A plant cell vesicle high-efficiency extraction method based on three-dimensional gradient micro-vortex and acoustic wave fluidization synergistic effect and application thereof

Abstract

This invention discloses a highly efficient extraction method for plant cell vesicles based on the synergistic effect of three-dimensional gradient microvortex and acoustic fluidization, and its application. The plant cell vesicle extraction method provided by this invention achieves high yield, high purity, and high activity of plant cell vesicles through a full-chain process of active enzymatic hydrolysis, acoustic lysis, and three-dimensional gradient separation, enabling large-scale production. This method overcomes the shortcomings of traditional extraction methods, such as low yield and purity, chemical contamination, low extraction efficiency, uncontrollable vesicle size and easily damaged structure, and long extraction time. It allows for the large-scale production of more cosmetics and pharmaceuticals containing plant cell vesicles. This invention systematically solves the global challenge of plant vesicle extraction and application through multidisciplinary innovation (acoustics, fluid mechanics, and molecular biomimetic), achieving high extraction efficiency, good activity, and leading technical parameters, which is of great significance for the extraction and development of plant cell vesicles.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, and more specifically, to a method for efficient extraction of plant cell vesicles based on the synergistic effect of three-dimensional gradient microvortex and acoustic fluidization, and its application. Background Technology

[0002] Plant-derived extracellular vesicles (PEVs) are nanoscale membrane structures secreted by plant cells, carrying various bioactive molecules such as proteins, lipids, and nucleic acids. They have broad potential for biomedical applications, including drug delivery, immune modulation, and disease treatment. For example, Centella asiatica-derived extracellular vesicles (CA-EVs) are nanoscale lipid bilayer structures (30–150 nm in diameter) secreted by the cell membrane. They are rich in triterpenoid saponins (such as asiaticoside and hydroxyasiaticoside), 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, demonstrating unique advantages in anti-aging, scar repair, and sensitive skin care.

[0003] At present, 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 centrifugations (e.g., 300×g→2000×g→10000×g→>100000×g), with a total time of more than 12 hours or even longer, resulting in a loss of more than 30% of the vesicle membrane surface protein due to oxidative stress; at the same time, the shear force generated by high-speed centrifugation (>100000×g) will destroy the vesicle structure, such as the leakage rate of asiaticoside as high as 45%. (2) Chemical pollution and safety hazards: Although the polyethylene glycol (PEG) precipitation method can shorten the extraction time to 4 hours, the residual PEG will damage the skin barrier function and induce contact dermatitis (clinical reports allergy rate >3%); although the ultrafiltration method (300 kDa membrane pack) has no chemical additives, 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 in 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, resulting in poor transdermal efficiency. Therefore, developing an efficient, low-cost, and high-purity plant cell vesicle extraction technology has significant scientific and application value. Summary of the Invention

[0004] The technical problem to be solved by this 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. This invention provides a highly efficient extraction method for plant cell vesicles based on the synergistic effect of three-dimensional gradient microvortex and acoustic fluidization and its application.

[0005] The first objective of this invention is to provide a method for efficiently extracting plant cell vesicles.

[0006] A second objective of this invention is to provide a plant cell vesicle.

[0007] The third objective of this invention is to provide a product.

[0008] A fourth objective of this invention is to provide applications of the above-mentioned plant cell vesicles and products.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention provides a method for extracting plant cell vesicles based on the synergistic effect of three-dimensional gradient microvortex and acoustic fluidization, comprising the following steps:

[0011] S1. Enzymatic hydrolysis pretreatment: The plant samples to be extracted are enzymatically hydrolyzed using cell wall degrading enzymes, and subjected to a water bath at 30~38℃, 100~150 rpm, for 1~3 h to obtain plant enzymatic hydrolysate.

[0012] S2, Acoustic fluidization lysis: The enzymatic hydrolysate is transferred to the reaction vessel, and low-frequency cavitation is first performed at a frequency of 15~25 kHz for 3~7 min, and then high-frequency penetration is performed at a frequency of 0.8~1.2 MHz for 8~12 min to obtain the reaction solution;

[0013] S3. Gradient centrifugation: The medium solution used consists of three layers of different densities, with the bottom layer solution having a density of 1.1~2 g / cm³. 3 The high-density barrier layer solution and the intermediate layer solution use a density of 1.1~1.5 g / cm³. 3 The isotonic capture layer solution and the upper layer solution have a density of 1~1.5 g / cm³. 3 The hypotonic protective layer solution was slowly added to the three-layer medium solution and centrifuged. The middle layer solution was collected to obtain plant cell vesicles.

[0014] Each medium solution was prepared by using at least two substances selected from diatrizoate meglumine, iodixanol, sucrose, trehalose, Ficoll 400, mannitol, cesium chloride, glycerol, histidine buffer, EDTA, cholesterol, saponins, phospholipids, Tris-HCl, NaCl, PBS, and ultrapure water.

[0015] This invention achieves highly efficient extraction of plant cell vesicles by combining specific active enzymatic hydrolysis, dual-modal acoustic fluidization technology, and three-dimensional gradient microvortex centrifugation, significantly improving the yield of plant cell vesicles to over 58 mg / g (compared to <31 mg / g by conventional extraction methods). The separation efficiency is significantly improved, with an activity retention rate of >90% and a separation purity of >95% (compared to <70% by ordinary centrifugation). The plant cell vesicles extracted using this method have a particle size distribution of 30–150 nm, which is significantly reduced and more uniform (compared to 100–400 nm by conventional extraction methods), facilitating skin absorption and utilization, thus resulting in a higher passive diffusion absorption rate through the stratum corneum. Furthermore, this method is applicable to the extraction of various plant extracellular vesicles; by adjusting the composition and ratio of the gradient medium, it can be adapted to the extraction of various plant extracellular vesicles, maintaining high yields and activity.

[0016] The extraction method provided by this invention is free of chemical pollution, avoiding residues of PEG and organic solvents, and complies with EC 1223 / 2009 regulations. Furthermore, it has low energy consumption, reducing the total centrifugation time to 2 hours, a 45% reduction compared to traditional differential ultra-high-speed centrifugation (>12 hours). It offers advantages in industrial efficiency and cost, enabling high-throughput processing with a single batch capacity of up to 20L, compared to <0.5L for ordinary centrifugation methods, allowing for large-scale extraction and preparation, suitable for large-scale cosmetic production. This invention provides a highly efficient method for extracting plant cell vesicles. Through optimized enzymatic hydrolysis-sonic lysis-three-dimensional gradient microvortex separation, it achieves high yield, high purity, high activity, and large-scale production of plant cell vesicles. This technology overcomes the shortcomings of traditional methods, such as chemical pollution and low efficiency, and can be mass-produced, showing broad application prospects in the preparation of anti-aging, whitening, and barrier repair cosmetics.

[0017] The principle analysis of the extraction method of this invention is as follows: First, active enzymatic hydrolysis is performed using cell wall-degrading enzymes to break down the plant cell wall, eliminating the cell wall's obstruction of vesicle release. Different enzymatic hydrolysis systems are used for extracting different plant cells, requiring optimization for each plant. For example, enzymatic hydrolysis systems may include cellulase, pectinase, lignin peroxidase, cutinase, ascorbic acid, lysozyme, etc., selecting different combinations of hydrolysates to meet the characteristics of different plant vesicle extraction. Alternatively, enzymatic hydrolysis can be performed according to conventional enzymatic hydrolysis methods in the field.

[0018] The "dual-modal acoustic fluidization technology" of this invention refers to the use of both low-frequency and high-frequency acoustic waves to treat plant liquids after enzymatic hydrolysis. This technology employs low-frequency cavitation and high-frequency directional penetration to treat the enzymatically hydrolyzed plant sample, facilitating better vesicle expulsion during vesicle preparation. First, low-frequency ultrasound generates transient cavitation bubbles in the liquid; upon bubble collapse, microjets (velocity >100 m / s) are released, mechanically shearing the cell walls. Then, high-frequency focused ultrasound is used, utilizing acoustic radiation to propel vesicles through cell membrane pores, avoiding excessive damage to the membrane structure. Simultaneously, a thermal effect inhibition strategy ensures a treatment temperature <10℃, maintaining the thermal stability of active substances in the plant's exovesicles (degradation rate <5%).

[0019] In the "three-dimensional gradient microvortex centrifugation system" of this invention, the three-dimensional gradient refers to the use of three different density gradient medium solutions for microvortex centrifugation. The centrifugation parameters (speed and time) set for each medium are not excessively high, allowing extraction to be completed in a relatively short time, thus achieving "microvortex centrifugation." Using the three-dimensional gradient microvortex centrifugation system, through the synergistic effect of the density gradient medium and the progressive centrifugal force control of the angular rotor, a density of 1.00~1.35 g / cm³ is achieved. 3 The triple gradient medium is precisely layered to avoid interference from impurities. For example, a sucrose-iodixanol-diatrizoate meglumine triple gradient medium is used to dynamically match different plant vesicle densities (e.g., Centella asiatica 1.08 g / cm³). 3 Myrrh 1.20 g / cm³ 3 The separation purity is >95% (compared to <70% by conventional centrifugation). Combined with progressive centrifugation to reduce membrane damage and protect high activity, for example, the retention rate of asiaticoside is 93% (HPLC detection), while traditional ultracentrifugation only achieves 53%. Furthermore, by adjusting the composition and ratio of the gradient medium solution (such as meglumine diatrizoate, iodixanol, sucrose, trehalose, Ficoll 400, mannitol, cesium chloride, glycerol, histidine buffer, EDTA, cholesterol, saponins, phospholipids, etc.), it can be used for the extraction of various plant extracellular vesicles. Therefore, the combination of enzymatic hydrolysis, ultrasonic fluidized bed lysis, and a three-dimensional gradient microvortex centrifugation system solves the problems of low yield, low purity, significant activity loss, and limited throughput of traditional centrifugation methods. Its comprehensive advantages in separation efficiency (increased yield), activity retention (>90%), and industrial adaptability make it the optimal choice for extracting active plant components.

[0020] Preferably, the plant sample in S1 is a plant cell cluster or cell pellet.

[0021] More preferably, the specific treatment method for plant samples is as follows: Fresh plant explants are taken, surface sterilized (75% ethanol for 2 min → soaked in 2% octane disinfectant for 8 min), 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), and cultured in the dark at 25°C for 21 days to induce callus tissue. The callus tissue is then transferred to liquid medium (with 0.5 mg / L 6-BAP added), cultured with shaking at 120 rpm for 10 days, and the wet weight cell clusters are harvested.

[0022] Preferably, the cell wall degrading enzyme in S1 is one or more of cellulase, pectinase, lignin peroxidase, cutinase, ascorbic acid, and lysozyme.

[0023] Preferably, the specific conditions for acoustic fluidized bed lysis in S2 are as follows: the enzymatic hydrolysate is transferred to the reaction vessel, and low-frequency cavitation is first performed at a frequency of 15~25 kHz, a power of 45~55 W, and a pulse period of 3~7 s on / 1~3 s off, for 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 / cm³. 2 The focused acoustic beam diameter is 2~4 mm, the scanning speed is 1~3 mm / s, covering the entire reaction vessel, the processing time is 8~12 min, and the processing temperature is maintained at <10℃ to obtain the reaction solution.

[0024] More preferably, the specific conditions for acoustic fluidized lysis in S2 are as follows: the enzymatic hydrolysate is transferred to the reaction vessel, and low-frequency cavitation is first performed at a frequency of 20 kHz, a power of 50 W, and a pulse period of 5 s on / 2 s off for 5 min; then high-frequency penetration is performed, switching to a high frequency of 1 MHz and a power density of 0.5 W / cm³. 2 The focused acoustic beam diameter is 3 mm, the scanning speed is 2 mm / s, covering the entire reaction vessel, the processing time is 10 min, and the processing temperature is maintained at <10℃ to obtain the reaction solution.

[0025] Preferably, the low-frequency cavitation treatment in S2 is set with a cavitation density of not less than 10. 5 per mL.

[0026] Preferably, the density of the bottom solution in S3 is 1.15~1.3 g / cm³. 3 It is prepared from at least two substances selected from diatrizoate meglumine, Ficoll 400, cesium chloride, cholesterol, mannitol, ascorbic acid, ultrapure water, NaCl, and PBS.

[0027] More preferably, the density of the intermediate layer solution is 1.1~1.2 g / cm³. 3It is prepared from at least two substances selected from iodixanol, sucrose, mannitol, histidine buffer, saponins, NaCl, and PBS.

[0028] More preferably, the density of the upper layer solution is 1.05~1.2 g / cm³. 3 It is prepared from at least two substances selected from sucrose, trehalose, Tris-HCl, glycerol, EDTA-Na2, soybean lecithin, ultrapure water, and PBS.

[0029] As an alternative preferred embodiment, when the extracted plant is Centella asiatica, the media solution is selected as follows: bottom layer solution: 1.16~1.2 g / cm³ 3 Diazochrome meglumine solution; intermediate layer solution: 1.10~1.14 g / cm³ 3 Iodixanol solution; upper layer: 1.06~1.09 g / cm³ 3 Sucrose-trehalose complex solution.

[0030] More preferably, the Centella asiatica extraction medium solution is: bottom layer solution: 1.18 g / cm³ 3 Diazometrine solution (60 g diazometrine dissolved in 400 mL ultrapure water, sterilized via 0.22 μm filter membrane); Middle layer solution: 1.12 g / cm³ 3 Iodixanol solution (40% iodixanol solution (OptiPrep™) mixed with PBS at a volume ratio of 3:1); Supernatant: 1.08 g / cm³ 3 Sucrose-trehalose complex solution (54 g sucrose and 18 g trehalose were dissolved in 500 mL PBS by magnetic stirring until completely dissolved).

[0031] As an alternative preferred embodiment, when the extracted plant is myrrh, the media solution is selected as follows: bottom layer solution: 1.18~1.22 g / cm³ 3 Diazochrome meglumine solution; intermediate layer solution: 1.13~1.16 g / cm³ 3 Iodixanol solution; upper layer: 1.08~1.12 g / cm³ 3 Sucrose solution.

[0032] More preferably, the myrrh extraction medium solution is: bottom layer solution: 1.20 g / cm³ 3 Diazochrome meglumine solution (65 g diazochrome meglumine dissolved in 400 mL ultrapure water, 5 g NaCl added to adjust osmotic pressure); Middle layer solution: 1.15 g / cm³ 3 Iodixanol solution (45% OptiPrep™ mixed with 0.25 M sucrose); upper layer: 1.10 g / cm³ 3Sucrose solution (50 g sucrose dissolved in 500 mL Tris-HCl buffer, pH 6.8).

[0033] As an alternative preferred embodiment, when the plant to be extracted is jasmine, the medium solution is selected as follows: bottom layer solution: 1.13~1.17 g / cm³ 3 Ficoll 400 solution; intermediate layer solution: 1.08~1.12 g / cm³ 3 Sucrose-mannitol complex solution; upper layer: 1.04~1.07 g / cm³ 3 Trehalose solution.

[0034] More preferably, the jasmine flower extraction medium solution is: bottom layer solution: 1.15 g / cm³ 3 Ficoll 400 solution (40% Ficoll 400 dissolved in PBS); Intermediate layer solution: 1.10 g / cm³ 3 Sucrose-mannitol complex solution (40 g sucrose + 10 g mannitol dissolved in 500 mL PBS); upper layer: 1.06 g / cm³ 3 Trehalose solution (30 g trehalose dissolved in 500 mL PBS, pH 5.0).

[0035] As an alternative preferred embodiment, when the plant to be extracted is *Rosa desertica*, the media solution is selected as follows: bottom layer solution: 1.28~1.3 g / cm³ 3 Diazochrome meglumine-cesium chloride complex solution; middle layer: 1.23~1.27 g / cm³ 3 Iodixanol solution; upper layer: 1.18~1.22 g / cm³ 3 Sucrose-glycerol complex solution.

[0036] More preferably, the desert rose extraction medium solution is: bottom layer solution: 1.30 g / cm³ 3 Diazometrine-cesium chloride composite solution (70 g diazometrine dissolved in 400 mL ultrapure water + 10 g CsCl, density calibrated); middle layer: 1.25 g / cm³ 3 Iodixanol solution (50% OptiPrep™ mixed with 0.5 M NaCl); upper layer of solution: 1.20 g / cm³ 3 Sucrose-glycerol complex solution (60 g sucrose + 20 mL glycerol dissolved in 500 mL PBS).

[0037] As an alternative preferred embodiment, when the extracted plant is *Edelweiss*, the media solution is selected as follows: bottom layer solution: 1.13~1.17 g / cm³. 3 Mannitol solution; intermediate layer solution: 1.08~1.12 g / cm³3 Sucrose-histidine buffer; upper layer: 1.03–1.07 g / cm³ 3 Trehalose-EDTA solution.

[0038] More preferably, the extract medium solution for *Edelweiss* is: bottom layer solution: 1.15 g / cm³ 3 Mannitol solution (55 g mannitol + 1 g ascorbic acid dissolved in 500 mL PBS); Intermediate layer solution: 1.10 g / cm³ 3 Sucrose-histidine buffer (45 g sucrose dissolved in 0.1 M histidine buffer, pH 6.5); Supernatant: 1.05 g / cm³ 3 Trehalose-EDTA solution (25 g trehalose + 0.5 g EDTA-Na2 dissolved in 500 mL ultrapure water).

[0039] As an alternative preferred embodiment, when extracting ginseng from plants, the medium solution is selected as follows: bottom layer solution: 1.23~1.27 g / cm³ 3 Diatrizoate meglumine-cholesterol complex solution; intermediate layer solution: 1.18~1.22 g / cm³ 3 Iodixanol-saponin buffer; upper layer: 1.13~1.17 g / cm³ 3 Sucrose-phospholipid solution.

[0040] More preferably, the myrrh extraction medium solution is: bottom layer solution: 1.25 g / cm³ 3 Diatrizoate meglumine-cholesterol complex solution (60 g diatrizoate meglumine + 2 g cholesterol dissolved in 400 mL PBS); intermediate layer solution: 1.20 g / cm³ 3 Iodixanol-Saponin Buffer (45% OptiPrep) TM + 0.1% ginsenoside Rb1, pH 7.0); upper layer solution: 1.15 g / cm³ 3 Sucrose-phospholipid solution (50 g sucrose + 0.5 g soybean phospholipid dissolved in 500 mL PBS).

[0041] Preferably, the centrifugation program in S3 is as follows: first stage 1800~2200×g, 15~25 min; second stage 6000~10000×g, 25~35 min; third stage 13000~17000×g, 40~50 min.

[0042] Traditional sucrose density gradient (1.10~1.30 g / cm³) 3Centrifugation can cause vesicles to shrink due to excessively high osmotic pressure. This invention employs three layers of different biocompatible media: the bottom layer uses a solution such as diatrizoate meglumine, which forms a dense interface to prevent organelle sedimentation; the middle layer uses a solution such as iodixanol, a non-ionic isotonic medium, to maintain osmotic pressure balance across the vesicle membrane; and the top layer uses a solution such as a sucrose-trehalose complex (molar ratio 3:1), which protects vesicle membrane proteins through hydroxyl groups. By using multiple media in combination, extraction of vesicles from different plant sources can be achieved. Combined with physical angular rotor dynamics, a three-dimensional spiral flow field is formed during centrifugation; radial velocity gradient, centrifugal force decreases from the tube wall to the center (2000×g→8000×g), large particles (>200 nm) preferentially settle to the bottom layer; tangential vortex effect, the tilt angle of the angular rotor induces the fluid to generate Coriolis force, driving 30~200 nm vesicles to accumulate in the middle layer along the spiral trajectory; axial stratification control, in the 15000×g stage, the high-density medium forms vertical resistance, so that the vesicles form a clear zone (bandwidth <2 mm) in the middle section of the liquid column.

[0043] As a more preferred embodiment, the present invention provides a more specific extraction method:

[0044] Step 1: Enzymatic pretreatment: The plant samples to be extracted are enzymatically hydrolyzed using cell wall degrading enzymes. The mixture is then in a water bath at 30-38℃, 100-150 rpm, for 1-3 h to obtain the plant enzymatic hydrolysate. For example, for Centella asiatica enzymatic hydrolysate, 0.5 U / mg cellulase is added per gram of wet cells to specifically hydrolyze β-1,4-glucan; 0.2 U / mg pectinase is added to decompose α-1,4-galacturonic acid; and 0.01% lysozyme is added (to target bacterial contamination of plant cell walls). The mixture is then shaken in a water bath at 37℃ (120 rpm) for 2 h to obtain the plant enzymatic hydrolysate.

[0045] Step 2: Acoustic Fluidization Decomposition: An acoustic reaction system (frequency range: 10 kHz ~ 2 MHz, maximum power 200 W) equipped with a titanium alloy amplitude transformer (6 mm diameter, 50 μm tip amplitude). The specific operating procedure is as follows:

[0046] 1. Low-frequency cavitation stage: Transfer the plant enzymatic hydrolysate to the reactor, controlling the liquid level to a depth of 10 mm when the amplitude transformer is immersed. Start the 20 kHz low-frequency mode at 50 W, with a pulse cycle of 5 s on / 2 s off, and process for 5 minutes. Monitor the cavitation cloud density using a high-speed camera (1000 fps), and adjust the power to stabilize the cavitation density at 10. 5 cells / mL;

[0047] 2. High-frequency penetration stage: Switch to 1 MHz high-frequency mode, power density 0.5 W / cm²2 The focused acoustic beam, with a diameter of 3 mm, was used in scanning mode (speed 2 mm / s) to cover the entire reactor. The high-frequency treatment lasted for 10 minutes, and the cooling system was started simultaneously to maintain the temperature below 10℃.

[0048] Step 3: Three-dimensional gradient centrifugation purification:

[0049] 1. Gradient medium preparation (different gradient media are selected according to the characteristics of different plants; the following preparation method uses Centella asiatica as an example):

[0050] (1) Bottom solution (1.18 g / cm³) 3 Dissolve 60 g of meglumine diatrizoate in 400 mL of ultrapure water and sterilize using a 0.22 μm filter membrane;

[0051] (2) Middle layer solution (1.12 g / cm) 3 ): Use 40% iodixanol solution (OptiPrep) TM Mix with PBS at a volume ratio of 3:1;

[0052] (3) Upper layer solution (1.08 g / cm³) 3 ): Dissolve 54 g of sucrose and 18 g of trehalose in 500 mL of PBS and stir magnetically until completely dissolved.

[0053] 2. Centrifugation procedure: Centrifuge the above acoustic lysis buffer at 1000×g for 10 min, and discard the precipitate (unlyzed cell clusters). Slowly add the supernatant to the upper layer of the gradient solution (drop by drop with the needle against the wall, flow rate 1 mL / min), and then centrifuge according to the following procedure;

[0054] (1) First stage (2000×g, 20 min): Removal of nuclear debris and organelle impurities;

[0055] (2) Second stage (8000×g, 30 min): Collect the light blue zone in the middle layer (crude vesicles), and transmission electron microscopy (TEM) shows that the proportion of vesicles is >90%;

[0056] (3) Third stage (15000×g, 45 min): High-purity vesicles are obtained in the middle layer with a particle size distribution of 30~150 nm.

[0057] 3. Vesicle zone identification and collection: After centrifugation, three distinct layers are visible in the solution:

[0058] Top layer: transparent (contains soluble protein);

[0059] Middle layer: pale blue opalescent band (2-3 mm wide, vesicle-rich area); under UV light (365 nm), the middle layer of iodixanol emits blue fluorescence.

[0060] Bottom layer: white precipitate (organelle fragments and other impurities).

[0061] 4. Puncture sampling: Frozen centrifuge tube (-20℃ 30 min) → Take a sample in the middle of the mesosphere zone with the puncture needle (avoid touching the interface). The obtained mesosphere solution is the plant cell vesicle sample.

[0062] To maintain the integrity and bioactivity of plant vesicles, the prepared plant cell vesicles can be further concentrated by ultrafiltration and freeze-dried to obtain freeze-dried powder. Liquid raw materials require freezing conditions for preservation and transportation, while freeze-dried powder can be preserved and transported at room temperature, which is more conducive to room temperature preservation and transportation.

[0063] This invention provides the application of the above method in improving the yield, purity, and activity of plant cell vesicles.

[0064] This invention provides a plant cell vesicle prepared by the above method.

[0065] Preferably, the cell vesicles are: Centella asiatica, myrrh, jasmine, desert rose, edelweiss, or ginseng cell vesicles.

[0066] This invention provides a product containing plant cell vesicles.

[0067] Preferably, the product is a freeze-dried powder, which can better maintain the integrity of plant vesicles and has more lasting and efficient biological activity. The preparation method is as follows: plant cell vesicles are concentrated by ultrafiltration, the concentrated vesicle suspension is mixed with a protective agent, and then freeze-dried to obtain plant cell vesicle freeze-dried powder.

[0068] Preferably, a protectant is used to maintain the structural integrity and biological activity of plant cell vesicles during freeze-drying. The protectant contains: 5% trehalose, 1% hyaluronic acid, and 0.1% Tween-80; or contains 0.1% gelatin hydrolysate and 100-400 mmol / L arginine.

[0069] As a more preferred embodiment, the present invention provides a method for ultrafiltration concentration and freeze-drying:

[0070] 1. Tangential flow ultrafiltration concentration: using Millipore Pellicon ® 3. The high-purity vesicles obtained above were concentrated using a 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, with the backflushing pump started every 10 min (pressure 3 bar, duration 30 s) to prevent membrane fouling. The vesicle suspension was obtained by ultrafiltration.

[0071] 2. Freeze-drying and protection: The concentrated vesicle suspension and the protectant are mixed at a volume ratio of 1:1. The protectant is 5% trehalose (glass transition temperature Tg=115℃) + 1% hyaluronic acid (molecular weight 50 kDa) + 0.1% Tween-80 (to reduce ice crystals piercing the vesicle membrane).

[0072] 3. Freeze-drying: Pre-freezing stage: -80℃ ultra-low temperature, 4 h; Main drying stage: vacuum freeze-drying at -50℃, 0.05 mbar, 18 h; Desorption drying stage: 25℃, 0.001 mbar, 6 h, finally obtaining plant cell vesicle freeze-dried powder.

[0073] More preferably, the product is an essence.

[0074] More preferably, the serum also contains fat-soluble whitening ingredients, such as niacinamide.

[0075] The present invention also provides the application of the above-mentioned plant cell vesicles or products in the preparation of cosmetics and pharmaceuticals.

[0076] The present invention also provides a whitening product containing the above-mentioned plant cell vesicles and fat-soluble whitening ingredients.

[0077] The present invention has the following beneficial effects:

[0078] (1) Breakthrough in technical barriers: By adopting the three-in-one method of "active enzymatic hydrolysis-sonic lysis-three-dimensional gradient separation", the size of plant cell vesicles is precisely controlled and the extraction efficiency is high for the first time. It can better retain active ingredients (such as asiaticoside >90%), and significantly improve the yield, purity and activity of plant cell vesicles. Compared with traditional technology, the extraction time is shortened by 60% (total time <4 h) and the energy consumption is reduced by 45% (sonic power <100 W). It solves 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.

[0079] (2) Cosmetic application: The plant cell vesicles prepared by this invention have better purity and activity, smaller particle size, and significantly improved transdermal absorption rate, which can be better applied to cosmetics; for example, when they are combined with fat-soluble whitening ingredients (such as niacinamide) to prepare an essence, the skin stratum corneum moisture content increases by 38%, the erythema index decreases by 52%, and the transdermal absorption rate increases to 22% after use, which has significant effects.

[0080] (3) Industrial application: The method provided by this invention can be directly carried out using existing acoustic and centrifugal equipment, which is convenient and efficient. The single batch processing capacity is up to 20 L, which can be used for large-scale production and meets the requirements of GMPC certification. The residual amount of organic solvent in the extracted waste liquid is <0.1 ppm (GC-MS detection), which meets the EU EC 1223 / 2009 cosmetics regulations and has better industrial application value. Attached Figure Description

[0081] Figure 1 Comparative electron micrographs of extracellular vesicles of Centella asiatica (A in the figure is Example 1, and B in the figure is Comparative Example 1).

[0082] Figure 2 Comparative electron micrographs of extracellular vesicles from myrrh plants (A in the figure is Example 2, and B in the figure is Comparative Example 2).

[0083] Figure 3 Comparative electron micrographs of extracellular vesicles in jasmine plants (A in the figure is Example 3, and B in the figure is Comparative Example 3).

[0084] Figure 4 Comparative electron micrographs of extracellular vesicles from the desert rose plant (A in the figure is Example 4, and B in the figure is Comparative Example 4).

[0085] Figure 5 Comparative electron micrographs of extracellular vesicles from Edelweiss (A in the figure is Example 5, and B in the figure is Comparative Example 5).

[0086] Figure 6 Comparative electron micrographs of extracellular vesicles in ginseng plants (A in the figure is Example 6, and B in the figure is Comparative Example 6).

[0087] Figure 7 The results of Centella Asiatica cell vesicle extract improving skin sensitivity and hydration.

[0088] Figure 8 The image shows the results of Centella Asiatica cell vesicle extract improving skin inflammation.

[0089] Figure 9 The result of Centella Asiatica cell vesicles improving skin sensitivity and hydration.

[0090] Figure 10 The result of Centella Asiatica cell vesicles improving skin inflammation. Detailed Implementation

[0091] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0092] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0093] The centrifuge used in the implementation was a Beckman Coulter Optima XE-90 with a rotor model of Type 70Ti.

[0094] Example 1: Extraction of Centella asiatica cell vesicles

[0095] Step 1: Cell Culture and Enzymatic Digestion Pretreatment

[0096] 1. Cell Expansion: Fresh explants of Centella asiatica were surface-sterilized (75% ethanol for 2 min → immersion in 2% octane disinfectant for 8 min), rinsed three times with sterile purified water, and inoculated into MS solid medium (containing 30 g / L sucrose, 2 mg / L 2,4-D, and 0.1 mg / L NAA). The cells were incubated in the dark at 25°C for 21 days to induce callus formation. The callus was then transferred to liquid medium (with 0.5 mg / L 6-BAP added) and cultured with shaking at 120 rpm for 10 days. Cell clusters with a wet weight of approximately 50 g were harvested.

[0097] 2. Complex enzymatic hydrolysis system: The cell clusters were washed three times with PBS (pH 5.5, containing 5 mM CaCl2), resuspended in the enzymatic hydrolysis solution, and shaken in a water bath at 37°C (120 rpm) for 2 h to obtain the enzymatic hydrolysis solution. Samples were taken every 30 min to detect the cell dissociation rate (trypan blue staining method, target >95%).

[0098] Preparation of enzymatic hydrolysate: Add 0.5 U / mg cellulase per gram of wet cells for specific hydrolysis of β-1,4-glucan; add 0.2 U / mg pectinase for decomposing α-1,4-galacturonic acid; and add 0.01% lysozyme to target bacterial contamination of plant cell walls.

[0099] Step 2: Acoustic fluidization decomposition

[0100] Equipment parameters: Utilizes an acoustic response system (frequency range: 10 kHz ~ 2 MHz, maximum power 200 W), equipped with a titanium alloy amplitude transformer (6 mm diameter, 50 μm tip amplitude). Specific operating procedures are as follows:

[0101] 1. Low-frequency cavitation stage: Transfer the enzymatic hydrolysate to the reactor, controlling the liquid level to a depth of 10mm when the amplitude transformer is immersed. Start the 20 kHz low-frequency mode at 50 W, with a pulse cycle of 5 s on / 2 s off, and process for 5 minutes. Monitor the cavitation cloud density using a high-speed camera (1000 fps), and adjust the power to stabilize the cavitation density at 10.5 per mL.

[0102] 2. High-frequency penetration stage: Switch to 1 MHz high-frequency mode, power density 0.5 W / cm² 2 A focused acoustic beam with a diameter of 3 mm was used to cover the entire reactor in scanning mode (speed 2 mm / s) for 10 minutes. Simultaneously, the cooling system was started to maintain the temperature <10℃, resulting in an acoustic fluidized treatment liquid.

[0103] Step 3: Three-dimensional gradient centrifugation purification

[0104] 1. Gradient medium preparation:

[0105] (1) Bottom solution (1.18 g / cm³) 3 Dissolve 60 g of meglumine diatrizoate in 400 mL of ultrapure water and sterilize using a 0.22 μm filter membrane;

[0106] (2) Middle layer solution (1.12 g / cm) 3 : 40% Iodixanol solution (OptiPrep) TM Mix with PBS at a volume ratio of 3:1;

[0107] (3) Upper layer solution (1.08 g / cm³) 3 ): Dissolve 54 g of sucrose and 18 g of trehalose in 500 mL of PBS and stir magnetically until completely dissolved.

[0108] 2. Centrifugation program:

[0109] The solutions of each concentration prepared above are sequentially laid in centrifuge tubes to form a layered density gradient; then centrifugation is performed according to the following procedure.

[0110] Sample pretreatment: Centrifuge the sonic fluidization solution at 1000×g for 10 min and discard the precipitate (unlyzed cell clusters). Slowly add the supernatant to the upper layer of the gradient solution (drop by drop with the needle against the wall at a flow rate of 1 mL / min).

[0111] (1) First stage (2000×g, 20 min): Removal of nuclear debris and organelle impurities;

[0112] (2) Second stage (8000×g, 30 min): Collect the light blue zone in the middle layer (crude vesicles, about 3 mL), and transmission electron microscopy (TEM) showed that the proportion of vesicles was >90%;

[0113] (3) Third stage (15000×g, 45 min): High-purity vesicles were obtained in the middle layer, and the particle size distribution was 30~150 nm (PDI=0.18) by nanoparticle tracking analysis (NTA).

[0114] 3. Vesicle zone identification and collection:

[0115] After centrifugation, the solution shows three distinct layers:

[0116] Top layer: transparent (contains soluble protein);

[0117] Middle layer: pale blue opalescent band (2-3 mm wide, vesicle-rich area); under UV light (365 nm), the middle layer of iodixanol emits blue fluorescence.

[0118] Bottom layer: white precipitate (organelle fragments and other impurities).

[0119] 4. Puncture sampling:

[0120] Freeze centrifuge tubes (-20℃ for 30 min) → Take a sample from the middle of the mesosphere using a puncture needle (avoid touching the interface). Sample volume: 2~3 mL (50 mL centrifuge tube). The obtained mesosphere solution is the plant cell vesicle sample.

[0121] Step 4: Ultrafiltration Concentration and Lyophilization

[0122] The prepared plant cell vesicles were further concentrated by ultrafiltration and freeze-dried to obtain freeze-dried powder, which maintained the integrity and biological activity of the plant vesicles and facilitated storage and transportation at room temperature.

[0123] 1. Tangential flow ultrafiltration: using Millipore Pellicon ® 3. The plant vesicles were further concentrated by filtration using a Cassette (300 kDa regenerated cellulose membrane). Operating parameters: transmembrane pressure (TMP) 1.5 bar, feed flow rate 20 mL / min; dynamic backwashing, with the backwash pump started every 10 min (pressure 3 bar, duration 30 s) to prevent membrane fouling. The concentrated vesicle suspension was obtained by tangential flow ultrafiltration.

[0124] 2. Vesicle protection: The concentrated vesicle suspension is mixed with a protectant at a volume ratio of 1:1. The protectant consists of 5% trehalose (glass transition temperature Tg=115℃) + 1% hyaluronic acid (molecular weight 50 kDa) + 0.1% Tween-80 (to reduce ice crystal puncture of the vesicle membrane). Additionally, 0.1% gelatin hydrolysate and arginine at 100~400 mmol / L can be added as protectants to maintain the structural integrity and biological activity of plant cell vesicles during freeze-drying.

[0125] 3. Freeze-drying: The protected plant vesicles prepared above were freeze-dried. The process included a pre-freezing stage: -80℃ ultra-low temperature for 4 h; a main drying stage: vacuum freeze-drying at -50℃ and 0.05 mbar for 18 h; and a desorption drying stage: 25℃ and 0.001 mbar for 6 h, to obtain CA-EVs freeze-dried powder.

[0126] Finally, the total protein content was determined by the BCA method, and the NTA count was used for conversion, resulting in a yield of 82 mg / g cells of Centella asiatica cells.

[0127] Comparative Example 1: Extraction of Centella Asiatica vesicles using conventional ultracentrifugation

[0128] 1. Cell lysis:

[0129] Centella asiatica callus cells (treated in the same way as in Example 1) were suspended in PBS (pH 7.4) and subjected to three freeze-thaw cycles in liquid nitrogen. The cells were then incubated at -4°C for 30 min and centrifuged (300×g, 10 min) to remove unbroken cells, yielding a cell lysate.

[0130] 2. Differential centrifugation:

[0131] The cell lysate was ultracentrifuged according to the following procedure:

[0132] First stage: 2000×g, 20 min, discard the precipitate (cell nuclei and debris).

[0133] Second stage: 10000×g, 30 min, collect supernatant;

[0134] Third stage: 100,000 × g, 2 h, precipitate resuspended in PBS.

[0135] 3. Purification:

[0136] Ultrafiltration concentration was performed using a 300 kDa membrane, 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.

[0137] The traditional ultracentrifugation method for extracting Centella asiatica vesicles takes longer than usual. High-speed centrifugation can cause vesicle membrane rupture. The extracted Centella asiatica cell vesicles were measured (using the same method as in Implementation 1), and the yield of cell vesicles was less than 19 mg / g cells. This method has the disadvantages of loss of activity and very low yield.

[0138] Example 2: Extraction of myrrh (Commiphora myrrha) leaf cell vesicles

[0139] The extraction method and steps are the same as in Example 1, except that adaptation adjustments are made for the plant raw materials. The specific differences are as follows:

[0140] Step 1: Enzymatic hydrolysate: 0.1% lignin peroxidase + 0.5% cellulase.

[0141] Step 2: Acoustic parameters: 20 kHz (60 W, 8 min) → 1 MHz (0.6 W / cm²) 2 12 min).

[0142] Step 3: To address the high density of myrrh vesicles, a stepped gradient medium was designed:

[0143] Bottom layer solution: 1.20 g / cm³ 3 Diazometrine solution (dissolve 65 g diazometrine in 400 mL of ultrapure water, add 5 g NaCl, and adjust the osmotic pressure);

[0144] Intermediate layer solution: 1.15 g / cm³ 3 Iodixanol solution (45% OptiPrep) TM (Mixed with 0.25 M sucrose).

[0145] Upper layer solution: 1.10 g / cm³ 3 Sucrose solution (50 g sucrose dissolved in 500 mL Tris-HCl buffer, pH 6.8).

[0146] The yield of myrrh cell vesicles was determined to be 68 mg / g cells.

[0147] Comparative Example 2: Extraction of myrrh cell vesicles by PEG precipitation

[0148] 1. Cell lysis:

[0149] Myrrh callus cells (treated in the same way as in Example 1) were suspended in PBS (pH 7.4) and subjected to three freeze-thaw cycles in liquid nitrogen. After standing at -4°C for 30 min, the cells were centrifuged (300×g, 10 min) to remove unbroken cells, yielding cell lysate. The myrrh cell suspension was then sonicated (20 kHz, 100 W, 5 min) and centrifuged (10000×g, 20 min) to collect the supernatant.

[0150] 2. PEG precipitation: Add 15% PEG8000 (final concentration) to the supernatant and let stand at 4℃ for 12 h; centrifuge (10000×g, 30 min), and resuspend the precipitate in PBS.

[0151] 3. Impurity removal: Large particles are removed by passing through a 0.45 μm filter membrane, followed by freeze drying.

[0152] The yield of myrrh cell vesicles after treatment with the traditional PEG precipitation method was 31 mg / g cells. At the same time, the extraction method using PEG precipitation method also has the disadvantages of (1) chemical pollution: PEG residue was 0.48% (GC-MS detection result), which may cause skin irritation after subsequent use; (2) poor selectivity: a large number of impurity proteins were co-precipitated (SDS-PAGE detection showed multiple non-target bands).

[0153] Example 3: Extraction of vesicles from jasmine (Jasminum sambac) leaf cells

[0154] The extraction method and steps are the same as in Example 1, except that adaptation adjustments are made for the plant raw materials. The specific differences are as follows:

[0155] Step 1: Enzymatic hydrolysate: 0.6% pectinase + 0.1% cellulase, pH 4.8.

[0156] Step 2: Acoustic parameters: 20 kHz (40 W, 3 min) → 1 MHz (0.3 W / cm²) 2 (10 min).

[0157] Step 3: To protect the volatile components of jasmine flowers, a low-osmotic-pressure Ficoll gradient system is used:

[0158] Bottom layer solution: 1.15 g / cm³ 3 Ficoll 400 solution (40% Ficoll 400 dissolved in PBS);

[0159] Intermediate layer solution: 1.10 g / cm³ 3 Sucrose-mannitol complex solution (40 g sucrose + 10 g mannitol dissolved in 500 mL PBS);

[0160] Upper layer solution: 1.06 g / cm³ 3 Trehalose solution (30 g trehalose dissolved in 500 mL PBS, pH 5.0).

[0161] The yield of jasmine cell vesicles was determined to be 73 mg / g cells.

[0162] Comparative Example 3: Extraction of Jasmine Flower Cell Vesicles by Organic Solvent Extraction

[0163] 1. Solvent extraction:

[0164] The freeze-dried jasmine leaf powder was mixed with ethyl acetate at a ratio of 1:10 (w / v) and extracted by shaking at 25°C for 48 h; the mixture was then filtered, and the filtrate was rotary evaporated (40°C, 0.09 MPa) until dry.

[0165] 2. Concentration and purification:

[0166] The dried residue was defatted with n-hexane and subjected to silica gel column chromatography (eluent: petroleum ether-ethyl acetate gradient); the target fraction was collected, concentrated again, and lyophilized to obtain jasmine cell vesicles.

[0167] The results showed that the yield of jasmine cell vesicles extracted by organic solvent extraction was 15 mg / g cells. This method has the disadvantage that the components are easily destroyed, and rotary evaporation will result in a 58% loss of methyl jasmonate; there is also a risk of solvent residue.

[0168] Example 4: Extraction of leaf cell vesicles from desert rose (Adenium obesum)

[0169] The extraction method and steps are the same as in Example 1, except that adaptation adjustments are made for the plant raw materials. The specific differences are as follows:

[0170] Step 1: Enzymatic hydrolysate: 0.2% keratinase + 0.5% cellulase, pretreatment for 3 h.

[0171] Step 2: Acoustic parameters: 25 kHz (70 W, 8 min) → 1 MHz (0.7 W / cm²) 2 (15 min).

[0172] Step 3: For the high-density hydrophobic proteins in desert rose, an ultra-dense gradient system is employed:

[0173] Bottom layer solution: 1.30 g / cm³ 3 Diazometrine-cesium chloride composite solution (70 g diazometrine dissolved in 400 mL ultrapure water + 10 g CsCl, density calibrated);

[0174] Intermediate layer of solution: 1.25 g / cm³ 3 Iodixanol solution (50% OptiPrep) TM (mixed with 0.5 M NaCl);

[0175] Upper layer of solution: 1.20 g / cm³ 3 Sucrose-glycerol complex solution (60 g sucrose + 20 mL glycerol dissolved in 500 mL PBS).

[0176] The yield of desert rose cell vesicles was determined to be 58 mg / g cells.

[0177] Comparative Example 4: Extraction of cell vesicles from Rosa desertica leaves using high-pressure homogenization

[0178] 1. High-pressure treatment:

[0179] The desert rose leaf cell suspension (lysed using the same method as Comparative Example 1) was circulated 10 times using a 1500 bar high-pressure homogenizer; then cooled in an ice bath to avoid temperatures exceeding 40°C.

[0180] 2. Centrifugal purification:

[0181] 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; the cells were then freeze-dried to obtain desert rose cell vesicles.

[0182] The yield of desert rose cell vesicles extracted by high pressure homogenization was 21 mg / g cells. However, this method has the disadvantage of protein denaturation: the high pressure shear force caused the hydrophobic protein (AQP5) to aggregate, which was not detected by Western blotting.

[0183] Example 5: Extraction of cell vesicles from Leontopodium alpinum

[0184] The extraction method and steps are the same as in Example 1, except that adaptation adjustments are made for the plant raw materials. The specific differences are as follows:

[0185] Step 1: Enzymatic hydrolysate: 0.1% ascorbic acid + 0.5% pectinase, pH 6.0.

[0186] Step 2: Acoustic parameters: 1 MHz intermittent ultrasound (0.4 W / cm²) 2 (2 seconds on / 1 second off).

[0187] Step 3: To avoid polyphenol oxidation, an antioxidant mediator system is used:

[0188] Bottom layer solution: 1.15 g / cm³ 3 Mannitol solution (55 g mannitol + 1 g ascorbic acid dissolved in 500 mL PBS);

[0189] Intermediate layer solution: 1.10 g / cm³ 3 Sucrose-histidine buffer (45 g sucrose dissolved in 0.1 M histidine buffer, pH 6.5);

[0190] Upper layer solution: 1.05 g / cm³ 3 Trehalose-EDTA solution (25 g trehalose + 0.5 g EDTA-Na2 dissolved in 500 mL ultrapure water).

[0191] The yield of Edelweiss cell vesicles was determined to be 66 mg / g cells.

[0192] Comparative Example 5: Obtaining Edelweiss Cell Vesicles by Hot Water Extraction

[0193] 1. Hot water extraction:

[0194] The freeze-dried powder of Edelweiss was mixed with deionized water at a ratio of 1:15 (w / v) and extracted by reflux at 80℃ three times, each time for 1 h; the filtrates were combined and centrifuged (5000×g, 10 min) to remove insoluble matter.

[0195] 2. Concentration and freeze-drying: The filtrate was concentrated under reduced pressure to 1 / 10 of its original volume, pre-frozen at -80℃, and then freeze-dried to obtain Edelweiss cell vesicles.

[0196] The yield of Edelweiss cell vesicles obtained by hot water extraction was 9 mg / g cells. According to existing research, this method has the disadvantages of heat-sensitive loss and activity destruction. The activation of polyphenol oxidase (PPO) leads to a decrease in total phenol content, and the tyrosinase inhibitory conformation is easily destroyed by high temperature.

[0197] Example 6: Vesicle extraction from ginseng (Panax ginseng) tissue culture

[0198] The extraction method and steps are the same as in Example 1, except that adaptation adjustments are made for the plant raw materials. The specific differences are as follows:

[0199] Step 1: Culture optimization: Add 0.5 mg / L methyljasmonic acid and culture for 28 days.

[0200] Step 3: Design a polar gradient system based on the saponin-membrane binding characteristics of ginseng:

[0201] Bottom layer solution: 1.25 g / cm³ 3 Diatrizoate meglumine-cholesterol complex solution (60 g diatrizoate meglumine + 2 g cholesterol dissolved in 400 mL PBS);

[0202] Intermediate layer solution: 1.20 g / cm³ 3 Iodixanol-Saponin Buffer (45% OptiPrep) TM +0.1% ginsenoside Rb1, pH 7.0);

[0203] Upper layer solution: 1.15 g / cm³ 3 Sucrose-phospholipid solution (50 g sucrose + 0.5 g soybean phospholipid dissolved in 500 mL PBS).

[0204] The yield of ginseng cell vesicles was determined to be 79 mg / g cells.

[0205] Comparative Example 6: Extraction of ginseng cell vesicles by ethanol reflux method

[0206] 1. Ethanol reflux:

[0207] Ginseng tissue culture was mixed with 70% ethanol at a ratio of 1:8 (w / v) and extracted by reflux at 85°C three times for 2 hours each time. The extracts were combined, filtered, and then the ethanol was removed by rotary evaporation.

[0208] 2. Purification with macroporous resin:

[0209] The sample was loaded onto an AB-8 macroporous resin column and eluted sequentially with 30%, 50%, and 70% ethanol. The 70% ethanol eluent was collected, concentrated, and freeze-dried to obtain ginseng cell vesicles.

[0210] The yield of ginseng cell vesicles obtained by the ethanol reflux method was determined to be 12 mg / g cells. The disadvantages of this method are low bioavailability and slow onset of action.

[0211] Comparative Example 7: Comparative Analysis of Different Treatment Methods

[0212] Based on the extraction method of Example 1, comparative groups with different treatment conditions were set up respectively: (1) The centrifugation medium was not layered, and no three-dimensional gradient was set in step 3. Sucrose was directly used for differential centrifugation (i.e., after treatment in step 1 and step 2, sucrose was directly used for centrifugation and then step 4 was performed without layered medium centrifugation); (2) Dual-mode acoustic fluidization treatment was performed alone (i.e., after treatment in step 2, ordinary centrifugation was performed and then step 4 was performed without enzymatic digestion in step 1 and three-dimensional gradient centrifugation in step 3); (3) Three-dimensional gradient microvortex centrifugation was performed alone (i.e., plant samples were directly subjected to three-dimensional gradient centrifugation in step 3 and step 4, without enzymatic digestion + acoustic pretreatment); (4) Enzymatic digestion in step 1 was not performed (after treatment in step 2 and 3, centrifugation was performed and then step 4 was performed); (5) Dual-mode acoustic fluidization treatment was not performed (i.e., dual-mode acoustic fluidization treatment in step 2 was not performed, after treatment in step 1, centrifugation was performed directly in step 3 and then step 4 was performed); (6) Sucrose solutions of different densities were used as the layering medium in step 3, specifically the bottom layer was 1.25 g / cm³. 3 Middle layer 1.15 g / cm 3 Upper layer 1.10 g / cm 3 The three-layer sucrose solution was processed in the same way as the others.

[0213] The control groups set up above were identical in procedure except for the method settings for differentiation. For groups not undergoing enzymatic hydrolysis, the samples were lysed according to Control Example 1 to obtain cell lysates, which were then processed further. Different control groups were used to extract asiatica from the plant cells. The yield (mg / g cells), activity retention rate, and other data of the extracted plant cell vesicles were measured and statistically analyzed for each group. The deficiencies of each method were also analyzed. Specific measurement methods are detailed in subsequent test examples. The activity retention rate was calculated by detecting the content of asiaticoside, an active ingredient, in the asiatica cell vesicles using HPLC.

[0214] 1. Analysis of the defects of different processing methods

[0215] (1) Defect of centrifugation medium not stratifying (traditional differential centrifugation): overlap of vesicle and cell debris densities (1.10~1.20 g / cm³). 3 The purity of the extracted material is <50%; multiple centrifugation cycles are required (total time >18 h), and the active ingredients are continuously lost (the retention rate of asiaticoside drops to 53%). This method is only suitable for crude extraction in the laboratory and cannot meet the purity requirements of cosmetic raw materials (additional ultrafiltration purification is required).

[0216] (2) Limitations of dual-mode acoustic fluidization alone: ​​wide particle size distribution (PDI>0.3), unable to separate vesicles from apoptotic bodies (yield of only 30~35 mg / g cells); unable to obtain the desired plant extracellular vesicles (30~150 nm).

[0217] (3) Defects of three-dimensional gradient microvortex centrifugation alone (without other pretreatment): If only three-dimensional gradient microvortex centrifugation is performed and enzymatic hydrolysis and acoustic treatment are omitted, irreversible process failure will occur; without enzymatic hydrolysis, the cell wall is not broken, the yield is extremely low (<20 mg / g), and it is completely ineffective for plants with thick cuticles such as desert rose; without acoustic treatment, high-density impurities (such as organelles) block the gradient interface, and the vesicle yield decreases by more than 40%.

[0218] (4) Defects of not performing enzymatic hydrolysis: Plant cell walls contain resistant structures such as cellulose / cuticle / lignin, which cannot be effectively broken down by physical force alone; mechanical disruption only releases 30% of vesicles (TEM shows that a large number of vesicles remain inside the cells); unreleased vesicles are discarded in subsequent centrifugation, thus reducing the vesicle yield. In samples without enzymatic hydrolysis, the density of vesicles overlaps with that of cell wall fragments (1.10~1.25 g / cm³). 3 After gradient centrifugation, the purity was significantly reduced; there was also protein aggregation, and residual pectin in the cell wall crosslinked with proteins on the surface of vesicles, forming aggregates that reduced the purification effect.

[0219] (5) Defects of not performing dual-modal acoustic fluidization: During acoustic fluidization, the low frequency (20kHz) is missing, which prevents the generation of cavitation bubbles to burst the cell wall, resulting in a reduction in vesicle release; the high frequency (1MHz) is missing, and the acoustic radiation force is insufficient to drive vesicles through the membrane pores, causing vesicles to remain inside the cell and reducing vesicle yield. The absence of the entire dual-modal acoustic fluidization step results in uneven vesicle particle size and an increased particle size range, which is not conducive to subsequent purification applications.

[0220] (6) Difference in three-dimensional gradient microvortex centrifugation medium: The three-layered medium of the present invention is replaced with sucrose solutions of different densities. This method has problems such as vesicle shrinkage and destruction of vesicle structure due to excessively high osmotic pressure of sucrose medium, resulting in reduced vesicle yield, loss of active ingredients, long centrifugation time (centrifugation time > 16 h), and incompatibility with the extraction of other plant cell vesicles. In contrast, the three-layered medium used in the method of the present invention is in one system, eliminating the need for separate centrifugation. The centrifugation conditions set for the medium solution are short and efficient, and can be used for the extraction of various plant cell vesicles.

[0221] The comparison results of the six different treatment methods with the method in Example 1 are shown in Table 1. The results show that all the different comparison groups have significant defects. For example, the yield and activity retention rate of the obtained cell vesicles are significantly reduced. Traditional differential centrifugation requires ultra-high speed centrifugation (greater than 120,000 g). The use of acoustic method or three-dimensional gradient centrifugation alone has limitations and is not suitable for the extraction of vesicles from some plants. Moreover, 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. Centrifugation media that are not stratified or use media of different densities will destroy the vesicle structure, resulting in a large loss of active ingredients and low yield and purity.

[0222] Table 1 Comparison of results from different extraction methods

[0223]

[0224] In Example 1, a combined process of active enzymatic hydrolysis, dual-modal acoustic fluidization technology, and a three-dimensional gradient microvortex centrifugation system was used to achieve efficient extraction of plant cell vesicles. The enzymatic hydrolysis protocol employed involved cellulase hydrolyzing β-1,4-glucan chains and pectinase degrading α-1,4-galacturonic acid, thus breaking down the thin-walled cytoskeleton. This targeted decomposition of the cell wall without damaging the vesicle membrane, through a dual mechanism of precisely breaking down the cell wall and protecting the active ingredients, aims to maintain the integrity of the vesicle membrane and promote the full release of intracellular vesicles, thereby increasing vesicle yield. The intact vesicle membrane maintains delivery function and vesicle stability, enhancing vesicle bioavailability.

[0225] The dual-modal acoustic fluidization technology employed first involves low-frequency ultrasound to generate transient cavitation bubbles in the liquid. When these bubbles collapse, they release microjets (velocities >100 m / s) that mechanically shear the cell walls. Then, high-frequency focused ultrasound is used to propel vesicles through cell membrane pores using acoustic radiation, 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 circulating cooling system (ethylene glycol-water solution, -10℃) ensure the processing temperature remains <10℃, maintaining the thermal stability of active substances in plant vesicles (degradation rate <5%). This maximizes efficient vesicle release, ensures the intact vesicle membrane maintains delivery function and vesicle stability, and increases vesicle bioavailability and extraction efficiency.

[0226] Finally, three-dimensional gradient microvortex centrifugation was performed. By dynamically matching different plant vesicle densities through a specific stratification medium, precise stratification was achieved, avoiding interference from impurities and ensuring accurate separation and activity retention. The set progressive centrifugal force reduced shear membrane damage and protected high activity. Through the synergistic effect of density gradient medium and progressive centrifugal force regulation by the angular rotor, the purity of Centella asiatica cell vesicles separated by the method of this invention was >95% (compared to <70% by ordinary centrifugation). HPLC analysis showed that the retention rate of the active component asiaticoside in Centella asiatica cell vesicles was 93%, a significant improvement compared to the 53% retention rate of traditional ultracentrifugation. This method solves the problems of low purity, large activity loss, and limited throughput of traditional centrifugation methods.

[0227] The method described in 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 traditional differential centrifugation), resulting in a 45% reduction in energy consumption. Furthermore, the method provided by this invention can effectively separate vesicles from apoptotic bodies (large vesicles with a diameter greater than 1-5 μm), and it is free from chemical contamination, avoiding PEG and organic solvent residues. It complies with EC 1223 / 2009 regulations, providing safer and more efficient raw materials for cosmetics. It is suitable for large-scale production and has comprehensive advantages in industrial adaptability, making it the optimal choice for extracting plant active ingredients. Therefore, the enzymatic hydrolysis-dual-modal acoustic wave-three-dimensional gradient centrifugation triple process is the only route that can simultaneously achieve high yield (>80 mg / g), high purity (>95%), and high activity (>90% retention), and all three processes are indispensable.

[0228] Test Example 1: Yield of plant cell vesicles

[0229] The total protein content of vesicles is quantified using the BCA method, and the particle count is statistically analyzed using the NTA technique. The vesicle yield per unit of raw material (mg / g cells) is then calculated using a formula to obtain vesicle yield data. The BCA-NTA combined method, through cross-validation of biochemical lysis quantification (BCA) and physical particle counting (NTA), provides high-reliability vesicle yield data within 1-2 hours, making it the gold standard for evaluating plant vesicle extraction processes. This method has been recommended by ISEV (International Society for Extracellular Vesicles) as a standardized quantitative protocol for plant vesicles (RSD < 5%).

[0230] The yields of plant cell vesicles extracted in Examples 1-6 and Comparative Examples 1-6 were determined respectively. The determination methods were as follows: (1) Vesicle lysis: 100 μL of vesicle suspension was added to an equal volume of 2% Triton X-100 and incubated at 37℃ for 30 min to completely release the protein. (2) BCA protein quantification: The absorbance at A562 nm was measured using a BCA kit after reacting at 60℃ for 30 min. 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 with PBS to 10 7 -10 9 particles / mL; three 60-second videos were captured using a MalvernNanoSight NS300 (detection threshold = 5, temperature 25℃) to analyze particle concentration (particles / mL).

[0231] The formula for calculating the yield is as follows:

[0232] ;

[0233] The results are shown in Table 2. The results show that the yield of plant cell vesicles extracted by the enzymatic hydrolysis-sonic fluidization-three-dimensional gradient microvortex synergistic method adopted in the example is significantly higher than that of the existing conventional extraction methods. The yield of cell vesicles is significantly improved, and plant cell vesicles can be extracted efficiently.

[0234] Table 2 Comparison of cell vesicle yield between Examples 1-6 and Comparative Examples 1-6

[0235]

[0236] Test Example 2: Detection of particle size distribution of plant cell vesicles

[0237] The particle size distribution of each sample was detected using nanoparticle tracking analysis (NTA), and the differences in the particle size distribution of plant cell vesicles in Examples 1-6 and Comparative Examples 1-6 were compared and analyzed. The equipment used was a Malvern NanoSight NS300 (configured with 405 nm / 638 nm laser and sCMOS camera).

[0238] Experimental procedure: (1) Sample pretreatment: vesicle suspension diluted with PBS to 1×10 7 -1×10 9 (1) Particles / mL (optimal signal-to-noise ratio). (2) Instrument calibration: Calibrate focal length and sensitivity using 100 nm latex standard (error <5%). (3) Detection parameter settings: Temperature 25±0.5℃, video recording 3×60 s (effective video frames >1000 frames / sample), detection threshold 5~8 (automatic optimization), camera level 13~16 (avoid oversaturation). (4) Data acquisition: Inject sample uniformly with syringe (flow rate: 20 AU), software tracks Brownian motion trajectory in real time. (5) Analysis output: Particle size distribution: Calculate hydrodynamic diameter based on Stokes-Einstein equation. Concentration report is particle number / mL (error <10%). Polydispersity index (PDI): <0.3 indicates monodisperse system.

[0239] The results are shown in Table 3. The plant cell vesicles extracted by the method of the embodiment have smaller particle sizes, with a particle size distribution in the range of 30~150nm. The plant cell vesicles extracted by the method of the comparative example have larger particle sizes, with a particle size distribution in the range of 100~400nm. The plant cell vesicles extracted by the present invention have a smaller and more uniform average particle size distribution, which is beneficial to absorption.

[0240] Table 3 Comparison of particle size distribution between Examples 1-6 and Comparative Examples 1-6

[0241]

[0242] Test Example 3: Comparison of the purity of plant cell vesicles prepared by different extraction methods

[0243] Based on data such as SDS-PAGE analysis of contaminating proteins, NTA particle size uniformity, and TEM membrane integrity, combined with other detection methods, such as chromatographic mass spectrometry (LC-MS), quantitative RNase-treated fluorescence (Qubit) detection, hydrophobic protein Western blotting (AQP5) detection, polyphenol-Folin reagent colorimetric correction detection, HPLC detection of free saponin ratio, etc., the purity of plant vesicles extracted by the methods in Examples 1-6 and Comparative Examples 1-6 was comprehensively determined (purity = target vesicle ratio).

[0244] The test results are shown in Table 4 below, which shows that the plant cell vesicles extracted in the example have higher purity. The extraction method of the example can extract plant cell vesicles more efficiently, significantly increase the purity of plant cell vesicles, and facilitate subsequent research and use of plant cell vesicles.

[0245] Table 4. Comparison of plant vesicle purity between Examples 1-6 and Comparative Examples 1-6

[0246]

[0247] Test Example 4: Transdermal Absorption Rate of Plant Cell Vesicles

[0248] A brief description of the standard procedure for detecting transdermal absorption of plant vesicles using the Franz diffusion cell: Isolated mouse skin (≤500 μm thick) is immobilized in a Franz diffusion cell, with the dermis facing the receiving cell (containing PBS medium at 32±0.5℃, PBS (pH 7.4) + 0.5% BSA); a suspension of plant vesicles labeled with a fluorescent dye (e.g., DiR) is added to the supply cell, maintaining magnetic stirring (600 rpm) and funnel conditions (vertical to the Franz cell, effective diffusion area 0.64 cm²). 2 The receiving cell volume is 5.0 mL; samples of the receiving solution are taken at regular intervals (e.g., 0, 1, 2, 4, 8, 12, 24 h) every 24 hours, and an equal volume of medium is added simultaneously. The sampled solution is stored at 4℃ protected from light. The intensity of the liquid fluorescence through the skin is detected. The DiR fluorescence parameters (Ex / Em=748 / 780 nm) are detected by an ELISA reader. Quantification is performed using a standard curve (linear range 0.01~10 μg / mL). The transdermal absorption rate (%) is calculated according to the following formula. The results are expressed as mean ± SD (n=3).

[0249] ;

[0250] Where: Total dosage: Total amount of drug added to the supply pool / total amount of vesicles (μg or ng);

[0251] Q 24 ×A: Total amount (μg or ng) permeated through the skin within 24 hours;

[0252] A: Effective skin diffusion area (cm²) 2 ).

[0253] The test results are shown in Table 5. The results show that there are significant differences in the transdermal absorption rate of plant cell vesicles prepared by different extraction methods. The transdermal absorption rate of plant cell vesicles prepared by the active enzymatic hydrolysis-sonic lysis-three-dimensional gradient separation method in the example is significantly improved.

[0254] Table 5. Comparison of transdermal absorption rates of plant vesicles between Examples 1-6 and Comparative Examples 1-6

[0255]

[0256] Test Example 5: Antioxidant Effect

[0257] Oxygen free radical scavenging experiments were conducted using plant cell vesicles extracted in Examples 1-6 and Comparative Examples 1-6, respectively, to compare their antioxidant effects. The method and steps for conducting the DPPH free radical scavenging experiment are as follows:

[0258] Reagent preparation: DPPH stock solution, accurately weigh 2.5 mg DPPH (1,1-diphenyl-2-trinitrophenylhydrazine), and dilute to 100 mL with anhydrous ethanol (concentration 0.025 mg / mL), and store protected from light (4℃).

[0259] Experimental procedure: (1) Sample preparation: Dissolve the sample to be tested (such as vesicle extract) in ethanol or buffer solution to prepare a 1 mg / mL stock solution. Serial dilution to test concentrations of 0.1, 0.2, 0.5, and 1.0 mg / mL (3 replicates for each concentration). (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 (subtracting background color). (3) Reaction conditions: Shake well in the dark, react at 25℃ in the dark for 30 min. (4) Absorbance detection: Measure the absorbance (A) at 517 nm using an ELISA reader.

[0260] The formula for calculating the free radical scavenging rate is shown below:

[0261] ;

[0262] In the formula: A sample : Sample well absorbance (sample + DPPH); A blank : Sample background absorbance (sample + ethanol); A control : Control group absorbance (DPPH + ethanol).

[0263] The test results are shown in Table 6 below. The results show that the plant cell vesicles extracted in the examples have better antioxidant effects. The extraction method of the examples can extract plant cell vesicles more efficiently, retain their plant active ingredients, and make them more effective, which is beneficial to the subsequent research and application of plant cell vesicles.

[0264] Table 6. Comparison of oxygen free radical scavenging rates between Examples 1-6 and Comparative Examples 1-6

[0265]

[0266] Test Example 6: Electron micrographs of plant cell vesicles

[0267] Transmission electron microscopy (TEM) was used to photograph the plant cell vesicles extracted in Examples 1-6 and Comparative Examples 1-6, respectively. TEM was used for ultra-microscopic imaging of nanoscale structures to compare the differences in the outer vesicles of each group. The instrument used was a Hitachi HT7800 transmission electron microscope (accelerating voltage 80-120 kV, resolution 0.2 nm).

[0268] The operation steps are as follows: (1) Sample pretreatment and fixation: Take 20 μL of vesicle suspension + 20 μL of 4% glutaraldehyde (fix at 4℃ for 2h) → rinse 3 times with PBS (5 min / time). Negative staining: Uranium acetate method: add 2% uranium acetate staining solution (pH 4.5) and stain for 60 s → blot dry with filter paper. (2) Sample preparation and copper mesh pretreatment: carbon support membrane copper mesh glow discharge treatment (30 s) to enhance hydrophilicity. Sample loading: add 5 μL of sample to copper mesh → let stand for 2 min → blot off excess liquid with filter paper → dry at room temperature. (3) TEM imaging parameters: accelerating voltage 80 kV (biological sample friendly) to avoid electron beam damage to vesicle membrane, magnification 20000~50000× to clearly display vesicle structure (diameter 30~500 nm), exposure time 1-2 s to prevent sample drift, imaging mode is low-dose mode to reduce electron radiation damage. (4) Image acquisition and vesicle enrichment region location: capture multi-field images (≥5 fields of view / sample). Key structure verification: bilayer membrane structure (cup-shaped or cup-shaped characteristic structure), particle size uniformity detection.

[0269] TEM results of different plant cell vesicles as follows Figures 1-6 As shown, the extracellular vesicles of Centella asiatica, Myrrh, Jasmine, Rosa desertica, Edelweiss, and Ginseng prepared by the extraction method of the present invention have the following advantages compared with the comparative example: (1) Better structural integrity. The present invention replaces chemical / mechanical violent cell disruption with biological enzymatic hydrolysis + physical field synergy (enzymatic hydrolysis + acoustic wave + three-dimensional gradient centrifugation), which significantly reduces the membrane damage rate; (2) Accurate preparation of vesicle size. 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 range and scattered distribution; (3) More active ingredients are retained. The vesicles prepared by the present invention have significantly less activity loss, while the traditional method has greater activity loss; (4) Improved vesicle purity. The purity of the vesicles prepared by the present invention is significantly higher than that of the traditional method, with clear background and fewer impurities.

[0270] Test Example 7: Cosmetic Effects of Plant Cell Vesicles

[0271] The test sample used an essence containing 0.5% Centella Asiatica cell vesicles, combined with a fat-soluble whitening ingredient (such as niacinamide). The essence was prepared as follows: the Centella Asiatica CA-EVs lyophilized powder prepared in Example 1 was reconstituted in deionized water, mixed with ceramide NP (2% w / v) and Pluronic F127 (5% w / v), and circulated 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 set up to apply 0.5% Centella Asiatica cell vesicles alone.

[0272] Subjects underwent Corneometer testing before the repair process. ® and Mexameter ® Imaging analysis showed that the skin had obvious inflammation and significant dehydration. During the repair period, the subjects used only this essence or Centella Asiatica cell vesicles and did not use other skin care products. The skin stratum corneum moisture content and erythema index of the subjects were measured at 7 days and 28 days after application and repair.

[0273] After applying Centella Asiatica cell vesicle essence for 7 days, the subjects experienced reduced skin sensitivity and increased skin hydration. Figure 7 and Figure 8 As shown, the inflammation has significantly subsided; after 28 days of use, Corneometer... ® The skin's stratum corneum moisture content increased by 38%, according to Mexameter. ® The erythema index decreased by 52%. Applying 0.5% Centella Asiatica plant vesicle extract alone also showed a good effect in reducing skin sensitivity, such as... Figure 9 and Figure 10 As shown, it has good repair and anti-inflammatory effects.

[0274] The transdermal absorption rate of the serum was increased to 22% according to the Franz diffusion cell test, indicating that the skin care products prepared using Centella Asiatica cell vesicles have a good repair effect on the skin. They can be used to prepare more anti-aging, whitening and barrier repair cosmetics to better exert the effects of plant cell vesicles.

[0275] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for extracting plant cell vesicles, characterized in that, Includes the following steps: S1. Enzymatic pretreatment: The plant sample to be extracted is enzymatically hydrolyzed using cell wall degrading enzymes, and subjected to a water bath at 30-38℃, 100-150 rpm, for 1-3 hours to obtain plant enzymatic hydrolysate. S2, Acoustic fluidization lysis: The enzymatic hydrolysate is transferred to the reaction vessel, and low-frequency cavitation is performed at a frequency of 15~25kHz for 3~7 minutes. Then, high-frequency penetration is performed at a frequency of 0.8~1.2MHz for 8~12 minutes to obtain the reaction solution. The specific conditions for acoustic fluidization decomposition are as follows: first, low-frequency cavitation is performed at a frequency of 15~25kHz, a power of 45~55W, a pulse period of 3~7s on / 1~3s off, and processing for 3~7 minutes; then, high-frequency penetration is performed, switching to a high frequency of 0.8~1.2MHz, with a power density of 0.3~0.7W / cm³. 2 The focused acoustic beam diameter is 2~4mm, the scanning speed is 1~3mm / s, covering the entire reaction vessel, the processing time is 8~12min, and the processing temperature is maintained at <10℃ to obtain the reaction solution; The cavitation density set for low-frequency cavitation treatment should not be less than 10. 5 cells / mL; S3. Gradient centrifugation: The medium solution used consists of three layers of different densities, with the bottom layer solution having a density of 1.1~2 g / cm³. 3 The high-density barrier layer solution and the intermediate layer solution use a density of 1.1~1.5 g / cm³. 3 The isotonic capture layer solution and the upper layer solution have a density of 1~1.5 g / cm³. 3 The hypotonic protective layer solution was slowly added to the three-layer medium solution and centrifuged. The middle layer solution was collected to obtain plant cell vesicles. The centrifugation program is as follows: First stage: 1800~2200×g, 15~25min; The second stage is 6000~10000×g, 25~35min; the third stage is 13000~17000×g, 40~50min. Each medium solution was prepared by using at least two substances selected from diatrizoate meglumine, iodixanol, sucrose, trehalose, Ficoll 400, mannitol, cesium chloride, glycerol, histidine buffer, EDTA, cholesterol, saponins, phospholipids, Tris-HCl, NaCl, PBS, and ultrapure water.

2. The method according to claim 1, characterized in that, The plant sample mentioned in S1 is a cluster of plant cells.

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

4. The method according to claim 1, characterized in that, The bottom solution used in S3 is prepared from at least two substances selected from diatrizoate meglumine, Ficoll 400, 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, saponins, 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.

5. The application of the method according to any one of claims 1 to 4 in improving the yield, purity, and activity of plant cell vesicles.

6. A plant cell vesicle, characterized in that, It is prepared by the method described in any one of claims 1 to 4.

7. A product characterized in that, Contains the plant cell vesicles as described in claim 6.

Images