A method for extracting plant leaf vesicles from exosomes based on heterologous plant material exosomes

Abstract

This invention provides a method for extracting plant leaf vesicles from exosomes using heterologous plant materials, belonging to the field of plant vesicle preparation technology. The method provided by this invention significantly improves the yield of target vesicles and better maintains their intact morphology and membrane integrity. By optimizing the extraction and preservation system, the aggregation and degradation of vesicles during preparation and storage are effectively inhibited, ensuring their long-term stability. The vesicles obtained by this method exhibit significantly enhanced physiological activity and therapeutic potential in disease model experiments, such as superior effects in improving fatty liver pathology and promoting endothelial cell repair. This provides a high-quality material basis and reliable technical solution for developing innovative drugs or functional products based on plant vesicles.

Description

Technical Field

[0001] This invention relates to the field of plant exovesicle preparation technology, and in particular to a method for extracting plant leaf exovesicles based on exolymph from heterologous plant materials. Background Technology

[0002] Exovesicles are a class of nanoscale vesicles with lipid bilayer membranes, actively released by cells, and widely found in animal body fluids and plant sap. They carry bioactive molecules such as proteins, nucleic acids, and lipids, playing a crucial role in intercellular communication. In recent years, plant-derived exovesicles have shown great application potential in the fields of biomedicine, nutrition and health care, and cosmetics due to their wide availability, high safety, and unique plant active ingredients. Leaves, as important plant organs, are considered a rich source of exovesicles with specific functions.

[0003] Apoplastic fluid, the fluid within the intercellular spaces of plant cells, contains a unique environment of ions, metabolites, and signaling molecules, theoretically providing a natural and stable matrix for exovesicles originating from this region. Existing methods for extracting plant exovesicles mostly use phosphate buffers or homologous plant homogenates directly. These methods have significant limitations: buffers lack protective components, easily leading to vesicle rupture under mechanical force or oxidative stress; while homologous homogenates may contain high concentrations of phenols, pigments, and active enzymes, which can interfere with vesicle integrity and reduce their activity. Therefore, developing an extraction strategy that can both mimic the natural microenvironment of vesicles and avoid interference from harmful substances is crucial for improving the yield, purity, and functional activity of plant exovesicles, and represents a critical bottleneck that urgently needs to be overcome in this technological field. Summary of the Invention

[0004] The purpose of this invention is to provide a method for extracting plant leaf vesicles from exogenous fluids of heterologous plant materials, which solves the technical problems in the prior art of low vesicle extraction efficiency, easy damage to vesicle structure, loss of biological activity, and poor long-term storage stability caused by improper extraction environment when extracting plant leaf-derived vesicles.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for extracting leaf-like exovesicles, wherein the extract used in the extraction method contains devesaccharified ED-AWF from a heterologous plant, and the volume ratio of the devesaccharified ED-AWF from the heterologous plant in the extract is 20~30% v / v.

[0007] Preferably, the extract further includes an osmotic pressure regulator, a pH buffer, an antioxidant, and an anti-aggregation agent.

[0008] Preferably, the osmotic pressure regulator is mannitol, with a final concentration of 0.28~0.32 M.

[0009] Preferably, the pH buffer is ethanesulfonic acid with a final concentration of 10-20 mM.

[0010] Preferably, the antioxidant is carnosine, with a final concentration of 1-3 mM.

[0011] Preferably, the anti-aggregation agent comprises inositol and trehalose, wherein the final concentration of inositol is 5-20 mM and the final concentration of trehalose is 10-30 mM.

[0012] Preferably, the preparation of the devesicated ED-AWF includes the following steps: immersing plant leaves in an infiltration buffer and vacuuming them, collecting the exudate by centrifugation, clarifying it by step centrifugation, removing vesicles by ultracentrifugation, and filtering and removing phenols.

[0013] Preferably, the infiltration buffer contains mannitol and ethanesulfonic acid, and has a pH of 6.45 to 6.55.

[0014] Preferably, the process also includes pretreatment of the leaf samples, including cleaning and disinfection, cutting into pieces and weighing.

[0015] It also includes a homogenization step, in which the extract is added and homogenized in an ice bath.

[0016] It also includes a purification step, which includes differential centrifugation for impurity removal, tangential flow filtration concentration, and size exclusion chromatography separation.

[0017] Preferably, in the size exclusion chromatography separation, the fraction collected with an elution volume of 15-25 mL corresponds to vesicles with a particle size of 50-200 nm.

[0018] The beneficial effects of this invention are:

[0019] The method provided by this invention significantly improves the yield of target exovesicles and better maintains their intact morphology and membrane integrity. By optimizing the extraction and preservation system, the aggregation and degradation of vesicles during preparation and storage are effectively inhibited, ensuring their long-term stability. Exovesicles obtained by this method exhibit significantly enhanced physiological activity and therapeutic potential in disease model experiments, such as superior effects in improving fatty liver pathology and promoting endothelial cell repair. This provides a high-quality material basis and reliable technical solution for developing innovative drugs or functional products based on plant exovesicles. Attached Figure Description

[0020] Figure 1TEM images (scale bar: 100 nm) of the control group (PBS), the homologous ED-AWF (lotus leaf source, 25% v / v) group, and the heterologous ED-AWF (lettuce source, 25% v / v) group.

[0021] Figure 2 The following graphs are used to evaluate the effects of different extract components on the stability of leaf exovesicle size, including: (A) NTA plots of particle size distribution in the control group (PBS), the fixed component control group, and experimental groups 1-4 at 0 day; (B) Trend plots of average particle size changes in the control group (PBS), the fixed component control group, and experimental groups 1-4 at 0 day, 7 day, 14 day, and 28 day (*P<0.05, **P<0.01, ***P<0.001).

[0022] Figure 3 The diagram shows the validation of the optimal combination of extracted components based on zeta potential and particle size analysis, including: (A) NTA spectra of particle size distribution of the control group (PBS), experimental group 1, and experimental group 2 at 0 day; (B) percentage distribution of zeta potential of the control group (PBS), experimental group 1, and experimental group 2; and (C) trend of average particle size change of the control group (PBS), experimental group 1, and experimental group 2 at 0, 7, 14, and 28 days (*P<0.05, **P<0.01, ***P<0.001).

[0023] Figure 4 The image shows the therapeutic efficacy of lotus leaf vesicles extracted using the optimal process on NAFLD model mice. (A) HE staining results of liver tissue from the control group, model group, vesicle intervention group (PBS), and vesicle intervention group (ED-AWF); (B) Oil Red O staining results of liver tissue from each group; (C) Comparison of triglyceride (TRIGL) levels among the groups; (D) Comparison of low-density lipoprotein cholesterol (LDLC) levels among the groups; (E) Comparison of total cholesterol (CHOL) levels among the groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: no statistically significant difference).

[0024] Figure 5 The diagram shows the effect of Ginkgo biloba vesicles extracted using the optimal process on promoting endothelial cell migration. Among them: (A) Cell scratch experiment images of the control group, the external vesicle intervention group (PBS), and the external vesicle intervention group (ED-AWF) at 0h, 6h, 12h, 18h, and 24h; (B) Trend diagram of relative scratch density changes of each group at time points of 0h, 6h, 12h, 18h, and 24h. Detailed Implementation

[0025] This invention provides a method for extracting leaf-like exovesicles. The extraction solution system used in this method includes devesaccharified ED-AWF from a heterologous plant as a key matrix component. In this invention, "heterologous plant" refers to a plant species different from the leaf from which the exovesicles are to be extracted, such as, but not limited to, common leafy vegetables like lettuce, spinach, rapeseed, and cabbage. These plants are widely available, inexpensive, and their apoplast fluid has a mild chemical environment, which is beneficial for maintaining the structural stability of the exovesicles. The "devesaccharified ED-AWF" is a clear liquid obtained by physically removing the plant's own vesicle components from its apoplast fluid. It retains natural ions, small molecule metabolites, and polysaccharides, providing a biomimetic inert protective environment for the exogenous leaf exovesicles. The volume ratio of the devesaccharified ED-AWF from the heterologous plant in the extraction solution is 20% to 30% v / v, for example, 20%, 22%, 25%, 28%, 30%, etc. More preferably, the volume ratio is 22% to 28% v / v; more preferably, it is 24% to 26% v / v; and most preferably, it is about 25% v / v.

[0026] In a preferred embodiment of the present invention, the extract, in addition to containing devesaccharified ED-AWF from a heterologous plant, also includes one or more of an osmotic pressure regulator, a pH buffer, an antioxidant, and an anti-aggregating agent. The synergistic effect of these components helps maintain the integrity, dispersibility, and bioactivity of the exovesicles during extraction and subsequent processing.

[0027] Further preferably, the osmotic pressure regulator is selected from one or more of sugars, polyols, or inorganic salts. Its function is to regulate the osmotic pressure of the extract to make it similar to the osmotic environment inside plant cells, thereby reducing osmotic damage to the exovesicles during the extraction process. In a specific embodiment of the present invention, the osmotic pressure regulator is mannitol. Mannitol is a commonly used osmotic pressure protectant with stable chemical properties and is not easily involved in metabolic reactions. Its final concentration in the extract is 0.28 M to 0.32 M, for example, 0.28 M, 0.29 M, 0.30 M, 0.31 M, 0.32 M, etc. Further preferably, it is 0.29 M to 0.31 M; more preferably, it is about 0.30 M.

[0028] Further preferably, the pH buffer is used to maintain the pH stability of the extraction solution system and prevent denaturation of the outer vesicle membrane structure or surface proteins due to pH fluctuations. Commonly used biological buffers include, but are not limited to, HEPES, MES, PIPES, MOPS, and ethanesulfonic acid. In a specific embodiment of the present invention, the pH buffer is ethanesulfonic acid. Ethanolonic acid has good buffering capacity in a near-neutral pH range. Its final concentration in the extraction solution is 10 mM to 20 mM, for example, 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, etc. Further preferably, it is 12 mM to 18 mM; more preferably, it is about 15 mM.

[0029] Further preferably, the antioxidant is used to scavenge reactive oxygen species that may be generated during extraction and processing, reducing oxidative stress damage to the lipid membrane of external vesicles and bioactive molecules. Commonly used antioxidants include, but are not limited to, glutathione, ascorbic acid, lipoic acid, and carnosine. In a specific embodiment of the present invention, the antioxidant is carnosine. Carnosine is a dipeptide composed of β-alanine and L-histidine, and has strong antioxidant and metal ion chelating abilities. Its final concentration in the extract is 1 mM to 3 mM, for example, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, etc. Further preferably, it is 1.5 mM to 2.5 mM; more preferably, it is about 2 mM.

[0030] Further preferably, the anti-aggregation agent is used to prevent non-specific aggregation of the exovesicles during concentration and storage, maintaining the dispersed state of the nanoparticles. Commonly used anti-aggregation agents include sugars (such as trehalose and sucrose), polyols (such as sorbitol), amino acids (such as glycine), and cyclic alcohols (such as inositol). In a specific embodiment of the present invention, the anti-aggregation agent includes inositol and trehalose. Inositol (cyclohexanehexyl) and trehalose are both biocompatible protective agents that can stabilize the structure of biomacromolecules through hydrogen bonding. The final concentration of inositol in the extract is 5 mM to 20 mM, for example, 5 mM, 8 mM, 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, etc. Further preferably, it is 8 mM to 15 mM; more preferably, it is about 10 mM. The final concentration of trehalose in the extract is 10 mM to 30 mM, for example, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, etc. More preferably, it is 15 mM to 25 mM; more preferably, it is about 15 mM or about 25 mM.

[0031] In this invention, the devesicated ED-AWF can be prepared using conventional plant apoplast extraction and clarification methods in the art. A preferred preparation method includes the following steps: First, the selected heterologous plant leaves are completely immersed in pre-cooled infiltration buffer and subjected to vacuum treatment (e.g., vacuum degree of approximately -0.09 ± 0.01 MPa gauge pressure) to promote the infiltration of the buffer into the intercellular spaces of the leaves; then, the liquid exudated from the leaves is collected by low-speed centrifugation (e.g., 1000×g); next, the collected exudate is subjected to stepwise centrifugation to gradually remove cell debris and large particulate impurities, for example, centrifugation at 500×g, 3000×g, and 10000×g in sequence; then, the plant-derived vesicle components in the liquid are removed by ultracentrifugation (e.g., 100,000×g) to obtain a devesac-free apoplastic fluid supernatant; finally, the supernatant can be filtered (e.g., through 0.45 μm and 0.10 μm filter membranes in sequence) and treated to remove phenols (e.g., by adding polyvinylpyrrolidone (PVPP) and adsorption resin XAD-7HP), and its pH and osmotic pressure are adjusted to the desired range.

[0032] Further preferably, the infiltration buffer used in the preparation of devesaccharified ED-AWF comprises an osmotic pressure regulator and a pH buffer. In one specific embodiment, the infiltration buffer comprises mannitol and ethanesulfonic acid. Its pH value is adjusted to 6.45 to 6.55, for example, 6.45, 6.48, 6.50, 6.52, 6.55, etc. Further preferably, it is 6.48 to 6.52; more preferably, it is about 6.50.

[0033] The extraction method of the present invention also includes routine pretreatment steps for the plant leaf samples to be treated. These steps typically include: collecting fresh, disease-free plant leaves; washing with sterile water to remove surface contaminants; briefly immersing the leaves in a suitable disinfectant (e.g., 75% ethanol) for disinfection; rinsing again with sterile water to remove residual disinfectant; blotting the surface moisture with sterile filter paper; cutting or trimming the leaves into appropriately sized pieces (e.g., about 0.5 cm × 0.5 cm) and keeping them at a low temperature on ice to prevent degradation; and finally, accurately weighing the leaves to prepare the volume for the extraction solution.

[0034] After pretreatment, a homogenization step is performed to break down the plant tissue and release its contents. The weighed leaf samples are mixed with the previously prepared extract at a certain ratio (e.g., a mass-to-volume ratio of 1:1) and homogenized at high speed using a tissue homogenizer under ice bath conditions. Homogenization is usually performed intermittently (e.g., homogenize for 30 seconds, rest for 30 seconds, repeat several times) to avoid excessive heat generation.

[0035] The homogenized mixture requires a series of purification steps to separate and enrich the target extravesicles. These purification steps typically include: first, removing cell debris, organelles, and large particulate impurities from the homogenate by differential centrifugation, for example, sequentially centrifuging at different speeds such as 1000×g, 3000×g, and 20000×g; collecting the supernatant and further concentrating and replacing it with buffer using a tangential flow filtration system. The tangential flow filtration membrane is typically selected with a molecular weight cutoff of 100 kDa to 500 kDa (e.g., a 300 kDa polyethersulfone membrane). This step effectively concentrates the extravesicles and removes most soluble impurities; finally, the concentrated sample is finely separated using a size exclusion chromatography column, achieving purification based on the difference in hydrodynamic volume between the extravesicles and other impurity molecules.

[0036] More preferably, in the size exclusion chromatography separation step, the target leaf-like exovesicles are eluted within a specific elution volume range. The elution window of the target exovesicles can be determined based on preliminary experiments or column calibration results. In one specific embodiment, a fraction with an elution volume of 15 mL to 25 mL is collected, which mainly contains vesicle particles with hydrodynamic diameters in the range of 50 nm to 200 nm, such as 50 nm, 80 nm, 100 nm, 150 nm, and 200 nm. Furthermore, the particle size distribution and concentration of the collected fraction can be characterized by methods such as nanoparticle tracking analysis, dynamic light scattering, or transmission electron microscopy to confirm the enrichment of the target exovesicles.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] I. Special reagent combination (prepared in 1L system, pH 6.50±0.05 throughout, osmotic pressure 0.30-0.32Osm / kg)

[0040] (a) Infusion buffer (IB) (used for extracting devesicled heterologous exosome juice)

[0041] Components: Mannitol 0.30M (54.6g), ethanesulfonic acid 15mM (2.93g), ultrapure water to 1L;

[0042] Preparation: Adjust the pH to 6.50 with NaOH, pre-cool at 4℃, and then filter through a 0.22 μm PES membrane for later use;

[0043] Note: The final mannitol concentration of 0.30M is the total concentration of the system, which includes the mannitol naturally present in the devesicated ED-AWF and any additional added mannitol, and ensures an osmotic pressure of 0.30-0.32 Osm / kg.

[0044] (ii) Extraction / treatment of central nervous system buffer (EPB)

[0045] Components: Devesicated ED-AWF (10% v / v, 25% v / v, 50% v / v) of the same / different species (lettuce) , mannitol final concentration 0.30M, ethanesulfonic acid 15mM, inositol (10mM, 15mM), trehalose (15mM, 25mM), carnosine 2mM;

[0046] Preparation: After mixing, adjust the pH to 6.50 and filter through a 0.22μm PES membrane;

[0047] Note: The final mannitol concentration of 0.30M is the total concentration of the system, and the principle is the same as that of IB buffer.

[0048] (iii) SEC liquidity

[0049] Composition: Devesicated ED-AWF 10% v / v, HEPES 10mM, Mannitol 0.335M (calibrated to 0.30-0.32 Osm / kg using an osmometer);

[0050] Preparation: Adjust pH to 6.50 and filter through a 0.22μm PES membrane.

[0051] (iv) Storage / Final Solution Preparation

[0052] Components: Devesicated ED-AWF (determined based on final extraction efficiency) 25% v / v, ethanesulfonic acid 15mM, trehalose 25mM, carnosine 2mM, mannitol to supplement and adjust total osmotic pressure to 0.30-0.32 Osm / kg;

[0053] Preparation: Adjust pH to 6.50 and filter through a 0.22μm PES membrane.

[0054] II. Precision Preparation of Devected ED-AWF

[0055] (a) Infiltration steps

[0056] Procedure: Remove the midrib from leaves of the same species (lotus / ginkgo) or different species (lettuce), completely immerse them in cold IB solution, and apply a vacuum to -0.09±0.01 MPa (gauge pressure). Maintain this pressure for 5 minutes, then restore it to 0.1 MPa (atmospheric pressure) to promote the penetration of IB solution into the interstitial spaces of the leaves (i.e., 100 mL of cold IB solution corresponds to 100 g of leaves; after removing the midrib, the leaf weight refers to the net leaf weight, excluding coarse and hard veins). This example uses ginkgo leaves.

[0057] Temperature: Operate at 4℃ throughout.

[0058] (ii) Extrusion of AWF

[0059] Centrifugation parameters: 1000×g, 4℃ for 15 min, collect the lower exudate;

[0060] (III) Clarification Steps

[0061] Centrifugation parameters: Centrifuge at 500×g, 4℃ for 10 min sequentially → centrifuge at 3000×g, 4℃ for 15 min → centrifuge at 10,000×g, 4℃ for 30 min to gradually remove cell debris and large particles, and collect the supernatant.

[0062] (iv) Devesicle removal steps

[0063] Centrifugation parameters: 100,000×g, 4℃ for 90 min, collect the supernatant; repeat centrifugation at 100,000×g, 4℃ for 60 min to completely remove the vesicles in the AWF.

[0064] (v) Polishing, filtration and phenol / color removal

[0065] Filtration parameters: 0.45μm pre-filtration → 0.10μm polishing filtration to remove residual minute impurities;

[0066] Phenol removal treatment: Add 1% w / v PVPP + 30g / L XAD-7HP (wet weight), shake gently at 4℃ for 10min, and centrifuge at 500×g and 4℃ for 5min to remove the adsorbent;

[0067] Standardization: Detect pH and osmotic pressure, finely adjust to pH 6.50±0.05 and osmotic pressure 0.30-0.32Osm / kg using 1M MES / NaOH, 1.50M mannitol or sterile water, and store at 4℃ for ≤24h after final filtration at 0.22μm.

[0068] Example 2

[0069] The reagent preparation is as described in Example 1.

[0070] (a) Pretreatment of lotus leaf samples

[0071] Sampling: Fresh lotus leaves ≤24h after collection, free from mold and insect infestation;

[0072] Cleaning and disinfection: Rinse 3 times with sterile water (1 minute each time) → Soak in 75% ethanol for 30 seconds → Rinse 5 times with sterile water, and dry the surface with sterile filter paper.

[0073] Cut into pieces: Cut into 0.5cm x 0.5cm pieces, place on ice and set aside, weighing precisely 100g.

[0074] (ii) Activation with polyphenol removal reagent

[0075] PVPP activation: Add 10g PVPP to 50mL EPB, stir magnetically for 10min → centrifuge at 5000×g, 4℃ for 5min, discard the supernatant; wash twice with EPB to remove residual impurities;

[0076] XAD-7HP activation: Add 5g of wet resin to 50mL of EPB, soak for 30min, then stir magnetically for 10min → centrifuge at 5000×g, 4℃ for 5min, and discard the supernatant; wash 3 times with EPB to ensure that the activated resin is compatible with the EPB system.

[0077] (III) Homogenization and removal of polyphenols / anthocyanins

[0078] Homogenization parameters: Add 100 mL of EPB to 100 g of lotus leaf pieces and homogenize at 18,000 rpm for 3 x 30 s (30 s interval) in an ice bath.

[0079] Phenol removal treatment: Add 5% w / v activated PVPP and 30g / L XAD-7HP to the homogenate and shake gently at 4℃ for 15min (100rpm).

[0080] Remove adsorbent: Centrifuge at 8000×g, 4℃ for 20min, take the supernatant and filter it through a 0.45μm filter membrane to obtain a clear homogenate.

[0081] (iv) Differential centrifugation for impurity removal

[0082] Centrifugation parameters: 1000×g, 4℃ for 10 min → 3000×g, 4℃ for 30 min → 20,000×g, 4℃ for 60 min. The supernatant was filtered through a 0.22μm filter membrane to obtain crude LDEVs extract.

[0083] (V) TFF purification

[0084] Membrane specifications: 300kDa MWCO polyethersulfone (PES) hollow fiber membrane, membrane area 0.1m²;

[0085] Operating parameters: The flow path is designed as a circulating cross-flow mode; rinse 3 times with EPB and pre-cool at 4℃; concentration: pump in the crude extract, control the transmembrane pressure at 0.5-0.8 bar and the cross-flow rate at 15 mL / min, and concentrate to 1 / 10 of the original volume; replacement / dialysis: continuously replace 8 volumes (8 DVs) with EPB until the system conductivity and A280 value are stable, and collect the concentrate.

[0086] (vi) SEC polishing

[0087] Equipment configuration: The SEC system is equipped with a UV detector (wavelength 280nm) and a nanoparticle tracking analyzer (MalvernZetaView).

[0088] Elution volume range: Preliminary experiments have determined that the elution volume of LDEVs on the target SEC column (qEVoriginal column) is 15-25 mL;

[0089] Sample loading and elution: Load TFF concentrate according to column specifications, elute at a flow rate of 0.5 mL / min, and monitor the UV absorption peak and NTA particle size distribution in real time.

[0090] Collection and Validation: Collect the elution peak fractions corresponding to particle sizes of 50-200 nm, and use offline NTA (Malvern NanoSight) to confirm the particle size distribution, concentration and purity after merging, ensuring that the target vesicle ratio is ≥95%; one volume can be replaced with SEC mobile phase as needed.

[0091] (vii) UC washing and final dispensing

[0092] Centrifugation parameters: Aliquot the combined SEC peak into ultracentrifuge tubes, centrifuge at 100,000×g, 4℃ for 60 min (SW32Ti rotor, acceleration / deceleration = 5), discard the supernatant; gently resuspend the precipitate in a small amount of stock solution, combine with the supernatant in the same centrifuge tube; centrifuge again at 100,000×g, 4℃ for 60 min, discard the supernatant; resuspend the precipitate in stock solution to the target volume (10 mL), aliquot under ultra-clean conditions;

[0093] Storage conditions: Store at 4℃ for ≤14 days, or freeze once at -80℃ (thaw at 4℃ throughout, and do not repeatedly freeze and thaw).

[0094] Experimental Example

[0095] 1. Experimental Methods

[0096] Based on the schemes described in Examples 1 and 2, the following comparative test experiments were conducted:

[0097] Devescarinated ED-AWF from the same species (lotus leaf source) and devescarinated ED-AWF from a different species (lettuce source) were used as extract components, with volume ratios of 10% v / v, 25% v / v, and 50% v / v, respectively. A standard PBS group was used as a control to determine the optimal ED-AWF addition ratio. The number of LDEVs per 100g of lotus leaf raw material was quantitatively analyzed, and morphological characterization was performed using transmission electron microscopy (TEM). The results are shown in Table 1 and [Table data missing]. Figure 1 As shown.

[0098] Table 1 Quantitative results of LDEV particle number in each group

[0099] Group Extract composition and ratio LDEVs particle count / 100 g (mean ± SD) control group PBS <![CDATA[5.2×10 10 ± 4.3×10 9 particles]]> Same type ED-AWF 10% group Lotus leaf ED-AWF 10% v / v <![CDATA[8.5×10 10 ±6.7×10 9 particles]]> Same type ED-AWF 25% group Lotus leaf ED-AWF 25% v / v <![CDATA[1.9×10 11 ± 8.2×10 9 particles]]> Same type ED-AWF 50% group Lotus leaf ED-AWF 50% v / v <![CDATA[1.3×10 10 ±7.0×10 9 particles]]> Heterogeneous ED-AWF 10% group Lettuce source ED-AWF 50% v / v <![CDATA[7.8×10 10 ± 5.7×10 9 particles]]> Heterogeneous ED-AWF 25% group Lettuce source ED-AWF 50% v / v <![CDATA[2.3×10 11 ± 7.7×10 9 particles]]> Heterogeneous ED-AWF 50% group Lettuce source ED-AWF 50% v / v <![CDATA[5.2×10 10 ± 6.4×10 9 particles]]>

[0100] The results above show that when xenogeneic devescapulomorphized ED-AWF is used as the core component of the extract at a ratio of 25% v / v, the number of lotus leaf extravesicles (LDEVs) extracted per 100g of lotus leaf raw material reaches the highest level, which is 2.3×10¹¹ ± 7.7×10. 9 Electron microscopy results showed that the particles had a uniform particle size distribution. Compared with the control group, the heterologous ED-AWF (25%) group had a richer number of exovesicles with more intact morphology (mainly characterized by typical double-membrane vesicle structures). The core mechanism is that, compared with homologous devestigmatized ED-AWF, heterologous devestigmatized ED-AWF (such as lettuce) has milder chemical properties, forming an "inert" extraction environment with low phenol content, weak oxidizing properties, stable ion balance, and low enzyme activity. This reduces oxidative damage to the LDEV membrane structure and provides stable protection for the vesicles through its natural matrix (such as compatible polysaccharides and amino acids). At the same time, heterologous ED-AWF has a wide range of sources and higher batch stability, further ensuring extraction efficiency. In summary, 25% v / v heterologous devestigmatized ED-AWF is the optimal ratio to balance matrix protection and impurity interference, achieving efficient enrichment and structural preservation of LDEVs.

[0101] Optimization of the proportion of anti-aggregation components in leaf-like exovesicles:

[0102] 1. Experimental Methods

[0103] The extraction solution was designed with "synergistic effect of anti-aggregation and matrix stabilization" as the core, and the experimental design approach of "fixed core components and variable gradient optimization" was adopted. The specific scheme is as follows:

[0104] Fixed core ingredients and final concentrations: 0.30M mannitol (osmotic pressure regulator), 15mM ethanesulfonic acid (pH buffer), 2mM carnosine (antioxidant).

[0105] Optimized variables: inositol (10mM, 15mM), trehalose (15mM, 25mM), and finally introduced the optimal extraction matrix verified in Example 1—25% v / v heterologous (lettuce-derived) devesicated ED-AWF, while setting up a blank control group. The group design is shown in Tables 2-1 and 2-3.

[0106] Table 2-1 Group Design

[0107] Group Element control group PBS Fixed component control group 0.30M mannitol, 15mM ethanesulfonic acid, 2mM carnosine Experimental group 1 0.30M mannitol, 15mM ethanesulfonic acid, 2mM carnosine, 10mM inositol, 15mM trehalose Experimental group 2 0.30M mannitol, 15mM ethanesulfonic acid, 2mM carnosine, 10mM inositol, 25mM trehalose Experimental group 3 0.30M mannitol, 15mM ethanesulfonic acid, 2mM carnosine, 15mM inositol, 15mM trehalose Experimental group 4 0.30M mannitol, 15mM ethanesulfonic acid, 2mM carnosine, 15mM inositol, 25mM trehalose

[0108] In the experimental procedure, the LDEVs extracted from each combination of extracts were uniformly adjusted to the same initial concentration (1×10¹¹ particles / mL) and then stored in a sealed container at 4℃. Samples were taken at four time points: 0 days (initial state), 7 days (short-term storage), 14 days (medium- to long-term storage), and 28 days (long-term storage). The average particle size was measured using a nanoparticle tracking analyzer (Malvern NanoSight NS300), and the zeta potential was measured using a zeta potential analyzer (Malvern Zetasizer Nano ZS). The results are as follows: Figures 2-3 As shown in Tables 2-2 and 2-4.

[0109] Table 2-2 Time-dynamic effects of different component combinations on the particle size stability of LDEVs

[0110] Group / Time Point 0d average particle size (nm) 7-day average particle size (nm) 14d average particle size (nm) 28-day average particle size (nm) control group 165.4±4.1 193.5±6.2 213.0±8.5 225.5±8.8 Fixed component control group 160.5±3.8 170.3±5.7 179.2±7.1 195.0±8.3 Experimental group 1 138.3±4.0 148.2±4.8 175.7±6.3 189.0±6.8 Experimental group 2 135.8±3.9 155.5±4.2 179.3±5.6 186.2±6.3 Experimental group 3 145.5±4.2 156.0±4.5 173.5±5.1 193.4±6.9 Experimental group 4 123.2±3.7 137.4±5.5 155.5±7.5 163.0±6.4

[0111] The results show that different component combinations significantly regulate the particle size stability of LDEVs. The experimental groups (especially experimental group 4) exhibited a more concentrated particle size distribution and a significantly higher proportion of small particles compared to the control group. Furthermore, while the control group showed a sustained and substantial increase in particle size over time (0-28 days), the increase in the fixed-component control group was significantly reduced. The particle size growth in all experimental groups (especially experimental group 4) further slowed down, with experimental group 4 showing an average particle size of 163.0 ± 6.4 nm at 28 days, far lower than the control group's 225.5 ± 8.8 nm. The core mechanism lies in the fact that the experimental components (inositol, trehalose, etc.) effectively inhibited LDEV aggregation by maintaining colloidal osmotic pressure and stabilizing the membrane structure. Optimization of component concentration and ratio (experimental group 4) further enhanced this stabilizing effect, achieving particle size stability of LDEVs during long-term storage. In summary, the component combination in experimental group 4 represents the optimal solution for balancing the synergistic effect of LDEV particle size stability and anti-aggregation components, laying the groundwork for future experiments.

[0112] Table 2-3 Group Design

[0113] Group Element control group PBS Experimental group 1 0.30M mannitol, 15mM ethanesulfonic acid, 2mM carnosine, 15mM inositol, 25mM trehalose Experimental group 2 0.30M mannitol, 15mM ethanesulfonic acid, 2mM carnosine, 10mM inositol, 15mM trehalose, 25% v / v heterologous ED-AWF

[0114] Table 2-4 Time-dynamic effects of different component combinations on the particle size stability of LDEVs

[0115] Group / Time Point 0d average particle size (nm) 7-day average particle size (nm) 14d average particle size (nm) 28-day average particle size (nm) control group 168.2±4.3 196.8±6.5 216.5±8.7 228.3±6.5 Experimental group 1 128.6±4.0 142.3±5.1 156.8±7.0 165.5±6.1 Experimental group 2 120.5±3.5 129.2±4.8 135.3±6.2 142.1±5.7

[0116] The results above show that the optimized scheme (0.30M mannitol, 15mM ethanesulfonic acid, 2mM carnosine, 10mM inositol, 15mM trehalose, 25% v / v heterogeneous ED-AWF) is a better scheme to balance the particle size stability of LDEVs, the regulation of zeta potential and the anti-aggregation effect.

[0117] Enhancement of physiological activity of leaf exovesicles based on optimal combination of extracted components and its application in high-fat and endothelial cell models.

[0118] 1. Experimental Methods

[0119] 1.1 Lotus leaf exovesicles as a model for treating fatty liver disease (NAFLD)

[0120] To investigate the intervention effects of lotus leaf vesicles prepared with different extraction reagents on non-alcoholic fatty liver disease (NAFLD), this experiment used 6-8 week old male C57BL / 6 mice (Vitollife). In the experimental groups, except for the normal control group, all other mice were fed a high-fat diet (Research Diets, catalog number D12492, 60% fat energy) for 8 consecutive weeks to induce the NAFLD model. The model group, vesicle intervention group (PBS), and vesicle intervention group (ED-AWF) were established using the optimal combination of extraction components and the standardized operating procedure (SOP) for leaf vesicle extraction. Suspensions of appropriate volumes were prepared with sterile PBS, and each administration was freshly prepared. The control group received an equal volume of PBS. After 8 weeks of modeling, the model mice were treated with 800 µg / kg lotus leaf vesicles via tail vein injection for 4 weeks, administered every other day via tail vein injection. During this period, the model group mice maintained a high-fat diet; the control group and the vesicle intervention group received PBS as the extraction solution. The experimental endpoint was assessed by observing hepatocyte morphology with HE staining, observing lipid droplet deposition with Oil Red O staining, and measuring triglycerides (TRIGL), low-density lipoprotein cholesterol (LDLC), and cholesterol (CHOL) to evaluate the effect of lotus leaf exovesicles on NAFLD. Results are shown below. Figure 4 .

[0121] The above results indicate that the control group showed clear liver tissue structure and normal hepatocyte morphology; the model group showed extensive fatty degeneration of hepatocytes with widespread lipid droplet accumulation (vacuolization visible in HE staining and numerous red lipid droplets in Oil Red O staining); the hepatocyte fatty degeneration and lipid droplet deposition in the external vesicle intervention group (PBS) were somewhat improved, but a large number of lipid droplets were still present; the hepatocyte morphology in the external vesicle intervention group (ED-AWF) was close to normal, and lipid droplet deposition was significantly reduced, with a much better improvement in liver pathology than other groups. The model group showed significantly elevated levels of triglycerides (TRIGL), low-density lipoprotein cholesterol (LDLC), and total cholesterol (CHOL) (P<0.05, **P<0.001, ***P<0.0001); the external vesicle intervention group (ED-AWF) showed significantly lower levels of TRIGL, LDLC, and CHOL compared to the model group and the external vesicle intervention group (PBS), suggesting that the physiological activity of lotus leaf external vesicles protected by the optimal component combination can effectively regulate lipid metabolism disorders in the NAFLD model.

[0122] 1.2 Synergistic effect experiment of Ginkgo biloba exovesicles on promoting endothelial cell proliferation

[0123] Ginkgo biloba extracts (such as flavonoids and ginkgolides) have been shown to have pharmacological activities that improve vascular function and resist atherosclerosis. To investigate the effects of ginkgo biloba vesicles prepared with different extraction reagents on the migration ability of endothelial cells, this experiment used a cell scratch assay. The experiment included a control group, an external vesicle intervention group (PBS), and an external vesicle intervention group (ED-AWF). The external vesicles used in the ED-AWF group were prepared using the optimal combination of the above-mentioned extract components combined with the standardized operating procedure (SOP) for extracting leaf-type external vesicles. Human umbilical vein endothelial cells (HUVECs) were purchased from Wuhan Shangen Biotechnology and cultured in endothelial cell culture medium (ECM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics, and passaged in a constant temperature incubator at 37 °C and 5% CO2. Before the experiment, cells were seeded in 6-well plates and allowed to grow to 90%–100% confluence. Using a sterile 200 µL pipette tip, a straight, uniform scratch was made along the center of the cell monolayer. Cells were then gently washed twice with PBS to remove detached cells, and immediately replaced with serum-free medium to eliminate interference from cell proliferation factors. Working solutions for each group of Ginkgo biloba vesicles were prepared and adjusted to a final concentration of 50 µg / mL (based on total protein content of the vesicles). 2 mL of working medium containing the corresponding vesicles was added to each well of each group, and the mixture was gently shaken to ensure even distribution of the vesicles. At 0 h, 6 h, 12 h, 18 h, and 24 h after scratching, the same scratched area was photographed using an inverted microscope. ImageJ software was used to quantitatively analyze the scratch healing area. Results are shown below. Figure 5 .

[0124] The results of the cell scratch assay showed that the scratch healing rate of the external vesicle intervention group (ED-AWF) was significantly faster than that of the control group and the external vesicle intervention group (PBS). In terms of the relative scratch density trend, the relative scratch density of the external vesicle intervention group (ED-AWF) was significantly higher than that of other groups at 6h, 12h, 18h, and 24h, indicating that Ginkgo external vesicles protected by the optimal component combination can significantly promote the migration and proliferation of human umbilical vein endothelial cells (HUVECs), demonstrating a significant synergistic effect.

[0125] Based on the above-mentioned optimized combination of extracts from leaf-like exovesicles, the physiological activities of lotus leaf and ginkgo exovesicles can be effectively enhanced: in the NAFLD model, lotus leaf exovesicles can significantly improve liver pathological damage and lipid metabolism disorders; in the endothelial cell model, ginkgo exovesicles can efficiently promote endothelial cell proliferation and migration.

[0126] As demonstrated by the above embodiments, this invention provides an optimized overall scheme for the extraction and preservation of leaf-like exovesicles. This scheme determines the optimal ratio of devesicated exosome fluid from a different species as the core extraction matrix and screens out key component combinations that synergistically maintain vesicle particle size stability and resist aggregation effects. Lotus leaf and ginkgo exovesicles extracted using this scheme exhibit superior physiological activity and therapeutic efficacy compared to exovesicles prepared using traditional methods in both animal disease models and cell function models.

[0127] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for extracting leaf-like exovesicles, characterized in that, The extraction method uses a extract containing devesaccharified exosomes from a different plant species, and the volume ratio of the devesaccharified exosomes from the different plant species in the extract is 20-30% v / v.

2. The extraction method as described in claim 1, characterized in that, The extract also includes osmotic pressure regulators, pH buffers, antioxidants, and anti-aggregation agents.

3. The extraction method as described in claim 2, characterized in that, The osmotic pressure regulator is mannitol, with a final concentration of 0.28~0.32 M.

4. The extraction method as described in claim 2, characterized in that, The pH buffer is ethanesulfonic acid, with a final concentration of 10-20 mM.

5. The extraction method as described in claim 2, characterized in that, The antioxidant is carnosine, with a final concentration of 1-3 mM.

6. The extraction method as described in claim 2, characterized in that, The anti-aggregation agent includes inositol and trehalose, wherein the final concentration of inositol is 5-20 mM and the final concentration of trehalose is 10-30 mM.

7. The extraction method as described in claim 1, characterized in that, The preparation of the devesicated extraplasmic fluid includes the following steps: Plant leaves were immersed in an infiltration buffer and vacuumed. The exudate was collected by centrifugation, clarified by step centrifugation, and vesicles were removed by ultracentrifugation. The solution was then filtered and treated to remove phenol.

8. The extraction method as described in claim 7, characterized in that, The infiltration buffer contains mannitol and ethanesulfonic acid, with a pH of 6.45 to 6.

55.

9. The extraction method as described in claim 1, characterized in that, It also includes pretreatment of leaf samples, including cleaning, disinfection, cutting, and weighing; It also includes a homogenization step, in which the extract is added and homogenized in an ice bath. It also includes a purification step, which includes differential centrifugation for impurity removal, tangential flow filtration concentration, and size exclusion chromatography separation.

10. The extraction method as described in claim 9, characterized in that, In the size exclusion chromatography separation, fractions with an elution volume of 15-25 mL are collected, corresponding to vesicles with a particle size of 50-200 nm.

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