layer solution and the upper layer solution have a density of 1~1.5g / cm 3 The reaction solution was slowly added to the three-layer medium solution and centrifuged, and the middle layer solution was collected to obtain plant cell vesicles; Each medium solution was prepared by using at least two substances selected from diatrizoate, iodixanol, sucrose, trehalose, Ficoll 400, mannitol, cesium chloride, glycerol, histidine buffer, EDTA, cholesterol, saponin, phospholipids, Tris-HCl, NaCl, PBS, and ultrapure water.

[0010] This method combines specific active enzymatic hydrolysis, bimodal sonic fluidization technology, and three-dimensional gradient microvortex centrifugation to achieve efficient extraction of plant cell vesicles. This method significantly increases the yield of plant cell vesicles to over 58 mg / g (compared to <31 mg / g using conventional extraction methods). Separation efficiency is significantly improved, with activity retention exceeding 90% and purity exceeding 95% (compared to <70% using conventional centrifugation methods). The plant cell vesicles extracted using this method have a significantly smaller and more uniform particle size distribution between 30 and 150 nm (compared to the 100-400 nm distribution using conventional extraction methods), facilitating skin absorption and utilization, thereby increasing the passive diffusion absorption rate of the particles through the stratum corneum. Furthermore, this method is suitable for extracting extracellular vesicles from a variety of plants. By adjusting the composition and ratio of the gradient medium, it can be adapted to extract a variety of plant extracellular vesicles, achieving the same high yield and activity efficacy.

[0011] The extraction method provided by the present invention is chemical-free and avoids PEG and organic solvent residues, complying with the requirements of EC 1223 / 2009. Furthermore, the extraction energy consumption is low, with the total centrifugation time shortened to 2 hours, compared to conventional differential ultracentrifugation of >12 hours, and energy consumption reduced by 45%. The method has industrial efficiency and cost advantages, is capable of high-throughput processing, with a single batch processing volume of 20 L, compared to <0.5 L by conventional centrifugation, enabling large-scale extraction and preparation, and is suitable for large-scale production of cosmetics. The method for efficiently extracting plant cell vesicles provided by the present invention achieves high yield, high purity, high activity, and large-scale production of plant cell vesicles by optimizing the full-chain process of enzymatic hydrolysis-sonic lysis-three-dimensional gradient microvortex separation. This technology overcomes the shortcomings of conventional methods such as chemical pollution and low efficiency, is suitable for large-scale production, and has broad application prospects in the preparation of anti-aging, whitening, and barrier repair cosmetics.

[0012] The extraction method of the present invention utilizes active enzymatic hydrolysis, employing cell wall-degrading enzymes to decompose the plant cell wall and eliminate its barrier to vesicle release. Different enzymatic hydrolysis systems are used for extracting different plant cells and require optimization for each plant to be extracted. For example, enzymatic hydrolysis systems such as cellulase, pectinase, lignin peroxidase, cutinase, ascorbic acid, and lysozyme can be used. Different enzymatic hydrolysis solutions can be selected to meet the extraction requirements of different plant exosomes. Enzymatic hydrolysis can also be performed using conventional enzymatic hydrolysis methods and conditions in the art.

[0013] The "dual-modal acoustic fluidization technology" of this invention corresponds to the use of both low-frequency and high-frequency acoustic waves to treat plant liquids after enzymatic hydrolysis. Dual-modal acoustic fluidization technology uses low-frequency cavitation and high-frequency directional penetration to treat plant samples after enzymatic hydrolysis, allowing for better drainage during vesicle preparation. Low-frequency ultrasound is first applied to generate transient cavitation bubbles in the liquid. When the bubbles collapse, microjets (speed >100 m / s) are released, mechanically shearing the cell walls. High-frequency focused ultrasound is then applied, using acoustic radiation force to propel the vesicles through the cell membrane pores, preventing excessive damage to the membrane structure. Simultaneously, a thermal effect suppression strategy is combined to ensure that the treatment temperature is <10°C, maintaining the thermal stability of the active substances in the plant extracellular vesicles (degradation rate <5%).

[0014] The three-dimensional gradient in the "three-dimensional gradient micro-vortex centrifugation system" of the present invention refers to the use of three different density gradient medium solutions for micro-vortex centrifugation. The centrifugal parameters and centrifugal speed and time set for the corresponding medium will not be too large, and the extraction can be completed in a shorter time, realizing "micro-vortex centrifugation". The three-dimensional gradient micro-vortex centrifugation system is used, through the synergistic effect of the density gradient medium and the progressive centrifugal force control of the angular rotor, and the density is 1.00~1.35 g / cm 3 The triple gradient medium is precisely layered to avoid interference from impurities. For example, a sucrose-iodixanol-diatrizoate triple gradient medium is used to dynamically match different plant vesicle densities (such as Centella asiatica 1.08 g / cm 3 , myrrh 1.20 g / cm 3 ), achieving separation purity >95% (compared to <70% with conventional centrifugation). Combined with progressive centrifugal force to minimize membrane damage, this method offers high activity preservation, resulting in, for example, 93% retention of asiaticoside (HPLC assay), compared to only 53% with conventional ultracentrifugation. Furthermore, by adjusting the composition and ratio of the gradient media (e.g., diatrizoate, iodixanol, sucrose, trehalose, Ficoll 400, mannitol, cesium chloride, glycerol, histidine buffer, EDTA, cholesterol, saponin, phospholipids, etc.), it can be used to extract extracellular vesicles from a variety of plant species. Therefore, the combination of enzymatic hydrolysis, sonication, and a three-dimensional gradient microvortex centrifugation system overcomes the challenges of traditional centrifugation methods, including low yield, low purity, significant activity loss, and limited throughput. Its comprehensive advantages in separation efficiency (improved yield), activity retention (>90%), and industrial compatibility make it an optimal choice for the extraction of plant active ingredients.

[0015] Preferably, the plant sample in S1 is a plant cell pellet or a cell sediment.

[0016] More preferably, the specific processing method for plant samples is as follows: fresh plant explants are surface sterilized (75% ethanol for 2 minutes → soaking in 2% Octasil disinfectant for 8 minutes), rinsed three times with sterile purified water, inoculated onto MS solid medium (containing 30 g / L sucrose, 2 mg / L 2,4-D, and 0.1 mg / L NAA), and cultured in the dark at 25°C for 21 days to induce callus. The callus is then transferred to liquid medium (supplemented with 0.5 mg / L 6-BAP) and cultured with shaking at 120 rpm for 10 days, after which the wet weight cell pellet is harvested.

[0017] Preferably, the cell wall degrading enzyme in S1 is one or more of cellulase, pectinase, lignin peroxidase, cutinase, ascorbic acid, and lysozyme.

[0018] Preferably, the specific conditions for performing sonic fluidization lysis in S2 are as follows: the enzymatic hydrolyzate is transferred to the reactor, and low-frequency cavitation is first performed, with a frequency of 15-25 kHz, a power of 45-55 W, a pulse period of 3-7 s on / 1-3 s off, and a treatment time of 3-7 min; then high-frequency penetration is performed, switching to a high frequency of 0.8-1.2 MHz, and a power density of 0.3-0.7 W / cm 2 The focused acoustic beam diameter is 2~4 mm, the scanning speed is 1~3 mm / s, covering the entire reactor, the processing time is 8~12 min, and the processing temperature is simultaneously maintained at <10℃ to obtain a reaction liquid.

[0019] More preferably, the specific condition parameters of the sonication fluidization lysis in S2 are as follows: the enzymatic hydrolyzate is transferred to the reactor, and low-frequency cavitation is first performed, with a frequency of 20 kHz, a power of 50 W, a pulse period of 5 s on / 2 s off, and a treatment time of 5 min; then high-frequency penetration is performed, switching to a high frequency of 1 MHz and a power density of 0.5 W / cm 2 , the focused acoustic beam diameter is 3 mm, the scanning speed is 2 mm / s, covering the entire reactor, the processing time is 10 min, and the processing temperature is simultaneously maintained at <10°C to obtain a reaction liquid.

[0020] Preferably, the cavitation density of the low-frequency cavitation treatment in S2 is set to be not less than 10 5 pieces / mL.

[0021] Preferably, the density of the bottom solution in S3 is 1.15-1.3 g / cm 3 , prepared from at least two substances selected from diatrizoate, Ficoll 400, cesium chloride, cholesterol, mannitol, ascorbic acid, ultrapure water, NaCl, and PBS.

[0022] More preferably, the density of the middle layer solution is 1.1-1.2 g / cm 3, prepared from at least two substances selected from iodixanol, sucrose, mannitol, histidine buffer, saponin, NaCl, and PBS.

[0023] More preferably, the density of the upper solution is 1.05-1.2 g / cm 3 , prepared from at least two substances among sucrose, trehalose, Tris-HCl, glycerol, EDTA-Na2, soybean lecithin, ultrapure water, and PBS.

[0024] As an optional preferred embodiment, when the plant to be extracted is Centella asiatica, the medium solution is selected as follows: bottom solution: 1.16~1.2 g / cm 3 Diatrizoate solution; middle layer solution: 1.10~1.14 g / cm 3 Iodixanol solution; upper layer solution: 1.06-1.09 g / cm 3 Sucrose-trehalose complex solution.

[0025] More preferably, the extract medium solution of Centella asiatica is: bottom solution: 1.18 g / cm 3 Diatrizoate solution (dissolve 60 g of diatrizoate in 400 mL of ultrapure water and sterilize with a 0.22 μm filter); middle layer solution: 1.12 g / cm 3 Iodixanol solution (mix 40% iodixanol solution (OptiPrep™) with PBS at a volume ratio of 3:1); upper layer solution: 1.08 g / cm 3 Sucrose-trehalose complex solution (54 g sucrose and 18 g trehalose in 500 mL PBS, magnetic stirring until completely dissolved).

[0026] As an optional preferred embodiment, when the plant to be extracted is myrrh, the medium solution is selected as follows: bottom solution: 1.18~1.22 g / cm 3 Diatrizoate solution; middle layer solution: 1.13~1.16 g / cm 3 Iodixanol solution; upper layer solution: 1.08-1.12 g / cm 3 Sucrose solution.

[0027] More preferably, the myrrh extraction medium solution is: bottom solution: 1.20 g / cm 3 Diatrizoate solution (dissolve 65 g of diatrizoate in 400 mL of ultrapure water and add 5 g of NaCl to adjust the osmotic pressure); middle layer solution: 1.15 g / cm 3 Iodixanol solution (45% OptiPrep™ mixed with 0.25 M sucrose); upper layer: 1.10 g / cm 3Sucrose solution (50 g sucrose dissolved in 500 mL Tris-HCl buffer, pH 6.8).

[0028] As an optional preferred embodiment, when the plant to be extracted is jasmine, the medium solution is selected as follows: bottom solution: 1.13~1.17 g / cm 3 Ficoll 400 solution; middle layer solution: 1.08~1.12 g / cm 3 Sucrose-mannitol complex solution; upper layer solution: 1.04-1.07 g / cm 3 Trehalose solution.

[0029] More preferably, the jasmine flower extract medium solution is: bottom solution: 1.15 g / cm 3 Ficoll 400 solution (40% Ficoll 400 dissolved in PBS); middle layer solution: 1.10 g / cm 3 Sucrose-mannitol complex solution (40 g sucrose + 10 g mannitol dissolved in 500 mL PBS); upper layer solution: 1.06 g / cm 3 Trehalose solution (30 g trehalose dissolved in 500 mL PBS, pH 5.0).

[0030] As an optional preferred embodiment, when the plant to be extracted is Desert Rose, the medium solution is selected as follows: bottom solution: 1.28~1.3 g / cm 3 Diatrizoate meglumine-cesium chloride complex solution; solution middle layer: 1.23~1.27 g / cm 3 Iodixanol solution; upper layer of solution: 1.18-1.22 g / cm 3 Sucrose-glycerol complex solution.

[0031] More preferably, the Desert Rose extract medium solution is: bottom solution: 1.30 g / cm 3 Diatrizoate-cesium chloride complex solution (70 g diatrizoate dissolved in 400 mL ultrapure water + 10 g CsCl, density calibration); solution middle layer: 1.25 g / cm 3 Iodixanol solution (50% OptiPrep™ mixed with 0.5 M NaCl); upper layer: 1.20 g / cm 3 Sucrose-glycerol complex solution (60 g sucrose + 20 mL glycerol dissolved in 500 mL PBS).

[0032] As an optional preferred embodiment, when the extracted plant is alpine edelweiss, the medium solution is selected as follows: bottom solution: 1.13~1.17g / cm 3 Mannitol solution; middle layer solution: 1.08~1.12 g / cm3 Sucrose-histidine buffer; upper layer solution: 1.03~1.07 g / cm 3 Trehalose-EDTA solution.

[0033] More preferably, the alpine edelweiss extract medium solution is: bottom solution: 1.15 g / cm 3 Mannitol solution (55 g mannitol + 1 g ascorbic acid dissolved in 500 mL PBS); middle layer solution: 1.10 g / cm 3 Sucrose-histidine buffer (45 g sucrose dissolved in 0.1 M histidine buffer, pH 6.5); upper layer solution: 1.05 g / cm 3 Trehalose-EDTA solution (25 g trehalose + 0.5 g EDTA-Na2 dissolved in 500 mL ultrapure water).

[0034] As an optional preferred embodiment, when the plant to be extracted is ginseng, the medium solution is selected as follows: bottom solution: 1.23~1.27 g / cm 3 Diatrizoate-cholesterol complex solution; middle layer solution: 1.18~1.22 g / cm 3 Iodixanol-saponin buffer; upper layer solution: 1.13-1.17 g / cm 3 Sucrose-phospholipid solution.

[0035] More preferably, the myrrh extraction medium solution is: bottom solution: 1.25 g / cm 3 Diatrizoate-cholesterol complex solution (60 g diatrizoate + 2 g cholesterol dissolved in 400 mL PBS); middle layer solution: 1.20 g / cm 3 Iodixanol-saponin buffer (45% OptiPrep TM + 0.1% ginsenoside Rb1, pH 7.0); upper layer solution: 1.15 g / cm 3 Sucrose-phospholipid solution (50 g sucrose + 0.5 g soybean phospholipid dissolved in 500 mL PBS).

[0036] Preferably, the centrifugation procedure in S3 is: first stage 1800-2200×g, 15-25 min; second stage 6000-10000×g, 25-35 min; third stage 13000-17000×g, 40-50 min.

[0037] Traditional sucrose density gradient (1.10~1.30 g / cm 3Centrifugation can cause vesicle shrinkage due to excessive osmotic pressure. This method utilizes three different layers of biocompatible media. The bottom layer, such as diatrizoate meglumine, forms a dense interface to prevent organelle sedimentation. The middle layer, such as iodixanol, maintains osmotic pressure balance inside and outside the vesicle membrane. The top layer, such as a sucrose-trehalose complex (molar ratio 3:1), protects vesicle membrane proteins through hydroxyl groups. By combining multiple media, the extraction of exosomes from different plant sources can be achieved. Combined with the physical angle rotor dynamics, a three-dimensional spiral flow field is formed during the centrifugation process; the radial velocity gradient, the centrifugal force decreases from the tube wall to the center (2000×g→8000×g), and large particles (>200 nm) preferentially settle to the bottom layer; the tangential vortex effect, the angle rotor tilt angle induces the fluid to generate Coriolis force, driving 30-200 nm vesicles to enrich in the middle layer along the spiral trajectory; axial stratification control, at the 15,000×g stage, the high-density medium forms a vertical resistance, so that the vesicles form a clear zone in the middle of the liquid column (bandwidth <2 mm).

[0038] As a more preferred embodiment, the present invention provides a more specific extraction method: Step 1: Enzymatic pretreatment: The plant sample to be extracted is enzymatically hydrolyzed using cell wall degrading enzymes in a 30-38°C water bath at 100-150 rpm for 1-3 h to obtain a plant hydrolyzate. For example, for Centella asiatica hydrolysate, 0.5 U / mg of cellulase is added per gram of wet cells to specifically hydrolyze β-1,4-glucan; 0.2 U / mg of pectinase is added to decompose α-1,4-galacturonic acid; 0.01% lysozyme (for residual bacterial contamination in the plant cell wall) is added, and the solution is shaken in a 37°C water bath (120 rpm) for 2 h to obtain the plant hydrolyzate.

[0039] Step 2: Sonic fluidization lysis: Use a sonic reaction system (frequency range: 10 kHz-2 MHz, maximum power 200 W) equipped with a titanium alloy horn (6 mm diameter, 50 μm tip amplitude). The specific operation process is as follows: 1. Low-frequency cavitation stage: Transfer the plant enzymatic hydrolysate to the reactor, and control the liquid level so that the horn is immersed at a depth of 10 mm. Start the 20 kHz low-frequency mode, power 50 W, pulse cycle 5 s on / 2 s off, and process for 5 minutes. Monitor the cavitation cloud density with a high-speed camera (1000 fps) and adjust the power to keep the cavitation density stable at 10 5 / mL; 2. High-frequency penetration stage: switch to 1 MHz high-frequency mode, power density 0.5 W / cm 2The focused acoustic beam, 3 mm in diameter, was used in a scanning pattern (at a speed of 2 mm / s) to cover the entire reactor. The high-frequency treatment lasted 10 minutes, and the cooling system was activated simultaneously to maintain the temperature below 10°C.

[0040] Step 3: Purification by three-dimensional gradient centrifugation: 1. Preparation of gradient medium (different plants should use different gradient media according to their characteristics. The following preparation method uses Centella asiatica as an example): (1) Bottom solution (1.18 g / cm 3 ): Dissolve 60 g of diatrizoate in 400 mL of ultrapure water and sterilize with a 0.22 μm filter membrane; (2) Middle layer solution (1.12 g / cm 3 ): Using 40% iodixanol solution (OptiPrep TM ) was mixed with PBS at a volume ratio of 3:1; (3) Upper solution (1.08 g / cm 3 ): Dissolve 54 g sucrose and 18 g trehalose in 500 mL PBS and stir magnetically until completely dissolved.

[0041] 2. Centrifugation Procedure: Centrifuge the sonicated lysis solution at 1000 × g for 10 min. Discard the pellet (unlysed cell mass). Slowly add the supernatant to the top layer of the gradient solution (add dropwise with the needle against the wall at a flow rate of 1 mL / min). Then centrifuge according to the following centrifugation procedure. (1) Stage 1 (2000 × g, 20 min): removal of nuclear fragments and organelle impurities; (2) The second stage (8000 × g, 30 min): The light blue zone in the middle layer (crude vesicles) was collected, and transmission electron microscopy (TEM) showed that the vesicle ratio was >90%; (3) The third stage (15000×g, 45 min): High-purity vesicles are obtained in the middle layer, with a particle size distribution of 30~150 nm.

[0042] 3. Identification and collection of vesicle zones: After centrifugation, the solution can be divided into three layers: Upper layer: transparent (containing soluble protein); Middle layer: light blue opalescent band (2-3 mm wide, vesicle-rich area), the middle layer of iodixanol emits blue fluorescence when observed under ultraviolet light (365 nm); Bottom layer: white precipitate (organelle fragments and other impurities).

[0043] 4. Puncture sampling: Refrigerate centrifuge tube (-20℃ 30 min) → Place the puncture needle in the middle of the middle zone (avoid touching the interface) to sample. The obtained middle solution is the plant cell vesicle sample.

[0044] In order to maintain the integrity and biological activity of the plant vesicles, the prepared plant cell vesicles can be further subjected to ultrafiltration concentration and freeze-dried to obtain freeze-dried powder; liquid raw materials need to be stored and transported under frozen conditions, while freeze-dried powder can be stored and transported at room temperature, which is more conducive to room temperature storage and transportation.

[0045] The present invention provides application of the above method in improving the yield, purity and activity effect of plant cell vesicles.

[0046] The present invention provides a plant cell vesicle prepared by the above method.

[0047] Preferably, the cell vesicles are: Centella asiatica, Myrrha, Jasmine, Desert Rosa, Leontopodium alpinum, and Ginseng cell vesicles.

[0048] The present invention provides a product comprising plant cell vesicles.

[0049] Preferably, the product is a freeze-dried powder, which can better maintain the integrity of plant vesicles and the biological activity is more lasting and efficient. The preparation method is: ultrafiltration and concentration of plant cell vesicles, mixing the concentrated vesicle suspension with a protective agent, and freeze-drying to obtain plant cell vesicle freeze-dried powder.

[0050] Preferably, a protective agent is used to maintain the structural integrity and biological activity of the plant cell vesicles during the freeze-drying process. The protective agent comprises: 5% trehalose, 1% hyaluronic acid, 0.1% Tween-80; or 0.1% gelatin hydrolysate and 100-400 mmol / L arginine.

[0051] As a more preferred embodiment, the present invention provides a processing method for ultrafiltration concentration and freeze drying: 1. Tangential flow ultrafiltration concentration: using Millipore Pellicon ® The high-purity vesicles obtained above were concentrated using a 3 Cassette (300 kDa regenerated cellulose membrane) with a transmembrane pressure (TMP) of 1.5 bar and a feed flow rate of 20 mL / min. Dynamic backflushing was performed by starting the backflush pump (pressure 3 bar, duration 30 s) every 10 min to prevent membrane contamination, and ultrafiltration was used to obtain a vesicle suspension.

[0052] 2. Freeze-drying and protection: The concentrated vesicle suspension was mixed with a protective agent in a volume ratio of 1:1. The protective agent was 5% trehalose (glass transition temperature Tg = 115°C) + 1% hyaluronic acid (molecular weight 50 kDa) + 0.1% Tween-80 (to reduce ice crystals piercing the vesicle membrane).

[0053] 3. Freeze-drying: Pre-freezing stage: -80°C ultra-low temperature, 4 h; main drying stage: vacuum freeze drying -50°C, 0.05 mbar, 18 h; analytical drying stage: 25°C, 0.001 mbar, 6 h, and finally obtain plant cell vesicle freeze-dried powder.

[0054] More preferably, the product is a serum.

[0055] More preferably, the essence further contains a fat-soluble whitening ingredient, such as niacinamide.

[0056] The present invention also provides the use of the plant cell vesicles or products in the preparation of cosmetics and medicines.

[0057] The present invention also provides a whitening product containing the plant cell vesicles and a fat-soluble whitening component.

[0058] The present invention has the following beneficial effects: (1) Breakthrough in technical barriers: By adopting the three-in-one method of "active enzymatic hydrolysis-sonic lysis-three-dimensional gradient separation", the precise size control and high extraction efficiency of plant cell vesicles were achieved for the first time, which can better retain the active ingredients (such as Centella asiatica glycoside>90%) and significantly improve the yield, purity and activity effect of plant cell vesicles; compared with traditional technology, the extraction time was shortened by 60% (total time <4 h) and energy consumption was reduced by 45% (sonic wave power <100 W), solving the problems of low yield, low purity, low activity, low efficiency, chemical pollution, poor transdermal efficiency and inability to produce on a large scale in traditional plant vesicle extraction methods.

[0059] (2) Cosmetic application: The plant cell vesicles prepared by the present invention have better purity and activity effect, smaller particle size, and significantly improved transdermal absorption rate, and can be better used in cosmetics; for example, when it is loaded with fat-soluble whitening ingredients (such as niacinamide) to prepare essence, the water content of the skin stratum corneum is increased by 38%, the erythema index is reduced by 52%, and the transdermal absorption rate is increased to 22%, which has a significant effect.

[0060] (3) Industrial application: The method provided by the present invention can be directly carried out using existing ultrasonic and centrifugal equipment. It is convenient and efficient, with a single batch processing volume of up to 20 L. It can be used for large-scale production and meets the GMPC certification requirements. The residual organic solvent content in the waste liquid after extraction is <0.1 ppm (GC-MS detection), which meets the EU EC 1223 / 2009 cosmetics regulations and has better industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 These are comparison electron micrographs of extracellular vesicles of Centella asiatica plants (A in the figure is Example 1, and B in the figure is Comparative Example 1).

[0062] Figure 2 The figures are comparison electron micrographs of extracellular vesicles of myrrh plants (A in the figure is Example 2, and B in the figure is Comparative Example 2).

[0063] Figure 3 The figures are comparisons of electron micrographs of extracellular vesicles of Jasmine plants (A in the figure is Example 3, and B in the figure is Comparative Example 3).

[0064] Figure 4 The figures are comparison electron micrographs of extracellular vesicles of the desert rose plant (A in the figure is Example 4, and B in the figure is Comparative Example 4).

[0065] Figure 5 Comparison of electron microscopic images of extracellular vesicles of alpine edelweiss plants (A in the figure is Example 5, and B in the figure is Comparative Example 5).

[0066] Figure 6 Comparison of electron microscopic images of extracellular vesicles of ginseng plants (A in the figure is Example 6, and B in the figure is Comparative Example 6).

[0067] Figure 7 This is a picture showing the results of Centella Asiatica Cell Vesicle Essence improving the moisture of sensitive skin.

[0068] Figure 8 This is the result of Centella asiatica Cell Vesicle Essence improving skin inflammation.

[0069] Figure 9 This image shows the results of Centella asiatica cell vesicles improving moisture in sensitive skin.

[0070] Figure 10 This is a graph showing the results of Centella asiatica cell vesicles improving skin inflammation. DETAILED DESCRIPTION

[0071] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0072] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0073] The centrifuge used in the implementation was Beckman Coulter Optima XE-90, rotor model: Type 70Ti.

[0074] Example 1: Extraction of Centella asiatica Cell Vesicles Step 1: Cell culture and enzymatic pretreatment 1. Cell Expansion: Fresh explants of Centella asiatica were surface sterilized (75% ethanol for 2 minutes followed by soaking in 2% Octasil disinfectant for 8 minutes), rinsed three times with sterile purified water, and inoculated onto MS solid medium (containing 30 g / L sucrose, 2 mg / L 2,4-D, and 0.1 mg / L NAA). Callus was cultured in the dark at 25°C for 21 days to induce callus. The callus was transferred to liquid medium (supplemented with 0.5 mg / L 6-BAP) and cultured with shaking at 120 rpm for 10 days. Cell pellets weighing approximately 50 g were harvested.

[0075] 2. Complex enzymatic hydrolysis system: Wash the cell pellet three times with PBS (pH 5.5, containing 5 mM CaCl2), resuspend in enzymatic hydrolysis solution, and shake in a 37°C water bath (120 rpm) for 2 h to obtain enzymatic hydrolysis solution. Samples were taken every 30 min to detect the cell dissociation rate (trypan blue staining method, target >95%).

[0076] Preparation of enzymatic hydrolysate: Add 0.5 U / mg cellulase per gram of wet cells to specifically hydrolyze β-1,4-glucan; add 0.2 U / mg pectinase to decompose α-1,4-galacturonic acid; and add 0.01% lysozyme to target residual bacterial contamination in plant cell walls.

[0077] Step 2: Sonic Fluidized Lysis Equipment parameters: Use an acoustic wave reaction system (frequency range: 10 kHz to 2 MHz, maximum power 200 W) equipped with a titanium alloy horn (diameter 6 mm, tip amplitude 50 μm). The specific operation process is as follows: 1. Low-frequency cavitation stage: The enzymatic hydrolyzate was transferred to the reactor, and the liquid level was controlled so that the horn was immersed at a depth of 10 mm. The 20 kHz low-frequency mode was started, the power was 50 W, the pulse period was 5 s on / 2 s off, and the treatment was carried out for 5 min. The cavitation cloud density was monitored by a high-speed camera (1000 fps), and the power was adjusted to stabilize the cavitation density at 10 5 pieces / mL.

[0078] 2. High-frequency penetration stage: switch to 1 MHz high-frequency mode, power density 0.5 W / cm 2 The focused acoustic beam had a diameter of 3 mm and covered the entire reactor in a scanning mode (speed 2 mm / s). The treatment lasted for 10 min, and the cooling system was started simultaneously to maintain the temperature <10°C to obtain the sonic fluidized treatment liquid.

[0079] Step 3: Purification by three-dimensional gradient centrifugation 1. Gradient medium preparation: (1) Bottom solution (1.18 g / cm 3): Dissolve 60 g of diatrizoate in 400 mL of ultrapure water and sterilize with a 0.22 μm filter membrane; (2) Middle layer solution (1.12 g / cm 3 ): 40% iodixanol solution (OptiPrep TM ) was mixed with PBS at a volume ratio of 3:1; (3) Upper solution (1.08 g / cm 3 ): Dissolve 54 g sucrose and 18 g trehalose in 500 mL PBS and stir magnetically until completely dissolved.

[0080] 2. Centrifugation procedure: The solutions of various concentrations prepared above were sequentially placed in a centrifuge tube to form a layered density gradient; and then centrifuged according to the following procedure.

[0081] Sample pretreatment: Centrifuge the sonicated fluid at 1000 × g for 10 min, discard the pellet (unlysed cell mass), and slowly layer the supernatant onto the top layer of the gradient (add dropwise with the needle against the wall at a flow rate of 1 mL / min).

[0082] (1) Stage 1 (2000 × g, 20 min): removal of nuclear fragments and organelle impurities; (2) Second stage (8000 × g, 30 min): The light blue zone in the middle layer (crude vesicles, about 3 mL) was collected. Transmission electron microscopy (TEM) showed that the vesicles accounted for >90%; (3) The third stage (15,000 × g, 45 min): High-purity vesicles were obtained in the middle layer, and nanoparticle tracking analysis (NTA) showed a particle size distribution of 30–150 nm (PDI = 0.18).

[0083] 3. Vesicle zone identification and collection: After centrifugation, the solution can be divided into three layers: Upper layer: transparent (containing soluble protein); Middle layer: light blue opalescent band (2-3 mm wide, vesicle-rich area), the middle layer of iodixanol emits blue fluorescence when observed under ultraviolet light (365 nm); Bottom layer: white precipitate (organelle fragments and other impurities).

[0084] 4. Puncture sampling: Freeze the centrifuge tube (-20°C for 30 min) → Pierce the sample in the middle of the middle zone (avoid touching the interface). Sample volume: 2-3 mL (50 mL centrifuge tube). The obtained middle zone solution is the plant cell vesicle sample.

[0085] Step 4: Ultrafiltration concentration and freeze drying The prepared plant cell vesicles are further subjected to ultrafiltration concentration and freeze-drying to obtain freeze-dried powder, which maintains the integrity and biological activity of the plant vesicles and is convenient for storage and transportation at room temperature.

[0086] 1. Tangential flow ultrafiltration: using Millipore Pellicon ® 3 Cassette (300 kDa regenerated cellulose membrane) was used for filtration to further concentrate the plant vesicles. The operating parameters were: transmembrane pressure (TMP) of 1.5 bar, feed flow rate of 20 mL / min; dynamic backflushing, starting the backflush pump (pressure of 3 bar, duration of 30 s) every 10 minutes to prevent membrane fouling, and the concentrated vesicle suspension was obtained by tangential flow ultrafiltration.

[0087] 2. Vesicle protection: The concentrated vesicle suspension is mixed with a protective agent in a volume ratio of 1:1. The protective agent composition is: 5% trehalose (glass transition temperature Tg = 115°C) + 1% hyaluronic acid (molecular weight 50 kDa) + 0.1% Tween-80 (to reduce ice crystals piercing the vesicle membrane). At the same time, 0.1% gelatin hydrolysate and 100-400 mmol / L arginine can also be added as protective agents to maintain the structural integrity and biological activity of plant cell vesicles during the freeze-drying process.

[0088] 3. Freeze-drying: The protected plant vesicles prepared above were freeze-dried, first with a pre-freezing stage: ultra-low temperature of -80°C for 4 h; a main drying stage: vacuum freeze drying at -50°C, 0.05 mbar, for 18 h; and a desorption drying stage: 25°C, 0.001 mbar, for 6 h to obtain CA-EVs freeze-dried powder.

[0089] Finally, the total protein content was determined by the BCA method, and combined with NTA counting conversion, the yield of Centella asiatica cell vesicles was obtained to be 82 mg / g cell.

[0090] Comparative Example 1: Extraction of Centella asiatica vesicles by traditional ultracentrifugation 1. Cell lysis: Centella asiatica callus cells (treated in the same manner as in Example 1) were suspended in PBS (pH 7.4) and repeatedly frozen and thawed three times in liquid nitrogen; the cells were allowed to stand at -4°C for 30 min and centrifuged (300×g, 10 min) to remove unbroken cells to obtain a cell lysate.

[0091] 2. Differential centrifugation: Ultracentrifuge the cell lysate according to the following procedure: Stage 1: 2000 × g, 20 min, discard the pellet (nuclei and debris); Stage 2: 10,000 × g, 30 min, collecting the supernatant; The third stage: 100,000 × g, 2 h, and the pellet was resuspended in PBS.

[0092] 3. Purification: Ultrafiltration and concentration were performed using a 300 kDa membrane package, and the processing steps were the same as in Example 1. After obtaining the vesicle suspension, it was directly freeze-dried to finally obtain Centella asiatica cell vesicles.

[0093] The traditional ultracentrifugation method for extracting Centella asiatica vesicles takes a longer total processing time, and high-speed centrifugation can cause the vesicle membrane to rupture. The extracted Centella asiatica cell vesicles were measured (the measurement method is the same as in Example 1), showing that the yield of cell vesicles was less than 19 mg / g cell. This method has the disadvantages of activity loss and very low yield.

[0094] Example 2: Extraction of vesicles from Commiphora myrrha leaf cells The extraction method and steps are the same as those in Example 1, except that the adaptability is adjusted for the plant raw materials. The specific different conditions are as follows: Step 1: Enzymatic hydrolysis solution: 0.1% lignin peroxidase + 0.5% cellulase.

[0095] Step 2: Sonication parameters: 20 kHz (60 W, 8 min) → 1 MHz (0.6 W / cm 2 , 12 min).

[0096] Step 3: Design a step gradient medium based on the high density of myrrh vesicles: Bottom solution: 1.20 g / cm 3 Diatrizoate solution (dissolve 65 g of diatrizoate in 400 mL of ultrapure water and add 5 g of NaCl to adjust the osmotic pressure); Middle layer solution: 1.15 g / cm 3 Iodixanol solution (45% OptiPrep TM mixed with 0.25 M sucrose); Upper solution: 1.10 g / cm 3 Sucrose solution (50 g sucrose dissolved in 500 mL Tris-HCl buffer, pH 6.8).

[0097] The yield of myrrh cell vesicles was determined to be 68 mg / g cells.

[0098] Comparative Example 2: Extraction of Myrrh Cell Vesicles by PEG Precipitation 1. Cell lysis: Myrrh callus cells (treated as in Example 1) were suspended in PBS (pH 7.4) and repeatedly frozen and thawed in liquid nitrogen three times. The cells were allowed to stand at -4°C for 30 min and centrifuged (300 × g, 10 min) to remove unbroken cells to obtain a cell lysate. The myrrh cell suspension was then ultrasonically disrupted (20 kHz, 100 W, 5 min) and centrifuged (10,000 × g, 20 min) to obtain the supernatant.

[0099] 2. PEG precipitation: Add 15% PEG8000 (final concentration) to the supernatant and incubate at 4°C for 12 h. Centrifuge (10,000 × g, 30 min) and resuspend the precipitate in PBS.

[0100] 3. Impurity removal: filter through 0.45 μm membrane to remove large particles and freeze-dry.

[0101] The yield of myrrh cell vesicles after treatment with traditional PEG precipitation was determined to be 31 mg / g cells. However, the PEG precipitation method also has the disadvantages of (1) chemical contamination: PEG residue was 0.48% (GC-MS test results), which could easily cause skin irritation during subsequent use; and (2) poor selectivity: a large amount of miscellaneous proteins were co-precipitated (SDS-PAGE showed multiple non-target bands).

[0102] Example 3: Extraction of vesicles from Jasminum sambac leaf cells The extraction method and steps are the same as those in Example 1, except that the adaptability is adjusted for the plant raw materials. The specific different conditions are as follows: Step 1: Enzyme hydrolysis solution: 0.6% pectinase + 0.1% cellulase, pH 4.8.

[0103] Step 2: Sonication parameters: 20 kHz (40 W, 3 min) → 1 MHz (0.3 W / cm 2 , 10 min).

[0104] Step 3: To protect the volatile components of jasmine, a low osmotic pressure Ficoll gradient system was used: Bottom solution: 1.15 g / cm 3 Ficoll 400 solution (40% Ficoll 400 dissolved in PBS); Middle layer solution: 1.10 g / cm 3 Sucrose-mannitol complex solution (40 g sucrose + 10 g mannitol dissolved in 500 mL PBS); Upper solution: 1.06 g / cm 3 Trehalose solution (30 g trehalose dissolved in 500 mL PBS, pH 5.0). The yield of jasmine cell vesicles was determined to be 73 mg / g cells.

[0105] Comparative Example 3: Extraction of Jasmine Cell Vesicles by Organic Solvent Extraction 1. Solvent extraction: Lyophilized powder of jasmine leaves was mixed with ethyl acetate at a ratio of 1:10 (w / v) and extracted with shaking at 25°C for 48 h. The mixture was filtered and the filtrate was rotary evaporated (40°C, 0.09 MPa) to dryness.

[0106] 2. Concentration and purification: The dried residue was defatted with n-hexane and subjected to silica gel column chromatography (eluent: petroleum ether-ethyl acetate gradient); the target fractions were collected, concentrated and freeze-dried again to obtain jasmine cell vesicles.

[0107] It has been determined that the yield of jasmine cell vesicles extracted by organic solvent extraction is 15 mg / g of cells. This method has the disadvantage that the components are easily destroyed. Rotary evaporation will cause a loss of 58% of methyl jasmonate; there is also the risk of solvent residue.

[0108] Example 4: Extraction of vesicles from Adenium obesum leaf cells The extraction method and steps are the same as those in Example 1, except that the adaptability is adjusted for the plant raw materials. The specific different conditions are as follows: Step 1: Enzymatic hydrolysis solution: 0.2% cutinase + 0.5% cellulase, pretreatment for 3 h.

[0109] Step 2: Sonication parameters: 25 kHz (70 W, 8 min) → 1 MHz (0.7 W / cm 2 , 15 min).

[0110] Step 3: Using an ultra-dense gradient system for the high-density hydrophobic protein of Rosa deserticola: Bottom solution: 1.30 g / cm 3 Diatrizoate-cesium chloride complex solution (70 g of diatrizoate dissolved in 400 mL of ultrapure water + 10 g of CsCl, density calibration); Solution middle layer: 1.25 g / cm 3 Iodixanol solution (50% OptiPrep TM mixed with 0.5 M NaCl); Upper layer of solution: 1.20 g / cm 3 Sucrose-glycerol complex solution (60 g sucrose + 20 mL glycerol dissolved in 500 mL PBS).

[0111] The yield of desert rose cell vesicles was determined to be 58 mg / g cells.

[0112] Comparative Example 4: Extraction of Desert Rose Leaf Cell Vesicles by High-Pressure Homogenization 1. High pressure treatment: The desert rose leaf cell suspension (lysis method is the same as that of Comparative Example 1) was processed 10 times by a 1500 bar high-pressure homogenizer; then cooled in an ice bath to avoid the temperature being greater than 40°C.

[0113] 2. Centrifugal purification: The cell suspension after high-pressure treatment was centrifuged (8000×g, 20 min), and the supernatant was filtered through a 0.22 μm filter membrane; and freeze-dried to obtain Desert Rose cell vesicles.

[0114] It was determined that the yield of vesicles extracted from Desert Rose cells using the high-pressure homogenization method was 21 mg / g cells; this method has the disadvantage of protein denaturation: the high-pressure shear force caused the hydrophobic protein (AQP5) to precipitate, and Western Blot could not detect it.

[0115] Example 5: Extraction of vesicles from Leontopodium alpinum cells The extraction method and steps are the same as those in Example 1, except that the adaptability is adjusted for the plant raw materials. The specific different conditions are as follows: Step 1: Enzyme hydrolysis solution: 0.1% ascorbic acid + 0.5% pectinase, pH 6.0.

[0116] Step 2: Ultrasonic parameters: 1 MHz intermittent ultrasound (0.4 W / cm 2 , 2 s on / 1 s off).

[0117] Step 3: To avoid polyphenol oxidation, use an antioxidant medium system: Bottom solution: 1.15 g / cm 3 Mannitol solution (55 g mannitol + 1 g ascorbic acid dissolved in 500 mL PBS); Middle layer solution: 1.10 g / cm 3 Sucrose-histidine buffer (45 g sucrose dissolved in 0.1 M histidine buffer, pH 6.5); Upper solution: 1.05 g / cm 3 Trehalose-EDTA solution (25 g trehalose + 0.5 g EDTA-Na2 dissolved in 500 mL ultrapure water).

[0118] The yield of vesicles from Leontopodium alpinum cells was determined to be 66 mg / g cells.

[0119] Comparative Example 5: Hot water extraction method to obtain alpine edelweiss cell vesicles 1. Hot water extraction: Lyophilized powder of Leontopodium alpinum was mixed with deionized water at a ratio of 1:15 (w / v) and extracted three times under reflux at 80°C for 1 h each time. The filtrates were combined and centrifuged (5000×g, 10 min) to remove insoluble matter.

[0120] 2. Concentration and freeze-drying: The filtrate is concentrated under reduced pressure to 1 / 10 of the original volume, pre-frozen at -80°C and then freeze-dried to obtain Leontopodium alpinum cell vesicles.

[0121] It has been determined that the yield of alpine edelweiss cell vesicles obtained by hot water extraction is 9 mg / g cell; according to existing research records, this method has the disadvantages of heat-sensitive loss and activity destruction. The activation of its polyphenol oxidase (PPO) will lead to a decrease in total phenol content, and the tyrosinase inhibitory conformation is easily destroyed by high temperature.

[0122] Example 6: Extraction of vesicles from Panax ginseng tissue culture The extraction method and steps are the same as those in Example 1, except that the adaptability is adjusted for the plant raw materials. The specific different conditions are as follows: Step 1: Culture optimization: Add 0.5 mg / L methyl jasmonic acid and culture for 28 days.

[0123] Step 3: Design a polarity gradient system based on the saponin-membrane binding properties of ginseng: Bottom solution: 1.25 g / cm 3 Diatrizoate-cholesterol complex solution (60 g of diatrizoate + 2 g of cholesterol dissolved in 400 mL of PBS); Middle layer solution: 1.20 g / cm 3 Iodixanol-saponin buffer (45% OptiPrep TM +0.1% ginsenoside Rb1, pH 7.0); Upper solution: 1.15 g / cm 3 Sucrose-phospholipid solution (50 g sucrose + 0.5 g soybean phospholipid dissolved in 500 mL PBS).

[0124] The yield of ginseng cell vesicles was determined to be 79 mg / g cells.

[0125] Comparative Example 6: Extraction of Ginseng Cell Vesicles by Ethanol Reflux 1. Ethanol reflux: Ginseng tissue culture was mixed with 70% ethanol at a ratio of 1:8 (w / v) and extracted three times at 85°C for 2 h each time. The combined extracts were filtered and rotary evaporated to remove the ethanol.

[0126] 2. Macroporous resin purification: The sample was loaded onto an AB-8 macroporous resin column and gradient eluted with 30%, 50%, and 70% ethanol in sequence; the 70% ethanol eluate was collected, concentrated, and freeze-dried to obtain ginseng cell vesicles.

[0127] It has been determined that the yield of ginseng cell vesicles obtained by the ethanol reflux method is 12 mg / g cell. The disadvantages of this extraction method are low bioavailability and slow onset of effect.

[0128] Comparative Example 7: Comparative Analysis of Different Treatment Methods Based on the extraction method of Example 1, control groups with different treatment conditions were set up respectively: (1) the centrifugal medium was not layered, and no three-dimensional gradient was set in step 3, and sucrose was directly used for differential centrifugation (i.e., sucrose was directly centrifuged after step 1 and step 2, and step 4 was performed without using layered medium centrifugation); (2) dual-modal sonication fluidization treatment was performed alone (i.e., ordinary centrifugation was directly performed after step 2, and step 4 was performed, without enzymatic hydrolysis in step 1 and three-dimensional gradient centrifugation in step 3); (3) three-dimensional gradient microvortex centrifugation was performed alone (i.e., the plant sample was directly subjected to three-dimensional gradient centrifugation and step 4, without enzymatic hydrolysis + sonication pretreatment); (4) enzymatic hydrolysis in step 1 was not performed (step 4 was performed after centrifugation after steps 2 and 3); (5) dual-modal sonication fluidization treatment was not performed (i.e., dual-modal sonication fluidization in step 2 was not performed, and step 4 was performed after step 1, and step 3 was directly centrifuged); (6) sucrose solutions of different densities were used as the layered medium in step 3, specifically 1.25 g / cm 3 , middle layer 1.15 g / cm 3 , upper layer 1.10 g / cm 3 Three layers of sucrose solution, other processing steps are the same.

[0129] The control groups described above followed the same method steps, except for the difference in method settings. The groups not subjected to enzymatic hydrolysis were treated as in Comparative Example 1, with samples lysed to obtain cell lysis and subsequent processing. Using the different control groups, Centella asiatica was extracted, and the yield (mg / g cell) and activity retention rate of plant cell vesicles extracted from each group were measured and statistically analyzed. The deficiencies of each method were analyzed. Specific measurement methods are described in the subsequent test examples. The activity retention rate was calculated by measuring the active component asiaticoside content in Centella asiatica cell vesicles using HPLC.

[0130] 1. Analysis of defects in different treatment methods (1) Disadvantages of non-stratified centrifugation medium (conventional differential centrifugation): the density of vesicles and cell debris overlaps (1.10~1.20g / cm 3), separation purity <50%; multiple repeated centrifugations (total time >18 h) were required, resulting in continuous loss of active ingredients (retention of asiaticaside decreased to 53%). This method is only suitable for laboratory crude extraction and cannot meet the purity requirements of cosmetic raw materials (additional ultrafiltration purification is required).

[0131] (2) Limitations of dual-modal sonic fluidization alone: ​​wide particle size distribution (PDI>0.3), inability to separate vesicles from apoptotic bodies (yield is only 30-35 mg / g cell); inability to obtain the desired target plant extracellular vesicles (30-150 nm).

[0132] (3) Disadvantages of three-dimensional gradient microvortex centrifugation alone (without other pretreatment): When only three-dimensional gradient microvortex centrifugation is performed and enzymatic hydrolysis and sonication are omitted, irreversible process failure will occur; without enzymatic hydrolysis, the cell wall is not broken, and the yield is extremely low (<20 mg / g), which is completely ineffective for plants with thick cuticles such as Desert Rose; without sonication, high-density impurities (such as organelles) block the gradient interface, and the vesicle yield decreases by more than 40%.

[0133] (4) Disadvantages of not performing enzymatic hydrolysis: Plant cell walls contain resistant structures such as cellulose / cutin / lignin, which cannot be effectively broken by simple physical force; mechanical disruption only releases 30% of vesicles (TEM shows that a large number of vesicles are retained in the cells); unreleased vesicles are discarded in subsequent centrifugation, thereby reducing the vesicle yield. In samples without enzymatic hydrolysis, the density of vesicles and cell wall fragments overlaps (1.10-1.25 g / cm 3 ), the purity was significantly reduced after gradient centrifugation; there was also protein aggregation, and the residual pectin in the cell wall was cross-linked with the vesicle surface protein to form aggregates, which made the purification effect worse.

[0134] (5) Disadvantages of not performing dual-modal sonic fluidization: During sonic fluidization, the low frequency (20 kHz) is missing, which makes it impossible to generate cavitation bubbles to blast the cell wall, resulting in a reduced amount of vesicle release. The high frequency (1 MHz) is missing, and the acoustic radiation force is insufficient to drive the vesicles through the membrane pores, causing the vesicles to be retained inside the cell and reducing the vesicle yield. The lack of the entire dual-modal sonic fluidization step results in uneven vesicle particle size and an increased particle size span, which is not conducive to subsequent purification applications.

[0135] (6) Differences in three-dimensional gradient microvortex centrifugation media: Sucrose solutions of different densities are used to replace the three-layered media of the present invention. This method has the following problems: the sucrose medium has high osmotic pressure, which can cause vesicle shrinkage and damage to the vesicle structure, resulting in reduced vesicle yield, loss of active ingredients, long centrifugation time (centrifugation time > 16 hours), and is not suitable for the extraction of vesicles from other plant cells. However, the three-layer media used in the method of the present invention are in one system, and there is no need to centrifuge them separately. The centrifugation conditions and parameters set for the corresponding medium solutions are short in time and highly efficient, and can be used for the extraction of vesicles from a variety of plant cells.

[0136] Table 1 shows the comparison results of the six different treatment methods compared with the method of Example 1. The results show that all the comparison groups have significant defects. For example, the yield and activity retention of the obtained cell vesicles are significantly reduced. Traditional differential centrifugation requires ultrahigh-speed centrifugation (greater than 120,000 g). The use of sonication or three-dimensional gradient centrifugation alone has limitations and is not suitable for the extraction of some plant vesicles. The vesicle yield and activity retention rate are also significantly reduced. Without enzymatic hydrolysis, the vesicle yield is greatly reduced, which is not conducive to efficient production. The lack of stratification of centrifugation media or the use of media with different densities can destroy the vesicle structure, resulting in significant loss of active ingredients and low yield and purity.

[0137] Table 1 Comparison of results of different extraction methods

[0138] In Example 1, efficient extraction of plant cell vesicles was achieved through a combined process of active enzymatic hydrolysis, dual-modal sonic fluidization technology, and a three-dimensional gradient microvortex centrifugation system. The enzymatic hydrolysis scheme employed cellulase hydrolyzed β-1,4-glucan chains, while pectinase degraded α-1,4-galacturonic acid, disrupting the thin-walled cell skeleton. This dual mechanism of precisely rupturing the cell wall while protecting the active ingredients achieved targeted cell wall decomposition without damaging the vesicle membrane, thereby preserving its integrity. This promoted the full release of intracellular vesicles and increased vesicle yield. The intact vesicle membrane maintained delivery function and vesicle stability, enhancing vesicle bioavailability.

[0139] The dual-modal acoustic fluidization technology used is to first perform low-frequency ultrasound to generate transient cavitation bubbles in the liquid. When the bubbles collapse, microjets (speed >100 m / s) are released to mechanically shear the cell wall. High-frequency focused ultrasound is then performed to use acoustic radiation force to propel the vesicles through the cell membrane pores, avoiding excessive damage to the membrane structure. Combined with a thermal effect suppression strategy, a titanium alloy reactor (thermal conductivity 21.9 W / m·K) and an external circulation cooling system (ethylene glycol-water solution, -10°C) are used to ensure that the processing temperature is <10°C, maintaining the thermal stability of the active substances in the plant extracellular vesicles (degradation rate <5%), maximizing the efficient release of the vesicles, ensuring that the intact vesicle membrane maintains the delivery function and vesicle stability, and increasing the vesicle bioavailability and extraction efficiency.

[0140] Finally, three-dimensional gradient microvortex centrifugation is performed to dynamically match the densities of different plant vesicles through a specific stratification medium, achieve precise stratification, avoid interference from impurities, and achieve precise separation and activity retention. The set progressive centrifugal force can reduce shear membrane damage and provide high activity protection: the three-dimensional gradient microvortex centrifugation system achieves high purity of >95% (compared to <70% by conventional centrifugation) through the synergistic effect of density gradient medium and progressive centrifugal force regulation of the angular rotor. The HPLC method detects the retention rate of asiaticoside, an active component in the vesicles of the Centella asiatica, at 93%, which is significantly improved compared to the 53% retention rate of the traditional ultracentrifugation method, solving the problems of low purity, large activity loss, and limited throughput of a single technology in the traditional centrifugation method.

[0141] The method of Example 1 effectively improves separation efficiency (yield increased by 4.8 times) and activity retention (>90%), while also reducing energy consumption. The total centrifugation time is shortened to 2 hours (compared to >12 hours for conventional differential centrifugation), resulting in a 45% reduction in energy consumption. Furthermore, the method provided by the present invention can effectively separate vesicles from apoptotic bodies (large vesicles with diameters greater than 1-5 μm), is chemically contaminant-free, and avoids PEG and organic solvent residues, thus complying with the requirements of EC 1223 / 2009. This provides a safer and more efficient raw material for cosmetics, is amenable to scalable production, and offers comprehensive advantages in industrial suitability, making it an optimal choice for extracting plant active ingredients. Therefore, the triple process of enzymatic hydrolysis, dual-modal sonication, and three-dimensional gradient centrifugation is the only path that can simultaneously achieve high yield (>80 mg / g), high purity (>95%), and high activity (>90% retention). Each of these three approaches is essential.

[0142] Test Example 1 Yield of Plant Cell Vesicles Vesicle yield data is obtained by quantifying total vesicle protein using the BCA assay and counting particles using the NTA technique. The vesicle yield per unit of raw material (mg / g cells) is calculated using the formula. The combined BCA-NTA method, cross-validated by biochemical lysis quantification (BCA) and physical particle counting (NTA), provides highly reliable vesicle yield data within 1-2 hours. This method is the gold standard for evaluating plant vesicle extraction processes and has been recommended by the International Society for Extracellular Vesicles (ISEV) as a standardized quantification protocol for plant vesicles (RSD < 5%).

[0143] The yield of plant cell vesicles extracted from Examples 1 to 6 and Comparative Examples 1 to 6 was determined respectively. The determination method was as follows: (1) Vesicle lysis: 100 μL of vesicle suspension was added with an equal volume of 2% Triton X-100 and incubated at 37°C for 30 min to completely release the protein. (2) BCA protein quantification: a BCA kit was used to react at 60°C for 30 min and the absorbance at A562 nm was measured; the total protein amount (μg) was calculated based on the bovine serum albumin (BSA) standard curve (0-2000 μg / mL). (3) NTA particle counting: the vesicle stock solution was diluted to 10 7 -10 9 particles / mL; Malvern NanoSight NS300 was used to capture three 60 s videos (detection threshold = 5, temperature 25°C) and analyze particle concentration (particles / mL).

[0144] The yield calculation formula is as follows: ; The measurement results are shown in Table 2, which show that the yield of plant cell vesicles extracted by the enzymatic hydrolysis-sonic wave fluidization-three-dimensional gradient microvortex synergistic method adopted in the embodiment is significantly higher than that of the existing conventional extraction method, the cell vesicle yield is significantly improved, and plant cell vesicles can be efficiently extracted.

[0145] Table 2 Comparison of cell vesicle yields between Examples 1-6 and Comparative Examples 1-6

[0146] Test Example 2: Particle Size Distribution Detection of Plant Cell Vesicles Nanoparticle tracking analysis (NTA) was used to examine the particle size distribution of each sample and to compare the differences in the plant cell vesicle size distribution of Examples 1-6 with those of Comparative Examples 1-6. The instrument used was a Malvern NanoSight NS300 (configuration: 405 nm / 638 nm laser, sCMOS camera).

[0147] Experimental procedure: (1) Sample pretreatment: vesicle suspension was diluted to 1×10 7 -1×10 9particles / mL (optimal signal-to-noise ratio). (2) Instrument calibration: Calibrate focus and sensitivity using a 100 nm latex standard (error <5%). (3) Detection parameter settings: Temperature 25±0.5°C, video recording 3×60 s (effective video frame number >1000 frames / sample), detection threshold 5-8 (automatically optimized), camera level 13-16 (avoid oversaturation). (4) Data acquisition: Samples were injected into the syringe at a constant speed (flow rate: 20 AU), and the software tracked the Brownian motion trajectory in real time. (5) Analytical output: Particle size distribution: Calculate the hydrodynamic diameter based on the Stokes-Einstein equation. Concentration is reported as number of particles / mL (error <10%). Polydispersity index (PDI): <0.3 indicates a monodisperse system.

[0148] The results are shown in Table 3, which shows that the plant cell vesicles extracted by the example method have a smaller particle size, with a particle size distribution in the range of 30-150 nm. The plant cell vesicles extracted by the comparative example method have a larger particle size, with a particle size distribution widely in the range of 100-400 nm. The average particle size distribution of the plant cell vesicles extracted by the present invention is smaller and more uniform, which is conducive to absorption.

[0149] Table 3 Comparison of particle size distribution between Examples 1 to 6 and Comparative Examples 1 to 6

[0150] Test Example 3 Comparison of Purity of Plant Cell Vesicles Prepared by Different Extraction Methods Based on data such as SDS-PAGE protein analysis, NTA particle size uniformity, and TEM membrane integrity, combined with other detection methods, such as chromatography-mass spectrometry (LC-MS) detection, fluorescence quantitative analysis after RNAse treatment (Qubit) detection, hydrophobic protein Western Blot (AQP5) detection, polyphenol-Folin reagent color correction detection, HPLC detection of free saponin ratio, etc., the purity of the plant vesicles extracted by the methods of Examples 1 to 6 and Comparative Examples 1 to 6 was comprehensively determined (purity = target vesicle ratio).

[0151] The measurement results are shown in Table 4 below, which show that the plant cell vesicles extracted in the example have higher purity. The extraction method of the example can more efficiently extract plant cell vesicles, significantly increase the purity of the plant cell vesicles, and facilitate subsequent research and use of the plant cell vesicles.

[0152] Table 4 Comparison of the purity of plant vesicles in Examples 1 to 6 and Comparative Examples 1 to 6

[0153] Test Example 4 Transdermal Absorption Rate of Plant Cell Vesicles The standard procedure for detecting the transdermal absorption rate of plant vesicles based on Franz diffusion cell is briefly described as follows: ex vivo mouse skin (thickness ≤ 500 μm) is fixed in a Franz diffusion cell, with the dermis facing the receiving cell (containing PBS medium at 32 ± 0.5°C, PBS (pH 7.4) + 0.5% BSA); a plant vesicle suspension labeled with a fluorescent dye (such as DiR) is added to the donor cell, and magnetic stirring (600 rpm) and sink conditions (vertical Franz cell, effective diffusion area 0.64 cm) are maintained. 2 , receiving cell volume 5.0 mL); sampling of the receiving fluid was performed periodically over a 24-h run (e.g., 0, 1, 2, 4, 8, 12, and 24 h), and an equal volume of medium was added. The sampled solution was stored at 4°C in the dark. Fluorescence intensity of the fluid passing through the skin was measured using a microplate reader to measure DiR fluorescence parameters (Ex / Em = 748 / 780 nm). Quantification was performed using a standard curve (linear range 0.01–10 μg / mL). Transdermal absorption (%) was calculated according to the following formula. Results are expressed as mean ± SD (n = 3).

[0154] ; Where: Total drug amount: total amount of drug / vesicles added to the supply pool (μg or ng); Q 24 ×A: total amount permeating the skin within 24 h (μg or ng); A: Effective diffusion area of ​​skin (cm 2 ).

[0155] The test results are shown in Table 5, which show that there are significant differences in the transdermal absorption rates of plant cell vesicles prepared by different extraction methods. The transdermal absorption rate of the plant cell vesicles prepared by the method of active enzymatic hydrolysis-sonic lysis-three-dimensional gradient separation in the example is significantly improved.

[0156] Table 5 Comparison of transdermal absorption rates of plant vesicles in Examples 1 to 6 and Comparative Examples 1 to 6

[0157] Test Example 5 Antioxidant Effect The plant cell vesicles extracted from Examples 1 to 6 and Comparative Examples 1 to 6 were used to conduct oxygen free radical scavenging experiments to compare the antioxidant effects of the plant cell vesicles. The steps for conducting the DPPH free radical scavenging experiment were as follows: Reagent preparation: DPPH stock solution: Accurately weigh 2.5 mg of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine), dilute to 100 mL with anhydrous ethanol (concentration 0.025 mg / mL), and store in the dark (4°C).

[0158] Experimental procedure: (1) Sample preparation: Dissolve the sample to be tested (e.g., vesicle extract) in ethanol or buffer to prepare a 1 mg / mL stock solution. Serial dilutions are performed to 0.1, 0.2, 0.5, and 1.0 mg / mL test concentrations (three replicates per well). (2) Reaction system (200 μL / well): 100 μL sample solution + 100 μL DPPH ethanol solution (0.025 mg / mL), control group: 100 μL ethanol + 100 μL DPPH ethanol solution (0.025 mg / mL), blank group: 100 μL sample + 100 μL ethanol (to deduct the background color). (3) Reaction conditions: Shake and mix in the dark, incubate at 25°C in the dark for 30 min. (4) Absorbance detection: Measure the absorbance (A) at 517 nm using a microplate reader.

[0159] The calculation formula of free radical scavenging rate is as follows: ; Where: A sample : Absorbance of sample well (sample + DPPH); A blank : Sample background well absorbance (sample + ethanol); A control : Absorbance of control group (DPPH+ethanol).

[0160] The measurement results are shown in Table 6 below, which show that the plant cell vesicles extracted in the example have better antioxidant effects. The extraction method of the example can more efficiently extract plant cell vesicles, retain their plant active ingredients, and make them have more significant effects, which is beneficial to the subsequent research and application of plant cell vesicles.

[0161] Table 6 Comparison of oxygen free radical scavenging rates of Examples 1 to 6 and Comparative Examples 1 to 6

[0162] Test Example 6 Electron Microscope Image of Plant Cell Vesicles Transmission electron microscopy (TEM) was used to image the plant cell vesicles extracted from Examples 1-6 and Comparative Examples 1-6. TEM is used for ultramicroscopic imaging of nanoscale structures, and the differences between the exosomes in each group were compared. The equipment used was a Hitachi HT7800 transmission electron microscope (accelerating voltage 80-120 kV, resolution 0.2 nm).

[0163] The operation steps are as follows: (1) Sample pretreatment, fixation: take 20 μL of vesicle suspension + 20 μL of 4% glutaraldehyde (fix at 4℃ for 2h) → rinse with PBS three times (5 min / time). Negative staining: uranyl acetate method: add 2% uranyl acetate stain solution (pH 4.5) and stain for 60 s → absorb with filter paper. (2) Sample preparation, copper grid pretreatment: carbon support film copper grid glow discharge treatment (30 s) to enhance hydrophilicity. Sample loading: add 5 μL of sample to the copper grid → let it stand for 2 min → absorb excess liquid with filter paper → dry at room temperature. (3) TEM imaging parameters: acceleration voltage 80 kV (biological sample friendly) to avoid electron beam damage to the vesicle membrane, magnification 20000~50000× to clearly display the vesicle structure (diameter 30~500 nm), exposure time 1-2 s to prevent sample drift, imaging mode is low dose mode (LowDose) to reduce electron radiation damage. (4) Image acquisition and localization of vesicle-enriched areas: capture multiple field-of-view images (≥5 fields / sample). Key structural verification: double-layer membrane structure (saucer-like or cup-shaped characteristic structure), particle size uniformity detection.

[0164] TEM results of different plant cell vesicles Figures 1 to 6 As shown, the extracellular vesicles of Centella asiatica, Commiphora myrrha, Jasmine, Desert Rose, Leontopodium alpinum, and Ginseng prepared by the extraction method of the embodiment of the present invention have the following advantages compared with the comparative examples: (1) The structural integrity is better. The present invention replaces chemical / mechanical violent wall breaking by biological enzymatic hydrolysis + physical field synergy (enzymatic hydrolysis + sonic wave + three-dimensional gradient centrifugation), and the membrane damage rate is significantly reduced; (2) The vesicle size can be accurately prepared. The vesicles prepared by the present invention have a high proportion of vesicles with a particle size of 30-150 nm, while the vesicles prepared by the traditional method have a large size span and a scattered distribution; (3) More active ingredients are retained: the activity loss of the vesicles prepared by the present invention is significantly reduced, while the activity loss of the traditional method is greater; (4) The purity of the vesicles is improved: the purity of the vesicles of the present invention is significantly higher than that of the traditional method, with a clear background and less impurities.

[0165] Test Example 7: Beauty Benefits of Plant Cell Vesicles The test sample used an essence containing 0.5% Centella asiatica cell vesicles, compounded with a fat-soluble whitening ingredient (such as niacinamide). The essence was prepared by re-dissolving the lyophilized Centella asiatica CA-EVs powder prepared in Example 1 in deionized water, mixing it with ceramide NPs (2% w / v) and Pluronic F127 (5% w / v), and then circulating the mixture three times at 1500 bar using a high-pressure homogenizer (ATS AH-100D) to obtain the Centella asiatica cell vesicle essence. An experimental group was also established using only 0.5% Centella asiatica cell vesicles for application.

[0166] Before the repair, the subjects were examined by Corneometer. ® and Mexameter ® Imaging analysis showed obvious inflammation and dehydration of the skin. During the repair period, the subjects only used this essence or Centella asiatica cell vesicles and no other skin care products. The water content of the subjects' skin stratum corneum and erythema index were measured 7 days and 28 days after the application of the repair product.

[0167] After 7 days of application of Centella asiatica cell vesicle essence for repair, the subjects' skin sensitivity decreased and skin moisture increased. Figure 7 and Figure 8 As shown, inflammation was significantly eliminated; after 28 days of use, Corneometer ® Mexameter detects that the water content of the skin's stratum corneum has increased by 38%. ® The erythema index was measured to be reduced by 52%. The application of 0.5% Centella asiatica plant vesicle solution alone also showed a good effect in reducing skin sensitivity. Figure 9 and Figure 10 As shown, it has good repair and anti-inflammatory effects.

[0168] The Franz diffusion cell test showed that the transdermal absorption rate of the essence increased to 22%, indicating that skin care products prepared with Centella asiatica cell vesicles have a good repairing effect on the skin and can be used to prepare more anti-aging, whitening and barrier repair cosmetics to better exert the effects of plant cell vesicles.

[0169] Specific implementation methods The above embodiments are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for extracting plant cell vesicles, characterized in that: The following steps are involved: S1. Enzymatic pretreatment: The plant sample to be extracted is enzymatically hydrolyzed using a cell wall degrading enzyme in a water bath at 30-38°C, 100-150 rpm, for 1-3 hours to obtain a plant enzymatic hydrolyzate; S2, sonic fluidization lysis: The enzymatic hydrolyzate was transferred to a reactor and subjected to low-frequency cavitation at a frequency of 15-25 kHz for 3-7 minutes, and then switched to high-frequency penetration at a frequency of 0.8-1.2 MHz for 8-12 minutes to obtain a reaction solution; S3, gradient centrifugation: the medium solution used is a three-layer medium solution with different densities, of which the bottom solution has a density of 1.1~2g / cm 3 The high-density barrier layer solution and the middle layer solution adopt a density of 1.1~1.5g / cm 3 The isotonic capture layer solution and the upper layer solution have a density of 1~1.5g / cm 3 The reaction solution was slowly added to the three-layer medium solution and centrifuged, and the middle layer solution was collected to obtain plant cell vesicles; Each medium solution was prepared by using at least two substances selected from diatrizoate, iodixanol, sucrose, trehalose, Ficoll 400, mannitol, cesium chloride, glycerol, histidine buffer, EDTA, cholesterol, saponin, phospholipids, Tris-HCl, NaCl, PBS, and ultrapure water.

2. The method according to claim 1, characterized in that The plant sample in S1 is a plant cell mass or cell pellet.

3. The method according to claim 2, characterized in that The cell wall degrading enzyme in S1 is one or more combinations of cellulase, pectinase, lignin peroxidase, cutinase, ascorbic acid, and lysozyme.

4. The method according to claim 1, characterized in that The specific conditions for sonication in S2 are as follows: first, low-frequency cavitation is performed, with a frequency of 15-25 kHz, a power of 45-55 W, a pulse period of 3-7 seconds on / 1-3 seconds off, and a treatment time of 3-7 minutes; then, high-frequency penetration is performed, switching to a high frequency of 0.8-1.2 MHz, and a power density of 0.3-0.7 W / cm 2 , the focused acoustic beam diameter is 2~4mm, the scanning speed is 1~3mm / s, covering the entire reactor, the processing time is 8~12min, and the processing temperature is simultaneously maintained at <10℃ to obtain a reaction liquid.

5. The method according to claim 4, characterized in that: The cavitation density of the low-frequency cavitation treatment in S2 is not less than 10 5 pieces / mL.

6. The method according to claim 1, wherein The bottom solution used in S3 was prepared from at least two substances selected from diatrizoate, Ficoll400, cesium chloride, cholesterol, mannitol, ascorbic acid, ultrapure water, NaCl, and PBS; The middle layer solution is prepared from at least two substances selected from iodixanol, sucrose, mannitol, histidine buffer, saponin, PBS, and NaCl; the upper layer solution is prepared from at least two substances selected from sucrose, trehalose, glycerol, soybean lecithin, PBS, Tris-HCl, EDTA-Na2, and ultrapure water.

7. The method according to claim 6, characterized in that The centrifugation procedure in S3 was as follows: first stage, 1800–2200 × g, 15–25 min; second stage, 6000–10000 × g, 25–35 min; and third stage, 13000–17000 × g, 40–50 min.

8. Use of the method according to any one of claims 1 to 7 in improving the yield, purity and activity of plant cell vesicles.

9. A plant cell vesicle, characterized in that Prepared by the method according to any one of claims 1 to 7.

10. A product, characterized in that Containing the plant cell vesicles according to claim 9.

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