Methods of processing ginseng stem leaf exosome-like vesicles, compositions, and dressings

Abstract

The application embodiment provides a processing method of ginseng stem and leaf exosome-like vesicles, comprising the following steps: step 1, taking ginseng stems and leaves, crushing the ginseng stems and leaves to obtain a homogenate; step 2, subjecting the homogenate to multistage purification to obtain a vesicle layer; and step 3, based on the vesicle layer, performing a stabilization treatment to obtain a ginseng stem and leaf exosome-like vesicle freeze-dried powder. After the stabilization treatment in step 3, the ginseng stem and leaf exosome-like vesicle freeze-dried powder is obtained, the technical effect of obtaining a high-stability product after purifying the ginseng stem and leaf exosome-like vesicles is achieved, the application provides technical support for batch production of high-quality ginseng stem and leaf exosome products, and can be applied to industrial production.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to methods, compositions, and dressings for processing exosome-like vesicles from ginseng stem and leaf cells. Background Technology

[0002] Ginseng stems and leaves are the main byproducts of ginseng cultivation (accounting for 60%–70% of the plant's weight), but their current utilization rate is less than 30%, resulting in a serious waste of natural medicinal resources. Ginseng exosome-like vesicles (20 nm–200 nm in diameter), as natural nanocarriers, have advantages such as low immunogenicity and high biocompatibility, showing great potential in skin repair and disease treatment.

[0003] In existing technologies, the storage methods for ginseng cell exosome-like vesicle products are prone to membrane fusion during storage due to various reasons, such as the lack of vesicle membrane protectants. This results in unstable products and easy loss of activity. Summary of the Invention

[0004] This application provides a method, composition, and dressing for processing ginseng stem and leaf cell exosome-like vesicles, thereby achieving the technical effect of ensuring good product stability after purifying the ginseng stem and leaf cell exosome-like vesicles.

[0005] In a first aspect, embodiments of this application provide a method for processing exosome-like vesicles from ginseng stem and leaf cells, comprising the following steps:

[0006] Step 1: Take ginseng stems and leaves and crush them. Prepare a homogenate based on the crushed ginseng stems and leaves.

[0007] Step 2: The homogenate is subjected to multiple purification stages to obtain the vesicle layer;

[0008] Step 3: Stabilize the vesicle layer to obtain lyophilized ginseng stem and leaf cell exosome-like vesicle powder.

[0009] In one possible implementation, step 1 specifically includes:

[0010] After pulverizing ginseng stems and leaves, add a compound enzymatic hydrolysate and enzymatically hydrolyze at 37 ℃ for 2 hours. Then, perform ultrasonication in an ice bath at 0 ℃~4 ℃ for 10 minutes and filter to obtain a homogenate. The compound enzymatic hydrolysate consists of 0.2% cellulase and 0.1% pectinase.

[0011] In one possible implementation, step 2 specifically includes:

[0012] The homogenate was subjected to primary, secondary, and tertiary purification. Primary purification involved centrifugation followed by collection of the supernatant. Centrifugation conditions were: 4 °C, 8000 g centrifugation force, and 15 minutes. Secondary purification involved filtration through a 0.22 μm polyethersulfone membrane to obtain the filtrate. Tertiary purification involved mixing the filtrate with a 30% sucrose solution at a 1:1 volume ratio at 4 °C and centrifuging at a density gradient of 100,000 g for 2 hours to obtain the vesicle layer.

[0013] In one possible implementation, step 3 specifically includes:

[0014] After washing the vesicle layer with PBS, it was mixed with a lyophilization protectant and pre-frozen at -80 °C for 2 hours. Then, it was freeze-dried at -50 °C under vacuum of 10 Pa for 12 hours to obtain lyophilized ginseng stem and leaf cell exosome-like vesicle powder. The lyophilization protectant contained 5% sucrose and 2% mannitol.

[0015] In one possible implementation, the method further includes: step 4: preparing a skin-repairing composition based on the lyophilized powder of ginseng stem and leaf cell exosome-like vesicles.

[0016] In one possible implementation, step 4 specifically includes:

[0017] Prepare a ginseng stem and leaf cell exosome vesicle suspension from the freeze-dried powder of the ginseng stem and leaf cell exosome vesicles.

[0018] A 5% (w / v) to 20% (w / v) prepolymer solution was prepared by dissolving the methacrylamide gelatin hydrogel carrier in a PBS solution containing 0.25% (w / v) LAP photoinitiator at 40℃ to 50℃ in the dark.

[0019] The prepolymer solution was sterilized by filtration through a 0.22 μm microporous membrane and then mixed with a suspension of ginseng stem and leaf cell exosomes at a volume ratio of 1:5. The mixture was thoroughly mixed at 37 °C in the dark, and then injected into a well plate with an intensity of 6 mW·cm⁻¹. -2 ~10 mW·cm -2 Photocrosslinking and curing are completed by irradiation with a 405 nm wavelength light source for 30 seconds.

[0020] Secondly, embodiments of this application provide a skin-repairing composition obtained by the processing method described in the first aspect.

[0021] Thirdly, embodiments of this application provide a skin-repairing dressing, the components of which include the skin-repairing composition described in the second aspect.

[0022] Fourthly, embodiments of this application provide a lyophilized powder of ginseng stem and leaf cell exosome-like vesicles, obtained by the processing method described in the first aspect.

[0023] Fifthly, embodiments of this application provide a skin-repairing dressing, the components of which include the ginseng stem and leaf cell exosome-like vesicle dry powder described in the fourth aspect, wherein the dressing is one of a face cream or a serum.

[0024] This application provides a method for processing extracellular vesicles derived from ginseng stems and leaves, comprising: Step 1: pulverizing ginseng stems and leaves to obtain a homogenate; Step 2: subjecting the homogenate to multi-stage purification to obtain a vesicle layer; Step 3: stabilizing the vesicle layer to obtain a lyophilized powder of ginseng stem and leaf exosome-like vesicles. The stabilization process in Step 3 yields a lyophilized powder of ginseng stem and leaf exosome-like vesicles, achieving the technical effect of obtaining a highly stable product after purification of ginseng stem and leaf exosome-like vesicles. Attached Figure Description

[0025] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0026] Figure 1 A schematic flowchart illustrating a method for processing exosome-like vesicles from ginseng stem and leaf cells, provided in an embodiment of this application;

[0027] Figure 2 Electron micrographs of exosome-like vesicles obtained by the method for extracting exosome-like vesicles from ginseng stems and leaves provided in this application and by traditional methods for extracting exosome-like vesicles from plant sources;

[0028] Figure 3 Comparison of nanoparticle counts of the same mass of extracts from ginseng stem and leaf exosome-like vesicles obtained using the method for extracting exosome-like vesicles from ginseng stems and leaves provided in this application and traditional plant-derived exosome-like vesicle extraction methods;

[0029] Figure 4 The ZETA potential diagram of the ginseng stem and leaf cell exosome-like vesicles obtained by the extraction method of ginseng stem and leaf cells provided in this application and the traditional plant-derived extracellular vesicle extraction method was obtained by physicochemical property analysis using NTA.

[0030] Figure 5 The stability test results of ginseng stem and leaf cell exosome-like vesicles obtained by the ginseng stem and leaf cell exosome-like vesicle extraction method provided in this application after storage in PBS solution at 4℃ for 7 days are shown in the figure.

[0031] Figure 6The stability test results of ginseng stem and leaf cell exosome-like vesicles obtained by the ginseng stem and leaf cell exosome-like vesicle extraction method provided in this application after a freeze-drying-re-thawing process are shown in the figure.

[0032] Figure 7 The molecular weight distribution diagram of exosome-like vesicle proteins in ginseng stem and leaf cells provided for this application;

[0033] Figure 8 This application provides a distribution map of the coverage of exosome-like vesicle protein peptides in ginseng stem and leaf cells.

[0034] Figure 9 The biological process analysis diagram for GO enrichment analysis of exosome-like vesicle proteins in ginseng stem and leaf cells provided in this application;

[0035] Figure 10 The cellular component analysis diagram of GO enrichment analysis of exosome-like vesicle proteins in ginseng stem and leaf cells provided for this application;

[0036] Figure 11 The molecular functional analysis diagram of GO enrichment analysis of exosome-like vesicle proteins in ginseng stem and leaf cells provided for this application;

[0037] Figure 12 The KEGG functional enrichment analysis diagram of ginseng stem and leaf cell exosome-like vesicle protein provided in this application;

[0038] Figure 13 Fluorescence microscopy images of the ability of ginseng stem and leaf cells to reduce ROS by exosome-like vesicles provided in this application;

[0039] Figure 14 A quantitative statistical chart showing the ability of ginseng stem and leaf cell exosome-like vesicles to reduce ROS, provided in this application;

[0040] Figure 15 The effect of ginseng stem and leaf cell exosome-like vesicles on MDA levels provided in this application;

[0041] Figure 16 A diagram illustrating the effect of ginseng stem and leaf cell exosome-like vesicles on SOD content, provided in this application.

[0042] Figure 17 A diagram illustrating the effect of ginseng stem and leaf cell exosome-like vesicles on GSH-Px activity, provided in this application.

[0043] Figure 18 HE staining image of ginseng stem and leaf cell exosome-like vesicles promoting the repair of diabetic skin lesions, provided for this application;

[0044] Figure 19 Immunofluorescence image of anti-Tnf-α exosome-like vesicles from ginseng stem and leaf cells provided in this application;

[0045] Figure 20 A pathological scoring statistical chart showing that ginseng stem and leaf cell exosome-like vesicles promote the repair of diabetic skin lesions, provided in this application;

[0046] Figure 21 A quantitative statistical chart of the anti-Tnf-α activity of ginseng stem and leaf cell exosome-like vesicles provided in this application.

[0047] The accompanying drawings illustrate specific embodiments of this application, which will be described in more detail below. These drawings and descriptions are not intended to limit the scope of the concept in any way, but rather to illustrate the concept of this application to those skilled in the art through reference to particular embodiments. Detailed Implementation

[0048] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of systems and methods consistent with some aspects of this application as detailed in the appended claims.

[0049] In this invention, "exosome-like vesicles" refers to nanoscale extracellular vesicles secreted by cells into the extracellular space. The vesicles include exosomes and are used in the broadest sense, encompassing vesicles similar to exosomes in nanoscale vesicular structures and compositions.

[0050] In this invention, "ginseng stem and leaf cell exosome-like vesicles" refers to nanoscale exosome-like vesicles secreted by ginseng stem and leaf cells. These ginseng stem and leaf cell exosome-like vesicles can be separated from ginseng extracellular fluid, and can be completely or partially separated from existing tissues or cells.

[0051] Please see Figure 1 The present application provides a method for processing exosome-like vesicles from ginseng stem and leaf cells, comprising the following steps:

[0052] Step S101: Take ginseng stems and leaves and crush them. Prepare a homogenate based on the crushed ginseng stems and leaves.

[0053] It should be noted that the ginseng stems and leaves described in the embodiments of this application are fresh stems and leaves.

[0054] Step S102: The homogenate is subjected to multiple stages of purification to obtain the vesicle layer;

[0055] Step S103: Stabilize the vesicle layer to obtain lyophilized powder of ginseng stem and leaf cell exosomes;

[0056] Furthermore, the processing method may also include step S104: preparing a skin-repairing composition based on lyophilized ginseng stem and leaf cell exosome-like vesicle powder.

[0057] In one possible implementation, step S101 specifically includes:

[0058] After pulverizing ginseng stems and leaves, a compound enzymatic hydrolysate was added and hydrolyzed at 37 ℃ for 2 hours. Then, the mixture was subjected to ultrasonic treatment in an ice bath at 0 ℃–4 ℃ for 10 minutes. The resulting homogenate was filtered. The compound enzymatic hydrolysate contained 0.2% cellulase and 0.1% pectinase. Furthermore, the pulverized ginseng stems and leaves could be passed through a 60-mesh sieve.

[0059] The extracted ginseng stem and leaf cell exosome-like vesicles meet the following criteria: number of particles per unit mass ≥ 5 × 10⁻⁶ 8 / μg, with a clean background and uniform morphology under transmission electron microscopy, and hydrodynamic diameters ranging from 30 nm to 200 nm as determined by nanoparticle tracking analysis (NTA). The physicochemical properties are stable after 7 days of storage in phosphate-buffered saline (PBS) at 4 °C or 30 days of sealed storage at -20 °C (after freeze-drying and thawing). The proteomic characteristics of exosome-like vesicles from ginseng stem and leaf cells are as follows: they are rich in proteins with a wide molecular weight coverage, with 20kD-40kD proteins accounting for about 33%, 400kD-60kD proteins accounting for about 25%, and high molecular weight proteins above 100kD accounting for less than 10%, and the peptide coverage of most identified proteins is less than 50. These proteins have biological functions such as regulating cellular redox homeostasis, vesicle-mediated transport, GTPase-mediated signal transduction, proteolysis, intracellular protein transport, carbohydrate metabolism, protein phosphorylation, and redox processes. They also have molecular functions such as zinc ion binding, GTP binding, RNA binding, redox enzyme activity, chemical catalytic activity, nuclear protein kinase activity, nucleic acid binding, and ATP binding.

[0060] Ginseng stem and leaf cell exosome-like vesicles meet the following in vitro antioxidant capacity:

[0061] （1 ）活性氧（Reactive Oxygen Species, ROS）：以1×106个 / 孔密度接种RAW 264.7巨噬细胞，经4 mmol·L-1 H2O2处理 4小时 诱导氧化应激后，分别用1 μg ·mL -1 5 μg·mL -1 10 μg·mL -1When ginseng stem and leaf cell exosome-like vesicles were treated at the concentrations mentioned above for 12 hours, the ROS levels were reduced by approximately 30%, 50%, and 70% or more, respectively, compared to the PBS control group without the addition of ginseng stem and leaf cell exosome-like vesicles, and this reduction was dose-dependent.

[0062] (2) Regulation of oxidative stress-related indicators: Under the above intervention conditions, compared with the PBS control group, the ginseng stem and leaf cell exosome-like vesicles obtained by the method provided in the embodiments of this application can reduce the level of malondialdehyde (MDA), a lipid peroxidation product, while increasing the content of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), among which the medium and high doses (5 μg·mL) can reduce the level of malondialdehyde (MDA), a lipid peroxidation product, while increasing the content of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). -1 10 μg·mL -1 It has the most significant effect on reducing lipid peroxidation.

[0063] In one possible implementation, step S102 specifically includes:

[0064] The homogenate was subjected to primary, secondary, and tertiary purification. Primary purification involved centrifugation, followed by collection of the supernatant. The centrifugation conditions were: 4 °C, 8000 g centrifugation force, and 15 minutes. Secondary purification involved filtration through a 0.22 μm polyethersulfone membrane to obtain the filtrate. Tertiary purification involved mixing the filtrate with a 30% sucrose solution at a 1:1 volume ratio at 4 °C and centrifuging at a density gradient of 100,000 g for 2 hours to obtain the vesicle layer. Density gradient centrifugation establishes a sucrose concentration gradient in the centrifuge tube, causing exosomes to move and remain at a position with the same density as themselves (approximately a 30% sucrose layer) under ultracentrifugal force. This allows them to be separated from protein impurities, cell debris, etc., thus achieving purification.

[0065] In existing technologies, the extraction of ginseng cell exosome-like vesicles is mostly based on purification of ginseng roots. However, according to the embodiments of this application, even when purifying ginseng stems and leaves, which have low medicinal value and are often used as by-products, stable ginseng stem and leaf cell exosome-like vesicle products can be obtained, thereby improving the utilization rate of ginseng stems and leaves as natural medicinal resources. This solution provides technical support for the mass production of high-quality ginseng stem and leaf cell exosomes and can be applied to industrial production.

[0066] In one possible implementation, step S103 specifically includes:

[0067] After washing the vesicle layer with PBS, it was mixed with a freeze-drying protectant containing 5% sucrose and 2% mannitol, pre-frozen at -80 °C for 2 hours, and then freeze-dried at -50 °C under vacuum of 10 Pa for 12 hours to obtain ginseng stem and leaf cell exosome-like vesicle freeze-dried powder. The 5% sucrose mainly reduces damage to the active ingredients from ice crystals and maintains product stability; the 2% mannitol acts as both a freeze-drying protectant and an excipient, helping to protect the active substances and giving the freeze-dried product a loose, easily soluble block or powder appearance.

[0068] In one possible implementation, step S104 specifically includes:

[0069] A suspension of ginseng stem and leaf cell exosome vesicles was prepared by lyophilizing ginseng stem and leaf cell exosome vesicles. A 5%–20% (w / v) prepolymer solution was prepared by dissolving methacrylamide gelatin hydrogel carrier in PBS solution containing 0.25% (w / v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator at 40–50 °C in the dark. The prepolymer solution was sterilized by filtration through a 0.22 μm microporous membrane and then mixed with the ginseng stem and leaf cell exosome vesicle suspension at a volume ratio of 1:5. The mixture was stirred thoroughly at 37 °C in the dark and injected into well plates. The solution was then sprayed with a 6 mW·cm⁻¹ filter. -2 ~10 mW·cm -2 Photocrosslinking and curing are completed by irradiation with a 405 nm wavelength light source for 30 seconds.

[0070] This application also provides a skin repair composition, prepared by steps S101 to S104. This application also provides a skin repair dressing, in which this composition is added. This dressing can be a hydrogel patch, specifically a chronic diabetic wound repair dressing, each patch containing 5 μg to 10 μg of ginseng stem and leaf cell exosome-like vesicles.

[0071] This application also provides a lyophilized powder of ginseng stem and leaf cell exosome-like vesicles, obtained by the processing method described in steps S101 to S103. This application also provides a skin repair dressing, in which the aforementioned ginseng stem and leaf cell exosome-like vesicle powder is added. The dressing is either a face cream or a serum. The face cream or serum is applied to the face 1-2 times daily, with each application consisting of 0.5 mL to 1 mL (serum) or 0.2 g to 0.5 g (face cream).

[0072] The following describes the processing procedure of ginseng stem and leaf cell exosome-like vesicles in the embodiments of this application.

[0073] (1) Raw materials: stems and leaves of 4-year-old ginseng from Huanren, Liaoning (batch number 20230501), with a saponin content of 4.2% as tested;

[0074] It should be noted that this experiment uses fresh ginseng stems and leaves as an example.

[0075] (2) Pretreatment: Take 1 kg of ginseng stems and leaves, crush them through a 60-mesh sieve, add 5 L of compound enzymatic hydrolysate (0.2% cellulase and 0.1% pectinase) to every 500 g of sample, enzymatically hydrolyze at 37 ℃ for 2 hours (microscopic examination showed that 80% of the cell walls were ruptured), sonicate in an ice bath for 10 minutes (temperature 38 ℃), and filter to obtain 4.5 L of homogenate;

[0076] (3) Purification: First-stage purification: The homogenate was centrifuged at 4 ℃ and 8000 g for 15 minutes, and the supernatant was collected; Second-stage purification: The supernatant was filtered through a 0.22 μm polyethersulfone membrane, and the filtrate was collected; Third-stage purification: The filtrate was mixed with 30% sucrose solution at a volume ratio of 1:1, centrifuged at 4 ℃ and 100000 g for 2 hours, and the middle milky white vesicle layer was collected. After washing twice with PBS, it was mixed with 5% sucrose + 2% mannitol, pre-frozen at -80 ℃ for 2 hours, and then transferred to a freeze dryer at -50 ℃ (vacuum degree 10 Pa) for 12 hours to obtain 12.5 mg of lyophilized powder (yield 25 μg·g). -1 );

[0077] (4) Each experiment was repeated 3 times. Data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used for comparison between groups. P <0.05 indicates a statistically significant difference.

[0078] Next, we will further describe the process of acquiring transmission electron microscopy images of exosome-like vesicles from ginseng stem and leaf cells. The specific process is as follows:

[0079] (1) Take the purified ginseng stem and leaf cell exosome-like vesicle sample and dilute it to 1×10⁻⁶. 11 / ml, add 10 μL of sample to the sealing film; invert the carbon-coated nickel mesh onto the droplet and allow it to adsorb for 1 to 5 minutes to allow the ginseng stem and leaf cell exosome-like vesicles to attach;

[0080] (2) After absorbing excess liquid with filter paper, rinse with three drops of 10 μL triple-distilled water for a few seconds (repeat three times) and blot dry; then fix with 2% paraformaldehyde at room temperature for 10 minutes, rinse, and then stain with 2% uranium acetate for 10 minutes to enhance contrast.

[0081] (3) The prepared nickel mesh or slice is loaded into a transmission electron microscope and observed and images are taken under an accelerating voltage of 80 kV to 120 kV.

[0082] (4) Each experiment was repeated 3 times. Data are expressed as mean ± standard deviation. ANOVA was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0083] (5) such as Figure 2 As shown, ginseng stem and leaf cell exosome-like vesicles extracted using traditional methods exhibit more severe contamination from impurities such as soluble proteins and lipids, specifically manifested as large background shadow areas and non-uniform vesicle particle sizes. Compared to traditional extraction methods, the ginseng stem and leaf cell exosome-like vesicles extracted using the method provided in this application embodiment have a clean background and uniform morphology, demonstrating less impurity contamination and a stable yield of cell exosome-like vesicles derived from ginseng stems and leaves.

[0084] Next, we will further introduce the NTA detection process of ginseng stem and leaf cell exosome-like vesicles.

[0085] (1) The obtained ginseng stem and leaf cell exosome-like vesicle precipitate was resuspended in PBS; the suspension was then diluted to approximately 1 μg / mL. -1 Concentration, vortex mixing to avoid aggregation.

[0086] (2) Start NTA, clean the sample cell with deionized water and then inject 100 nm standard particle size beads to calibrate the system accuracy; take 600 μL of diluted sample and inject it into the sample cell (avoid air bubbles), set the parameters: camera sensitivity 5-7, gain 1.0, detection threshold 10, solvent viscosity 0.89 cps (aqueous phase), and capture the Brownian motion trajectory of the particles in 60 seconds × 5 repetitions.

[0087] (3) Calculate the hydrodynamic diameter based on the Stokes-Einstein equation using analysis software (such as ZetaView 8.04.02) to generate a particle size distribution map (typical range 30 nm to 200 nm) and a concentration report.

[0088] (4) Each experiment was repeated 3 times. Data are expressed as mean ± standard deviation. ANOVA was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0089] (5) such as Figure 3 As shown, for the two extraction methods, the same mass of extract was used for nanoparticle counting. The results showed that the number of extracellular vesicles extracted using the extraction method described in this application was greater than 5 × 10⁻⁶. 8 / μg, while traditional extraction methods can only yield approximately 1×10⁶. 8 / μg of vesicle particles. This indicates that the extraction method we adopted in this application effectively reduces contamination from extraneous proteins and improves the purity of the ginseng stem and leaf cell exosome-like vesicle extraction. Figure 4 As shown, the same mass of extracts from both extraction methods were analyzed for physicochemical properties using NTA. ZETA results showed that the extraction method used in this application, derived from ginseng stems and leaves, produced a greater number of exosome-like vesicles with a more concentrated particle size distribution. Compared to traditional extraction methods, this extraction method provides a more stable product yield.

[0090] Next, we will further introduce the process of detecting the stability of exosome-like vesicles in ginseng stem and leaf cells.

[0091] (1) The precipitate of exosome-like vesicles obtained from ginseng stem and leaf cells was resuspended in PBS; stored in a refrigerator at 4 ℃, and NTA was detected at regular intervals every day.

[0092] (2) A 5% sucrose and 2% mannitol mixed freeze-drying protectant was used and mixed with the ginseng stem and leaf cell exosome vesicle suspension at a volume ratio of 1:1. After pre-freezing at -80 ℃ for 2 hours, it was transferred to a -50 ℃ freeze dryer and dried under a vacuum of 10 Pa for 12 hours as a freeze-drying protectant.

[0093] (3) The obtained ginseng stem and leaf cell exosome-like vesicle precipitate was resuspended in PBS; mixed with lyophilization protectant, vortexed and placed in an ultra-low temperature freezer at -80 ℃ for pre-freezing. The pre-frozen sample was quickly transferred to the sample rack of a freeze dryer pre-cooled to below -50 ℃, and the vacuum pump was turned on to obtain a dry white cake or powder lyophilized product. The lyophilized sample was stored in a sealed state at -20 ℃ for 30 days and then thawed; NTA detection was then performed.

[0094] (4) Each experiment was repeated three times. Data are expressed as mean ± standard deviation. ANOVA analysis was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0095] (5) As shown in the figure Figure 5 and Figure 6 As shown, the stability of ginseng stem and leaf cell exosome-like vesicles was tested. The results showed that after 7 days in PBS solution at 4 ℃, the ginseng stem and leaf cell exosome-like vesicles still exhibited stable physicochemical properties; after undergoing a freeze-drying-thawing process, they could be stably stored at -20 ℃ for more than 30 days. This excellent stability gives ginseng stem and leaf cell exosome-like vesicles good market application potential.

[0096] The following section will further introduce the proteomic detection and analysis process of exosome-like vesicles from ginseng stem and leaf cells.

[0097] (1) Detection of exosome-like vesicle proteomics in ginseng stem and leaf cells

[0098] The proteomics sequencing was assisted by Novogene Biotechnology Co., Ltd., and the general steps are as follows:

[0099] ① First, the purified ginseng stem and leaf cell exosome-like vesicle protein precipitate was dissolved in PBS buffer containing 8M urea and subjected to reduction (5 mM tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), incubated at 37 ℃ for 1 hour) and alkylation (10 mM iodoacetamide, room temperature in the dark for 30 minutes); then, trypsin digestion was performed using the filter-aided sample preparation (FASP) method (enzyme:substrate ratio 1:50, incubated at 37 ℃ overnight). After enzymatic hydrolysis, the peptide fragments were desalted using a C18 StageTip microcolumn (equilibrated sequentially with methanol and 0.1% formic acid, followed by elution with acetonitrile-water solution containing 0.1% formic acid (80:20, v / v)). After drying, the following strategies were selected based on quantitative requirements: Label-free quantification: The peptide fragments were directly separated by nano-liquid chromatography (nanoLC) using the Acquity M-class system, with gradient elution of 0.1% formic acid (phase A) and 0.1% formic acid / 80% acetonitrile (phase B) (60 min–120 min, flow rate 300 nL per min).

[0100] ② Mass spectrometry detection was performed using a high-resolution mass spectrometer such as Q Exactive Plus, with data-dependent acquisition (DDA) mode set: first-stage scan resolution 70,000 (m / z 200), scan range 350 m / z~1800 m / z; the top 20 strongest ions were selected for HCD fragmentation (normalized collision energy 28%~32%), and the second-stage scan resolution was 17,500.

[0101] ③ For example Figure 7 and Figure 8 As shown, proteomic analysis of ginseng stem and leaf cell exosome-like vesicles revealed abundant proteins with broad molecular weight coverage. Approximately 33% of the proteins were concentrated between 20 kD and 40 kD, while the remaining 25% were concentrated between 40 kD and 60 kD. High molecular weight proteins were present in relatively low amounts, with proteins exceeding 100 kD accounting for less than 10%. Furthermore, the peptide coverage of most identified proteins from ginseng stem and leaf cell exosome-like vesicles was less than 50, reflecting the complexity of these proteins.

[0102] (2) Functional enrichment analysis

[0103] ① Based on proteomics data, protein identifiers are converted into standard gene identifiers through alignment using the Basic Local Alignment Search Tool for Proteins (BLASTP) or database mapping to ensure compatibility with enrichment analysis tools; then, functional annotation and enrichment analysis are performed using the online platform DAVID—submitting a protein list and setting a background dataset;

[0104] ② The enrichment significance of GO genes (covering three dimensions: biological processes, molecular functions, and cellular components) and the enrichment significance of the KEGG pathway were calculated using hypergeometric tests or Fisher's exact tests. P <0.05 and FDR correction <0.05 are used as thresholds to filter significant items;

[0105] ③ The results are output in a visual format. For example... Figures 9 to 12 As shown, through G0 enrichment analysis and KEGG functional enrichment analysis, we revealed the potential of ginseng stem and leaf cell exosome-like vesicle-forming proteins to regulate cellular redox homeostasis, vesicle-mediated transport, small GTPase-mediated signal transduction, proteolysis, intracellular protein transport, carbohydrate metabolism, redox processes, zinc ion binding, GTP binding, RNA binding, oxidoreductase activity, chemocatalytic activity, and ATP binding. Among these, ginseng stem and leaf cell exosome-like vesicle-forming proteins have significant potential for regulating oxidative stress.

[0106] The following describes the process of detecting the in vitro antioxidant capacity of exosome-like vesicles from ginseng stem and leaf cells.

[0107] (1) The experimental groups are as follows (n = 6)

[0108] PBS group: Normal medium Dulbecco's Modified Eagle's Medium (DMEM) was supplemented with 4 mmol·L⁻¹ of oxidative stress-inducing agent. -1 Macrophages were treated with H2O2 for 4 hours, followed by PBS intervention for 12 hours.

[0109] Low-dose group: 4 mmol·L⁻¹ of oxidative stress-inducing agent was added to normal DMEM culture medium. -1 Macrophages were treated with H2O2 for 4 hours, followed by treatment with a low dose of ginseng stem and leaf cell exosome-like vesicles (1 μg / mL). -1 Intervention treatment was administered for 12 hours at a low dose.

[0110] Medium-dose group: 4 mmol·L⁻¹ of oxidative stress-inducing agent was added to normal DMEM culture medium. -1 Macrophages were treated with H2O2 for 4 hours, followed by treatment with a low dose of ginseng stem and leaf cell exosome-like vesicles (5 μg·mL). -1 Intervention treatment (medium dose) for 12 hours.

[0111] High-dose group: 4 mmol·L⁻¹ of oxidative stress-inducing agent was added to normal DMEM culture medium. -1 Macrophages were treated with H2O2 for 4 hours, followed by treatment with a low dose of ginseng stem and leaf cell exosome-like vesicles (10 μg·mL). -1 Intervention treatment (high dose) for 12 hours.

[0112] (2) Detection of the ability of ginseng stem and leaf cells to reduce ROS by exosome-like vesicles

[0113] ① RAW264.7 macrophages were cultured at a concentration of 1×10⁻⁶. 6 The cells were seeded at a density of 10 cells / well in 6-well plates and pre-cultured for 24 hours before being divided into four groups: PBS treatment group, low-dose treatment group of ginseng stem and leaf cell exosome-like vesicles, medium-dose treatment group of ginseng stem and leaf cell exosome-like vesicles, and high-dose treatment group of ginseng stem and leaf cell exosome-like vesicles.

[0114] ② Discard the original culture medium from adherent cells in good growth condition and gently wash the cells twice with pre-warmed PBS. Next, dilute the stock solution of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) 1000 times to the working concentration (20 μM) with serum-free culture medium. Then, add an appropriate amount of the diluted probe working solution to each well to ensure complete coverage of the cell layer. Incubate the cell culture in a cell incubator at 37 ℃ in the dark for 20 to 45 minutes.

[0115] ③ Discard the probe working solution and wash the cells thoroughly three to four times with pre-warmed PBS to completely remove any residual probes that have not yet entered the cells. Then, administer different interventions based on the experimental design. Observe and image using a fluorescence microscope.

[0116] ④ Each experiment was repeated three times. Data are expressed as mean ± standard deviation. ANOVA was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0117] ⑤ For example Figure 13 and Figure 14As shown, compared with the PBS control group, macrophages treated with different concentrations of ginseng stem and leaf cell exosome-like vesicles all exhibited anti-ROS effects. Statistical analysis of ROS fluorescence quantification revealed that low-dose ginseng stem and leaf cell exosome-like vesicles reduced H2O2-induced ROS levels by approximately 30%, medium-dose reduced H2O2-induced ROS levels by approximately 50%, and high-dose reduced H2O2-induced ROS levels by over 70%. This demonstrates that ginseng stem and leaf cell exosome-like vesicles have a significant ability to reduce ROS levels in a dose-dependent manner.

[0118] (3) Detection of antioxidant capacity of exosome-like vesicles in ginseng stem and leaf cells

[0119] ① RAW264.7 macrophages were cultured at a concentration of 1×10⁻⁶. 6 The cells were seeded at a density of 10 cells / well in 6-well plates and pre-cultured for 24 hours before being divided into four groups: PBS treatment group, low-dose treatment group of ginseng stem and leaf cell exosome-like vesicles, medium-dose treatment group of ginseng stem and leaf cell exosome-like vesicles, and high-dose treatment group of ginseng stem and leaf cell exosome-like vesicles.

[0120] ② Discard the original culture medium from adherent cells in good growth condition, and gently wash the cells twice with pre-warmed PBS. Then, add LPS (100 ng / mL) to all groups. -1 The cells were cultured in DMEM medium and incubated with different concentrations of ginseng stem and leaf cell exosome-like vesicles according to different treatment groups.

[0121] ③ Collect the supernatant and centrifuge at 2000 g for 10 minutes at 4 ℃ to thoroughly remove cell debris. Follow the subsequent steps strictly according to the commercially available oxidative stress ELISA kit.

[0122] ④ Each experiment was repeated three times. Data are expressed as mean ± standard deviation. ANOVA was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0123] ⑤ For example Figures 15 to 17As shown, compared with the PBS control group, macrophages treated with different concentrations of ginseng stem and leaf cell exosome-like vesicles all exhibited reduced oxidative stress and enhanced antioxidant effects. Statistical analysis of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels revealed that low-dose ginseng stem and leaf cell exosome-like vesicles reduced lipid peroxidation, while medium- and high-dose treatments maximally reduced lipid peroxidation. Simultaneously, treatment with ginseng stem and leaf cell exosome-like vesicles significantly increased the levels of superoxide dismutase and glutathione peroxidase, demonstrating the excellent antioxidant capacity of ginseng stem and leaf cell exosome-like vesicles.

[0124] Next, we will introduce the process of detecting the in vivo skin damage repair ability of ginseng stem and leaf cell exosome-like vesicles.

[0125] (1) The experimental groups are as follows (n = 6)

[0126] Ctrl group: normal mice

[0127] PBS group: Skin lesions were created in mice, and PBS loaded with methacrylamide gelatin hydrogel was used for treatment. The treatment effect was evaluated after 7 consecutive days of application.

[0128] Exosomes group: Skin lesions were created in mice, and ginseng stem and leaf cell exosome-like vesicles (8 mg / mL) were loaded onto methacryloylgel-type hydrogel. -1 Treatment: Apply the medication continuously for 7 days and then assess the treatment effect.

[0129] (2) Methacrylated gelatin hydrogel loaded with ginseng stem and leaf cell exosome-like vesicles

[0130] Lyophilized GelMA (purchased from Suzhou Yongqinquan Intelligent Equipment) was prepared into a 5m%–20% (w / v) prepolymer solution using PBS solution containing 0.25% (w / v) LAP photoinitiator (dissolved at 40 ℃–50 ℃ in the dark). After sterilization through a 0.22 μm filter membrane, it was mixed with ginseng stem and leaf cell exosome-like vesicle suspension at a volume ratio of 1:5 and gently mixed at 37 ℃ in the dark. The mixture was immediately injected into well plates (50 μL–100 μL / well of a 96-well plate) and heated using a 405 nm light source (intensity 6 mW·cm). -2 ~10 mW·cm -2 Photocrosslinking and curing are completed by irradiation for 30 seconds.

[0131] (3) Construction of a mouse model of diabetic skin lesions

[0132] Eight-week-old SPF-grade male C57BL / 6 mice were selected and, after one week of acclimatization, fasted for 12 hours. Streptozotocin (STZ) was then injected intraperitoneally, dissolved in 0.1 mol·L⁻¹ solution, protected from light. -1 pH 4.5 sodium citrate buffer, concentration 5 mg / mL -1 Inducing diabetes, at 50 mg / kg -1 The dosage was administered continuously for 5 days. One hour after each injection, a 10% sucrose solution was administered. Normal fluid intake was resumed 48 hours later. Fasting blood glucose was monitored twice weekly, starting 72 hours after the last injection, with two consecutive readings equal to or greater than 16.7 mmol / L. -1 The criteria for successful diabetes modeling were as follows: 7 days after the diabetes was stable, mice were anesthetized with 2% isoflurane, the hair on their backs was shaved and disinfected with iodine, and full-thickness skin defects with a diameter of 8 mm to 10 mm were made by vertically applying pressure with a sterile spring-type punch. The mice were then housed in individual cages after the operation.

[0133] (4) HE section detection of the repair-promoting capacity of exosome-like vesicles in ginseng stem and leaf cells

[0134] Mice were euthanized on postoperative day 7. A 2 mm tissue block from the wound and surrounding tissue was immediately immersed in 4% neutral formalin (pH 7.4) at 4°C for 24 hours. The block was then subjected to gradient dehydration (1-2 hours each in 70% to 100% ethanol), xylene clearing (2 x 30 minutes), and paraffin impregnation at 60°C (2 x 1.5 hours) for embedding. After trimming, 5 μm continuous sections were cut and attached to poly-L-lysine slides. The slides were dried at ℃ for 2 hours; after dewaxing with xylene (2×10 minutes), followed by gradient hydration with ethanol, Harris hematoxylin staining for 5 minutes, running water blue staining for 10 minutes, counterstaining with 0.5% eosin-Y (containing 1% glacial acetic acid) for 1 minute, gradient dehydration, xylene clearing, and mounting with neutral resin; finally, the slides were observed and quantified under an optical microscope: the re-epithelialization rate (length of new epidermis / length of original surface × 100%), granulation tissue thickness (μm), inflammatory cell count at 400× field, and the proportion of collagen deposition in the eosin-stained area were measured. ImageJ software was used for analysis, and ANOVA was used for intergroup comparisons. P <0.05 is the significance standard), key quality controls include fixation timeliness (processing within 30 minutes of ex vivo), slide integrity (≥3 consecutive slides per sample) and double-blind pathological evaluation.

[0135] (5) Detection of anti-Tnf-α in exosome-like vesicles of ginseng stem and leaf cells

[0136] Paraffin sections were prepared from the skin lesions of mice in each treatment group. The sections were dewaxed with xylene, hydrated with graded ethanol, and autoclaved for 5 minutes with sodium citrate buffer (pH 6.0). After cooling, they were permeabilized with 0.1% Triton X-100 at room temperature for 20 minutes, washed three times with PBS, and blocked with 5% BSA for 1 hour. Anti-TNF-α primary antibody (1:200 dilution) was added and incubated overnight in a humidified chamber at 4°C. After rinsing with PBS, Cy3-labeled secondary antibody (1:400) was added and incubated at room temperature in the dark for 1 hour. The nuclei were stained with DAPI for 5 minutes, and the sections were mounted with anti-fluorescence quencher and observed under a laser confocal microscope.

[0137] (6) Each experiment was repeated 3 times. Data are expressed as mean ± standard deviation. ANOVA was used for comparisons between groups. P <0.05 indicates a statistically significant difference.

[0138] (7) For example Figure 18 and Figure 20 As shown, compared with the PBS control group, treatment with ginseng stem and leaf cell exosome-like vesicles loaded with methacrylamide gelatin hydrogel significantly reduced the area of ​​skin lesions in diabetic mice, alleviated inflammatory infiltration, and significantly improved tissue structure disorder. The data indicate that ginseng stem and leaf cell exosome-like vesicles significantly improve diabetic skin lesions in mice.

[0139] like Figure 19 and Figure 21 As shown, compared with the PBS control group, treatment with ginseng stem and leaf cell exosome-like vesicles loaded with methacrylamide gelatin hydrogel significantly reduced the expression of the inflammatory factor Tnf-α in the skin lesions of diabetic mice and significantly alleviated inflammatory translation. The data indicate that ginseng stem and leaf cell exosome-like vesicles can significantly improve the inflammatory response after diabetic skin lesions in mice. MFI refers to "mean fluorescence intensity".

[0140] In the attached image, "*" indicates... P <0.05, "**" indicates P <0.01, “***” indicates P <0.001 indicates a statistically significant difference.

[0141] Traditional ultracentrifugation methods (such as CN108135831B) lack specific separation mechanisms and cannot effectively remove contaminating proteins (molecular weight 20 kD to 60 kD) with similar densities to exosomes, resulting in a purity of less than 70%. Existing storage methods (such as CN120168653A) do not add vesicle membrane protectants, and vesicles are prone to membrane fusion when stored at 4 ℃, resulting in a loss of more than 50% of activity after 30 days.

[0142] This invention achieves high-purity and high-stability preparation of ginseng stem and leaf cell exosome-like vesicles through innovative "enzymatic hydrolysis-ultrasonic coupling pretreatment", "three-stage purification" and "gradient freezing stabilization" technologies.

[0143] The above description is merely a preferred embodiment of this application and is not intended to limit the invention. Various modifications and variations can be made to this invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for processing exosome-like vesicles from ginseng stem and leaf cells, characterized in that, Includes the following steps: Step 1: After crushing ginseng stems and leaves, add compound enzymatic hydrolysate and enzymatically hydrolyze at 37 ℃ for 2 hours. Then, perform ultrasonication in an ice bath at 0 ℃~4 ℃ for 10 minutes and filter to obtain a homogenate. The compound enzymatic hydrolysate consists of 0.2% cellulase and 0.1% pectinase. Step 2: The homogenate is subjected to multiple purification stages to obtain the vesicle layer; Step 3: After washing the vesicle layer with PBS, mix it with a freeze-drying protectant, pre-freeze it at -80 ℃ for 2 hours, and then freeze-dry it at -50 ℃ under vacuum of 10 Pa for 12 hours to obtain ginseng stem and leaf cell exosome-like vesicle freeze-dried powder, wherein the freeze-drying protectant contains 5% sucrose and 2% mannitol.

2. The processing method according to claim 1, characterized in that, Step 2 specifically includes: The homogenate was subjected to primary, secondary, and tertiary purification. Primary purification involved centrifugation followed by collection of the supernatant. Centrifugation conditions were: 4 °C, 8000 g centrifugation force, and 15 minutes. Secondary purification involved filtration through a 0.22 μm polyethersulfone membrane to obtain the filtrate. Tertiary purification involved mixing the filtrate with a 30% sucrose solution at a 1:1 volume ratio at 4 °C and centrifuging at a density gradient of 100,000 g for 2 hours to obtain the vesicle layer.

3. The processing method according to any one of claims 1 or 2, characterized in that, The method further includes: Step 4: preparing a skin repair composition based on the lyophilized powder of ginseng stem and leaf cell exosome-like vesicles.

4. The processing method according to claim 3, characterized in that, Step 4 specifically includes: Prepare a ginseng stem and leaf cell exosome vesicle suspension from the freeze-dried powder of the ginseng stem and leaf cell exosome vesicles. A 5% (w / v) to 20% (w / v) prepolymer solution was prepared by dissolving the methacrylamide gelatin hydrogel carrier in a PBS solution containing 0.25% (w / v) LAP photoinitiator at 40 ℃ to 50 ℃ in the dark. The prepolymer solution was sterilized by filtration through a 0.22 μm microporous membrane and then mixed with a suspension of ginseng stem and leaf cell exosomes at a volume ratio of 1:

5. The mixture was thoroughly mixed at 37 °C in the dark, and then injected into a well plate with an intensity of 6 mW·cm⁻¹. -2 ~10 mW·cm -2 Photocrosslinking and curing are completed by irradiation with a 405 nm wavelength light source for 30 seconds.

5. A skin-repairing composition, characterized in that, Obtained by the processing method described in any one of claims 3 or 4.

6. A dressing for repairing skin, characterized in that, The ingredients include the skin-repairing composition as described in claim 5.

7. A freeze-dried powder of exosome-like vesicles from ginseng stem and leaf cells, characterized in that, Obtained by the processing method described in any one of claims 1 or 2.

8. A dressing for repairing skin, characterized in that, The ingredients include the lyophilized powder of ginseng stem and leaf cell exosome-like vesicles as described in claim 7, wherein the dressing is one of a face cream or a serum.

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