Preparation method, product and application of baical skullcap cell extracellular vesicles

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a preparation method, product and application of a Scutellaria baicalensis Georgi extracellular vesicle. The preparation method comprises the following steps: preparing a homogenate from Scutellaria baicalensis Georgi raw materials, collecting supernatant after solid-liquid separation; filtering the supernatant through a microporous filter membrane; sequentially passing the filtrate through nanometer pore membranes with pore diameters of 180-220 nm and 25-35 nm, and collecting a concentrated solution; adding a dextran sulfate solution to the concentrated solution, and preparing a freeze-dried powder through vacuum freeze-drying. The present application also provides the Scutellaria baicalensis Georgi extracellular vesicle freeze-dried powder prepared by the method, application of the freeze-dried powder in preparing a product for inhibiting epithelial cell ferroptosis, and a Scutellaria baicalensis Georgi extracellular vesicle water-soluble inclusion compound. The present application uses nanometer pore membrane fractionation technology to gently and efficiently enrich vesicles, effectively maintains the natural activity of the vesicles, and significantly improves the stability of the freeze-dried product through a dextran sulfate protection system.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing extracellular vesicles of Scutellaria baicalensis, as well as the product and its application. Background Technology

[0002] In recent years, plant-derived extracellular vesicles (PDEVs) have attracted much attention in the fields of drug delivery and anti-inflammatory therapy due to their natural biological activity, low immunogenicity, and ability to cross biological barriers. PDEVs are phospholipid bilayer vesicles secreted by plant cells, with a particle size distribution of 30-200 nm. Their surface carries plant-specific proteins, and their interior encapsulates functional microRNAs, metabolites, and enzymatic active substances.

[0003] However, the large-scale preparation of PDEVs remains a core bottleneck restricting their clinical translation. While traditional differential ultracentrifugation is considered the "gold standard," its high centrifugal force (typically above 150,000 g) easily causes mechanical damage to the vesicle membrane structure, and the equipment is expensive and time-consuming. Polymer-based precipitation methods, although simple to operate, often face problems such as high levels of co-precipitated impurities, low vesicle recovery rates, and polymer residues. Furthermore, while sucrose density gradient ultracentrifugation yields high purity, it is complex to operate and takes tens of hours, making it difficult to meet the needs of large-scale production. Therefore, developing a separation and purification process that can maintain the natural activity of vesicles while possessing low cost and high efficiency is a pressing technical challenge in this field.

[0004] Scutellaria baicalensis Georgi, a classic medicinal plant, is rich in flavonoid active ingredients such as baicalin and baicalein, and is often used in traditional medicine to treat diarrhea caused by damp-heat in the intestines. Modern pharmacological research reveals that the occurrence of intestinal diseases such as ulcerative colitis (UC) is not only related to chronic inflammation, but recent studies have also shown that ferroptosis of intestinal epithelial cells is one of its important pathological mechanisms. Ferroptosis is an iron-dependent form of regulated cell death characterized by loss of glutathione peroxidase 4 (GPX4) activity and lipid peroxidation accumulation, leading to severe damage to the intestinal mucosal barrier. Although existing treatments (such as 5-aminosalicylic acid and monoclonal antibodies) can alleviate symptoms to some extent, long-term use carries the risk of drug resistance and side effects. Therefore, finding natural active agents that can target and inhibit epithelial cell ferroptosis and promote mucosal repair has significant clinical implications.

[0005] Chinese patent application CN115747133A discloses a method for extracting Scutellaria baicalensis exosomes and its application. The method involves juicing fresh Scutellaria baicalensis, followed by low-speed centrifugation, filtration through a 0.22 μm microporous membrane, and centrifugation at 10000-13000 g for 10-13 h to obtain a crude extract. This extract is then purified by ultracentrifugation with gradient sucrose solutions of 8%, 30%, 45%, and 60%, collecting the 30% sucrose layer to obtain the purified Scutellaria baicalensis exosomes. The drawbacks of this method are: it still relies on ultracentrifugation (10000-13000 g for 10-13 h) and sucrose density gradient centrifugation (10000-13000 g for 14-16 h), resulting in a total centrifugation time exceeding 24 h, leading to high energy consumption and low efficiency; the high centrifugal force may cause mechanical damage to the vesicle membrane structure; and sucrose residue may affect subsequent applications.

[0006] Currently, there is a lack of targeted and mild process optimization for the preparation of extracellular vesicles from Scutellaria baicalensis. In particular, existing preservation methods mostly use simple PBS buffer or conventional lyophilization protectants, which are difficult to effectively prevent structural disintegration and loss of activity of vesicles during freezing. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method, product and application for preparing Scutellaria baicalensis extracellular vesicles that can maintain the natural activity of vesicles and have the characteristics of low cost and high efficiency.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing extracellular vesicles derived from Scutellaria baicalensis, characterized by comprising the following steps: (1) Prepare a homogenate from the raw material of Scutellaria baicalensis, filter it and collect the filtrate; (2) Centrifuge the filtrate to remove large particulate impurities and collect the supernatant; (3) The supernatant was filtered sequentially through 0.45 μm and 0.22 μm microporous membranes, and the filtrate was collected; (4) The filtrate is sequentially passed through a first ultrathin nanoporous membrane with a pore size of 180-220 nm and a second ultrathin nanoporous membrane with a pore size of 25-35 nm for size exclusion separation, and the extracellular vesicle concentrate is collected. (5) The extracellular vesicle concentrate is mixed with the sulfated dextran solution and then freeze-dried under vacuum to obtain Scutellaria baicalensis extracellular vesicle freeze-dried powder.

[0009] Another technical solution of the present invention is: a method for preparing a water-soluble inclusion complex of extracellular vesicles of Scutellaria baicalensis, comprising the following steps: S1: Dissolve cyclodextrin in pure water by heating at 37-45℃ to obtain solution A; S2: Add the lyophilized powder of Scutellaria baicalensis extracellular vesicles prepared by the above method to the solution A, and stir and react at 37-45℃ for 1-3 h to obtain the inclusion solution; S3: The inclusion solution is freeze-dried under vacuum to obtain a water-soluble inclusion complex of Scutellaria baicalensis extracellular vesicles.

[0010] Another technical solution of the present invention is to provide a product for inhibiting epithelial cell ferroptosis, wherein the product is a drug, health food or functional food, and the product comprises lyophilized powder of Scutellaria baicalensis extracellular vesicles prepared by the above method.

[0011] Another technical solution of the present invention is to provide an application of the freeze-dried powder of Scutellaria baicalensis extracellular vesicles prepared by the above method in the preparation of products that inhibit epithelial cell ferroptosis.

[0012] The beneficial effects of this invention are as follows: Existing Scutellaria baicalensis exosome extraction techniques (such as CN115747133A) rely on differential centrifugation combined with ultracentrifugation and sucrose gradient centrifugation, which is not only cumbersome and time-consuming, but also prone to mechanical damage to the vesicle membrane structure due to high-intensity centrifugation force; in terms of preservation, existing technologies only use PBS buffer for low-temperature preservation, which makes it difficult to maintain the structural integrity and biological activity of vesicles for a long time.

[0013] This invention employs a mild separation and purification route combining microfiltration for impurity removal and nanoporous membrane size exclusion. Its advantages are twofold: first, by replacing high-shear centrifugation with a physical sieving mechanism, it fundamentally avoids the mechanical damage to the vesicle lipid bilayer membrane caused by strong centrifugal force, thus preserving the natural structure of the vesicles intact; second, it significantly shortens the preparation cycle (from 24 hours in the prior art). The preparation time has been reduced from 29 hours to just a few hours, significantly improving efficiency, reducing equipment costs and energy consumption, and making it more suitable for large-scale production. Furthermore, this invention creatively introduces a sulfated dextran solution as a lyophilization protectant, enabling vesicles to be stably preserved in lyophilized powder form for extended periods, overcoming the limitations of existing technologies that can only use PBS suspensions. The limitation of short-term storage at 80℃ (not exceeding 90 days and not subject to repeated freeze-thaw cycles) has laid the formulation foundation for the industrial application of Scutellaria baicalensis extracellular vesicles. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the morphology of extracellular vesicles derived from Scutellaria baicalensis under transmission electron microscopy, as shown in Example 1 of this invention. Figure 2 Transmission electron microscopy (TEM) image of extracellular vesicle size and potential of Scutellaria baicalensis from Example 1 of this invention. Figure 3 The effect of Scutellaria baicalensis extracellular vesicles on the relative cell viability of epithelial cells induced by ferroptosis, as described in Example 2 of this invention. Figure 4 The effect of Scutellaria baicalensis extracellular vesicles on the expression level of ferroptosis-related proteins in epithelial cells in Example 2, a specific embodiment of the present invention; Figure 5 The effect of Scutellaria baicalensis extracellular vesicles on the expression level of epithelial cell junction protein induced by ferroptosis, as described in Example 2 of this invention. Detailed Implementation

[0015] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0016] This invention addresses the problems in existing plant extracellular vesicle preparation techniques, such as easy damage to vesicle structures, poor preservation stability, and lack of process optimization for Scutellaria baicalensis raw materials, by providing a complete and mild separation technology. Specifically, this invention uses nanoporous membrane fractionation technology to replace the traditional ultracentrifugation method, achieving mild and efficient enrichment of vesicles; and uses dextran sulfate as a freeze-drying protectant to achieve long-term stable preservation of vesicles.

[0017] This invention provides a method for preparing extracellular vesicles derived from Scutellaria baicalensis, comprising the following steps: (1) Prepare a homogenate from the raw material of Scutellaria baicalensis, and collect the filtrate after solid-liquid separation; (2) Centrifuge the filtrate to remove large particulate impurities and collect the supernatant; (3) Filter the supernatant through at least two microporous membranes with different pore sizes and collect the filtrate; (4) Pass the filtrate through a 180mm pore size sequentially. 220 nm and 25 35 nm nanoporous membrane, Collect concentrated fluid rich in extracellular vesicles; (5) Add sulfated dextran solution to the concentrated solution and freeze dry under vacuum to obtain Scutellaria baicalensis extracellular vesicle freeze-dried powder.

[0018] Furthermore, in the above-mentioned method for preparing extracellular vesicles derived from Scutellaria baicalensis, step (3) specifically involves: filtering the supernatant sequentially through 0.45 μm and 0.22 μm microporous membranes and collecting the filtrate.

[0019] As described above, the specific pore size and filtration sequence of the microporous membranes are further defined, constructing a stepwise fine pre-filtration system of 0.45 μm and 0.22 μm. The 0.45 μm membrane first traps cell debris, large tissue particles, and micron-sized suspended matter, while the 0.22 μm membrane further removes bacteria, fine particles, and submicron-sized impurities, providing a high-quality feed solution for the precise size exclusion process of the subsequent nanoporous membranes. This "coarse-to-fine" stepwise filtration strategy effectively prevents large particles from clogging the subsequent nanoporous membranes, extending the lifespan of the nanoporous membrane assembly, while ensuring that the separation efficiency of the nanoporous membranes remains undisturbed. Existing technologies (such as CN115747133A) only use a single-stage 0.22 μm filtration, lacking 0.45 μm pre-filter protection, which easily leads to rapid membrane clogging. This invention improves the stability and repeatability of the process through the division of labor and synergy of the two-stage microporous membranes.

[0020] Furthermore, in the above-mentioned method for preparing extracellular vesicles derived from Scutellaria baicalensis, the volume ratio of the concentrated solution to the dextran sulfate solution is (2-4):1; and the mass concentration of the dextran sulfate solution is 1-3 wt%.

[0021] As described above, dextran sulfate, as an anionic polymeric polysaccharide, can undergo electrostatic interactions between its sulfated ester groups and cationic proteins / lipids on the vesicle membrane surface, inducing a slight flocculation state in the vesicles. This allows the nanoscale vesicles to form a uniform solid matrix before freeze-drying, preventing random aggregation of vesicles during the freezing process. Simultaneously, during freeze-drying, dextran sulfate acts as a polymeric framework supporting the porous structure of the freeze-dried cake, reducing physical damage to the vesicle membrane caused by ice crystal formation. The concentration range of 3 wt% (preferably 2 wt%) and the volume ratio of (2-4):1 (preferably 3:1) are the optimal parameter ranges obtained by the inventors through systematic screening based on the characteristics of Scutellaria baicalensis extracellular vesicles, using freeze-drying reconstitution rate, particle size recovery rate, and vesicle activity retention as evaluation indicators. In existing technologies, plant exosomes are mostly preserved at low temperatures using PBS buffer (CN115747133A) or conventional sugar protectants (such as trehalose and sucrose). However, conventional sugars lack interaction with the vesicle membrane, making it difficult to effectively prevent structural disintegration and activity loss of vesicles during freeze-drying, and the freeze-dried cake is prone to collapse. The sulfated dextran protection system of this invention has both "membrane interaction" and "skeleton support" functions, which is one of the core innovations distinguishing it from existing technologies.

[0022] Furthermore, in the above-mentioned method for preparing extracellular vesicles derived from Scutellaria baicalensis, in step (1), the Scutellaria baicalensis raw material is pre-cooled at -20°C to -10°C before homogenization.

[0023] As described above, pre-cooling treatment causes water in Scutellaria baicalensis tissue to form tiny ice crystals. These ice crystals can pierce cell walls and vacuolar membranes during subsequent homogenization, promoting the release of intracellular vesicles. Simultaneously, the low-temperature environment inhibits the activity of endogenous enzymes (such as polyphenol oxidase and peroxidase), preventing the enzymatic oxidation and degradation of flavonoid active components in Scutellaria baicalensis during homogenization. Existing technologies (such as CN115747133A) directly perform homogenization without pre-cooling treatment. This invention achieves the dual technical effects of "gentle cell wall disruption promoting release + inhibition of enzymatic oxidation" through a pre-cooling step, making it particularly suitable for Scutellaria baicalensis medicinal materials rich in heat-sensitive / oxygen-sensitive flavonoid active components.

[0024] Furthermore, in the above-mentioned method for preparing extracellular vesicles derived from Scutellaria baicalensis, in step (1), the homogenate is homogenized at 36.5-37.5°C.

[0025] As described above, Scutellaria baicalensis is rich in thermosensitive flavonoid active ingredients such as baicalin and baicalein. The selection of extraction temperature needs to strike a balance between "vesicle release efficiency" and "active ingredient stability." Excessively high temperatures (e.g., above 45℃) easily accelerate the oxidative degradation of flavonoids, and high temperatures may damage the vesicle membrane structure; excessively low temperatures (e.g., 4℃ or room temperature) significantly reduce the release rate and amount of vesicles from plant tissue. This invention limits the homogenization temperature to near body temperature, ensuring both efficient and gentle release of vesicles from Scutellaria baicalensis tissue and avoiding thermal degradation of flavonoid active ingredients. Simultaneously, this temperature condition helps maintain the fluidity of the vesicle membrane, reducing vesicle aggregation and fusion during extraction. Existing technologies (such as CN115747133A) use a temperature of 3... Extraction at a low temperature of 5℃, while beneficial for protecting the vesicle structure, sacrifices extraction efficiency. Generally, plant exosome extraction is carried out at 4℃ or room temperature. This invention has carried out specialized temperature optimization for the characteristics of Scutellaria baicalensis raw material, demonstrating the targeted and innovative nature of the process.

[0026] Furthermore, in the above-mentioned method for preparing extracellular vesicles derived from Scutellaria baicalensis, in step (5), the conditions for vacuum freeze-drying are: temperature -30℃, vacuum degree 5 Pa, and time 36 h.

[0027] As can be seen from the above description, A freezing temperature of 30°C ensures that the vesicle suspension forms fine and uniform ice crystals during freezing, avoiding puncture damage to the vesicle membrane structure from large ice crystals. A high vacuum of 5 Pa provides sufficient pressure differential, ensuring that the ice crystals sublimate directly below the eutectic point temperature, shortening the initial drying time and avoiding the freeze-concentration effect caused by excessively low vacuum. A drying time of 36 hours ensures that the product's moisture content is reduced to an extremely low level (typically below 3%), ensuring the stability of the freeze-dried product during long-term storage. These parameters work synergistically with the dextran sulfate protection system: dextran sulfate provides skeletal support, and the optimized freeze-drying curve ensures that ice crystal formation and sublimation are mild and controllable, together maximizing the preservation of the vesicle's natural structure and bioactivity. Existing technologies (such as CN115747133A) only use PBS buffer. Storage at 80°C is neither suitable for long-term stable preservation nor feasible for large-scale production. This invention achieves solid-state formulation of vesicles through an optimized freeze-drying process, representing a key technological breakthrough for the industrial application of Scutellaria baicalensis extracellular vesicles.

[0028] Another technical solution of the present invention is: a method for preparing a water-soluble inclusion complex of extracellular vesicles of Scutellaria baicalensis, comprising the following steps: S1: Dissolve cyclodextrin in pure water by heating at 37-45℃ to obtain solution A; S2: Add the freeze-dried powder of Scutellaria baicalensis extracellular vesicles prepared by the preparation method described in claims 1-6 to the solution A, and stir the reaction at 37-45℃ for 1-3 h to obtain the inclusion solution; S3: The inclusion solution is freeze-dried under vacuum to obtain a water-soluble inclusion complex of Scutellaria baicalensis extracellular vesicles.

[0029] As described above, cyclodextrin possesses a truncated conical cavity structure with an internal hydrophobic and external hydrophilic coating. In aqueous solution, it can undergo intermolecular inclusion complexation with the hydrophobic regions on the surface of the phospholipid bilayer of the vesicle membrane and with baicalein-like flavonoid aglycones carried within the vesicles, encapsulating the vesicles within its hydrophilic shell. This inclusion complexation brings three technical benefits: firstly, it significantly improves the dispersibility and resolution rate of vesicles in aqueous media, solving the defect of easy agglomeration when directly reconstituted lyophilized vesicle powder; secondly, the cyclodextrin shell provides "molecular armor" protection to the vesicle membrane, resisting adverse factors such as physical shock and high temperatures during storage and transportation; and thirdly, the inclusion complex form expands the application scenarios of the product, allowing for convenient preparation into different dosage forms such as oral liquids, solid beverages, and injections. 37 The reaction temperature was 45℃ and 1 The 3-hour reaction time is an optimized condition that ensures the inclusion reaction proceeds fully while avoiding damage to the vesicle structure caused by prolonged high-temperature treatment. There is currently no technical guidance available for preparing plant-derived extracellular vesicles into cyclodextrin inclusion complexes.

[0030] Furthermore, in the above preparation method, the cyclodextrin is selected from one or a combination of two of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin; the mass ratio of the cyclodextrin to the freeze-dried powder of Scutellaria baicalensis extracellular vesicles is (1-3):1.

[0031] As described above, hydroxypropyl β Cyclodextrin and sulfobutyl β All cyclodextrins are β-cyclodextrins Chemical derivatives of cyclodextrin, through the introduction of hydroxypropyl or sulfonyl substituents, break the β-chain. The strong hydrogen bonds between cyclodextrin molecules reduce its water solubility from that of the parent β-molecule. The concentration of cyclodextrin was significantly increased from approximately 1.8 g / 100 mL to over 50 g / 100 mL, while retaining the unique cavitary inclusion capacity of cyclodextrin. Hydroxypropyl... β Cyclodextrin is the preferred choice due to its good biocompatibility and low nephrotoxicity. The mass ratio of cyclodextrin to lyophilized vesicle powder (1:1) The 3:1 (preferably 1:1) ratio ensures that the number of cyclodextrin cavities is sufficient to form a stable inclusion complex with the vesicles and their carried active ingredients. A ratio that is too low (<1:1) will result in insufficient inclusion, leaving some vesicles unprotected; a ratio that is too high (>3:1) will lead to cyclodextrin waste, excessive formulation volume, and the possibility that excessive cyclodextrin may competitively adsorb the active ingredients on the vesicle membrane surface. This ratio range balances inclusion efficiency, formulation quality, and cost-effectiveness.

[0032] Another technical solution of the present invention is: to provide a product for inhibiting epithelial cell ferroptosis, wherein the product is a drug, health food or functional food, and the product comprises the freeze-dried powder of Scutellaria baicalensis extracellular vesicles prepared by the above preparation method.

[0033] As described above, this invention is the first to target the application of Scutellaria baicalensis extracellular vesicles in inhibiting epithelial cell ferroptosis, representing a novel functional exploration of this traditional Chinese medicine resource at the molecular pharmacological level. As shown in the experimental data of Example 2, Scutellaria baicalensis extracellular vesicles exhibit IEC inhibition in RSL3 (a specific inducer of ferroptosis). In a rat small intestinal epithelial cell ferroptosis model, the drug exhibited a triple pharmacodynamic effect: First, it significantly reversed the RSL3-induced decrease in cell viability (P<0.05), demonstrating its protective effect against ferroptosis-induced cell damage; second, it upregulated the expression of the ferroptosis-inhibiting core proteins GPX4 and FTH1 (P<0.05) and downregulated the expression of the ferroptosis-promoting protein ACSL4 (P<0.01), clarifying its signaling pathway regulatory mechanism for inhibiting ferroptosis at the molecular level; third, it upregulated the expression of the intestinal epithelial tight junction proteins Occludin and ZO. The expression of 1 (P<0.05) indicates that it also has the additional function of repairing the intestinal mucosal barrier. Based on the above mechanism, products containing this vesicle can be used to prevent and / or treat ulcerative colitis, intestinal mucosal barrier damage and other related diseases with intestinal epithelial cell ferroptosis as the core pathological mechanism, opening up a new application direction for Scutellaria baicalensis resources in the treatment of inflammatory bowel disease.

[0034] Another technical solution of the present invention is to provide an application of the freeze-dried powder of Scutellaria baicalensis extracellular vesicles prepared by the above preparation method in the preparation of products that inhibit epithelial cell ferroptosis.

[0035] In summary, this invention introduces an ultrathin nanoporous membrane fractionation technique for purifying extracellular vesicles of Scutellaria baicalensis, replacing the traditional high-shear ultracentrifugation strategy. This process achieves efficient vesicle enrichment at low speeds through a gentle physical sieving mechanism, effectively avoiding the mechanical damage to the vesicle membrane structure caused by strong centrifugation, and significantly improving vesicle yield and natural biological activity. Compared with the sucrose density gradient ultracentrifugation method in prior art CN115747133A, which has a total centrifugation time exceeding 24 hours, the nanoporous membrane fractionation process of this invention has a shorter operation time, lower equipment requirements, and does not require a density gradient medium, making it more suitable for large-scale preparation.

[0036] This invention also sets the extraction temperature of Scutellaria baicalensis homogenate to 36.5-37.5℃, which is an optimized parameter that balances the stability of the heat-sensitive components of Scutellaria baicalensis flavonoids with the gentle release of vesicles from plant tissues. Scutellaria baicalensis is rich in heat-sensitive active flavonoid components such as baicalin and baicalein. Excessive temperature can easily promote oxidation / degradation, while excessively low temperature will affect the vesicle release efficiency, which is different from the conventional practice of extracting plant exosomes at 4℃ or room temperature.

[0037] This invention employs a 2 wt% dextran sulfate solution and vesicle concentrate mixed at a 3:1 volume ratio, followed by vacuum freeze-drying. The dextran sulfate interacts with cationic proteins / lipids on the vesicle surface to form a protective matrix, and also acts as a polymeric framework to form a porous freeze-dried cake with the vesicles. This protective system is the optimal solution determined through screening of freeze-drying reconstitution rate, particle size recovery, and activity retention for Scutellaria baicalensis samples, differing from existing preservation methods using simple PBS buffer or conventional freeze-drying protectants.

[0038] This invention utilizes cyclodextrin to form inclusion complexes with the extracellular vesicles of Scutellaria baicalensis, which improves the dispersibility and stability of the vesicles in water, expands the application forms of the product, and facilitates the preparation of various products such as oral preparations and health foods.

[0039] This invention, validated in vitro using the IEC-6 cell model, demonstrates that the extracellular vesicles of *Scutellaria baicalensis* prepared in this invention can significantly antagonize RSL3-induced ferroptosis. It effectively inhibits epithelial cell ferroptosis by upregulating the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) and downregulating the level of acyl-CoA synthase long chain family member 4 (ACSL4). Simultaneously, it promotes the expression of intestinal epithelial connexins Occludin and ZO-1, repairing the intestinal mucosal barrier, demonstrating significant clinical application potential in the treatment of inflammatory diseases such as ulcerative colitis.

[0040] Example 1: Extraction, purification and identification of extracellular vesicles from Scutellaria baicalensis A method for preparing extracellular vesicles derived from Scutellaria baicalensis includes the following steps: (1) Take 500 g of fresh Scutellaria baicalensis, wash it clean, and pre-cool it at -20℃ for 2 h. Use a high-speed blender to homogenize the pre-cooled Scutellaria baicalensis sample. The homogenization medium is PBS buffer (pH 7.4, 0.01 M). The homogenization temperature is controlled at 37℃ and the homogenization time is 3 min. Collect the initial filtrate after coarse filtration through multiple layers of gauze.

[0041] (2) Place the filtrate at 4°C and centrifuge at 12,000×g for 10 min. Discard the lower precipitate and suspended impurities, and keep the supernatant for later use.

[0042] (3) The supernatant was filtered under reduced pressure through 0.45 μm and 0.22 μm microporous membranes in sequence to trap and remove micron-sized particles and cell debris, and the filtrate was collected.

[0043] (4) Add the filtrate to the filtration chamber and pass it through an ultrathin nanoporous membrane assembly with pore sizes of 180-220 nm and 25-35 nm in sequence. Due to size exclusion, collect the concentrated solution containing extracellular vesicles with a diameter of 20-180 nm.

[0044] (5) The obtained extracellular vesicle concentrate was mixed with 2 wt% sulfated dextran solution at a volume ratio of 3:1. After mixing, the mixture was freeze-dried under vacuum (temperature -30℃, vacuum degree 5 Pa, time 36 h) to obtain Scutellaria baicalensis extracellular vesicle freeze-dried powder. The product was sealed and stored at -80℃ for a long time.

[0045] (6) The extracellular vesicle concentrate of Scutellaria baicalensis obtained in step (4) was characterized and analyzed by transmission electron microscopy, zeta potential detection and nanoparticle tracking analysis (NTA).

[0046] Experimental results Extracellular vesicles derived from Scutellaria baicalensis were identified using transmission electron microscopy, and the results were as follows: Figure 1 As shown, the extracellular vesicles derived from Scutellaria baicalensis are round or oval in shape and have a complete membrane structure. Zeta View was used to detect the particle size and potential of the extracellular vesicles derived from Scutellaria baicalensis, and the results are as follows. Figure 2 As shown, the extracted extracellular vesicles mostly ranged in size from 50 to 400 nm (mainly distributed in the range of 20 to 180 nm), with a Zeta potential of approximately -45 mV. These results indicate that the extracellular vesicles isolated from Scutellaria baicalensis in this invention meet the standard characteristics, laying a solid foundation for subsequent pharmacodynamic studies.

[0047] Example 2 (Validation Example): Experimental Study on the Inhibition of Epithelial Cell Ferric Death by Extracellular Vesicles Derived from Scutellaria baicalensis (1) Rat small intestinal epithelial cells (IEC-6) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator using standard methods. When the cells reached the logarithmic growth phase, they were digested with 0.25% trypsin and counted. Cells were then cultured at 2 × 10⁻⁶ cells / year. 5 Seeds were placed at a density of 100 μL / well in 96-well plates or 2 mL / well in 6-well plates and cultured for 24 h. Subsequent experiments were conducted after the cells had adhered to the plates at a rate of 70%-80%.

[0048] (2) The cells were randomly divided into the following three groups: Control group: Culture medium containing only an equal volume of PBS was added; Model group (RSL): ferroptosis was induced by adding RSL3 at a final concentration of 0.1 μM; Intervention group (EV): Added Scutellaria baicalensis extracellular vesicle lyophilized powder obtained in Example 1 Extracellular vesicles of Scutellaria baicalensis at concentrations of 80 μg / mL, 40 μg / mL, and 20 μg / mL, and RSL3 at a final concentration of 0.1 μM were co-incubated.

[0049] The dosage concentrations of 20 μg / mL, 40 μg / mL, and 80 μg / mL for the extracellular vesicles of Scutellaria baicalensis refer to the final total protein concentration of the vesicle preparation. These concentrations were determined by BCA method using the concentrated solution obtained in Example 1, followed by dilution with DMEM high-glucose medium containing 10% fetal bovine serum. The concentration gradients are geometrically increasing relationships of 1×, 2×, and 4×, and all are within the IEC range. 6. Within the range of cell tolerance.

[0050] Each group has 6 replicate wells, and the experiment is repeated 3 times.

[0051] (3) After 24 h of drug treatment, add 10 μL of CCK-8 reagent to each well and incubate at 37℃ and 5% CO2 for 1 h in the dark. Measure the absorbance (OD value) at 450 nm using a microplate reader. Calculate the relative cell viability using the following formula: Relative cell viability = [OD (experimental group) - OD (blank group)] / [OD (control group) - OD (blank group)] × 100% (4) Collect IEC-6 cells from each group after treatment, add RIPA lysis buffer (containing protease inhibitor) to extract total protein, and determine protein concentration by BCA method. Take 30 μg of protein, load it onto a PVDF membrane after separation by SDS-PAGE electrophoresis. After blocking with 5% skim milk powder for 1 h, add primary antibodies: GPX4, ACSL4, FTH1, Occludin, ZO-1 and internal control β-actin, and incubate overnight at 4℃. After washing the membrane with TBST, add HRP-labeled goat anti-rabbit secondary antibody, incubate at room temperature for 1 h, develop ECL and acquire images, and analyze the gray values ​​of the bands using ImageJ software.

[0052] Experimental results (1) Cell viability results as follows Figure 3 As shown in the figure, the relative cell viability of the RSL3 model group was significantly decreased compared with that of the control group (P<0.01); however, after pretreatment with 100 μg / mL Scutellaria baicalensis extracellular vesicles, the relative cell viability was significantly restored (P<0.05), indicating that Scutellaria baicalensis extracellular vesicles can effectively reverse RSL3-induced cell damage.

[0053] (2) Results of ferroptosis-related protein expression are as follows Figure 4 As shown in the figure. Compared with the control group, the expression of ferroptosis proteins GPX4 and FTH1 was significantly downregulated in the RSL3 model group (P<0.05), while the expression of ferroptosis protein ACSL4 was significantly upregulated (P<0.001). After intervention with Scutellaria baicalensis extracellular vesicles, the expression of GPX4 and FTH1 proteins was significantly restored (P<0.05), while the expression of ACSL4 was significantly inhibited (P<0.01).

[0054] (3) Results of connexin expression are as follows Figure 5 As shown in the figure. Compared with the control group, the expression levels of connexins Occludin and ZO-1 in the RSL3 model group were significantly decreased (P<0.01), and the expression level of connexins was significantly upregulated after intervention with Scutellaria baicalensis extracellular vesicles (P<0.05).

[0055] The above results indicate that the extracellular vesicles of Scutellaria baicalensis prepared in this invention can effectively inhibit epithelial cell ferroptosis by upregulating the expression of GPX4 and FTH1 and downregulating the expression of ACSL4, and promote the expression of intestinal epithelial connexins Occludin and ZO-1, thus having the potential to repair the intestinal mucosal barrier.

[0056] Example 3: Preparation of water-soluble inclusion complexes of extracellular vesicles from Scutellaria baicalensis A method for preparing a water-soluble inclusion complex of extracellular vesicles from Scutellaria baicalensis includes the following steps: 1 g of hydroxypropyl-β-cyclodextrin was dispersed in 20 mL of pure water, heated to 37°C, and magnetically stirred until completely dissolved to obtain solution A. Under magnetic stirring, 1 g of lyophilized Scutellaria baicalensis extracellular vesicle powder prepared in Example 1 (cyclodextrin to lyophilized powder mass ratio 1:1) was slowly added to solution A, and the mixture was stirred at 37°C for 2 h to obtain an inclusion complex. The resulting inclusion complex was then freeze-dried under vacuum (temperature -30°C, vacuum degree 5 Pa, time 36 h) to obtain the water-soluble inclusion complex.

[0057] The resulting water-soluble inclusion complex is a loose powder with good dispersibility and solubility in water, making it convenient for preparing functional products in various dosage forms such as oral liquids, capsules, and tablets.

[0058] Example 4: Comparative Experiment of Different Lyophilization Protectant Formulations To verify the superiority of the freeze-drying protectant formulation of this invention, the following comparative experiment was conducted: Scutellaria baicalensis extracellular vesicle concentrate was prepared according to the method in Example 1, and was treated using the following three methods: (1) The present invention group: the concentrated solution and 2 wt% dextran sulfate solution were mixed at a volume ratio of 3:1 and then freeze-dried; (2) Control group 1: The concentrate was directly freeze-dried (without a preservative). (3) Control group 2: The concentrate and 5 wt% sucrose solution were mixed at a volume ratio of 1:1 and then freeze-dried.

[0059] After lyophilization and reconstitution, the particle size recovery rate and relative activity of each group of vesicles were determined (with the un-lyophilized concentrate as 100%).

[0060] The results showed that the particle size recovery rate and relative bioactivity of vesicles in each group were detected after reconstitution. The specific data are shown in Table 1.

[0061] Table 1. Effects of different freeze-drying protectant formulations on the freeze-drying recovery of Scutellaria baicalensis extracellular vesicles.

[0062] Note: Particle size recovery rate = (average particle size of vesicles after reconstitution / average particle size of the original solution before lyophilization) × 100%; relative activity is calculated as 100% relative viability of ferroptosis cells in the unlyophilized concentrate, using IEC standards. Determination of the 6 / RSL3 ferroptosis model.

[0063] Control group 1 (directly lyophilized) vesicle structures during freezing The particles were severely damaged during dehydration, with both particle size recovery rate and relative activity below 50%, indicating that the extracellular vesicles of Scutellaria baicalensis could not tolerate freeze-drying stress without a protectant. Control group 2 (5 wt% sucrose 1:1) showed some protective effect, with a particle size recovery rate of 70.5% and relative activity of 72.3%, but significant membrane fusion and activity loss still occurred. The experimental group (concentrate: 2 wt% dextran sulfate solution 3:1) achieved a particle size recovery rate of 92.4% and a relative activity of 94.7%, showing no significant difference from the un-freeze-dried concentrate, fully validating the protective effect of dextran sulfate. The 3:1 volume ratio system demonstrates significant superiority in maintaining vesicle integrity and inhibiting epithelial cell ferroptosis.

[0064] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing extracellular vesicles derived from Scutellaria baicalensis, characterized in that, Includes the following steps: (1) Prepare a homogenate from the raw material of Scutellaria baicalensis, and collect the filtrate after solid-liquid separation; (2) Centrifuge the filtrate to remove large particulate impurities and collect the supernatant; (3) Filter the supernatant through at least two microporous membranes with different pore sizes and collect the filtrate; (4) Pass the filtrate sequentially through a pore size of 180 mm. 220 nm and 25 35 nm nanoporous membrane, Collect concentrated fluid rich in extracellular vesicles; (5) Add sulfated dextran solution to the concentrated solution and freeze dry under vacuum to obtain Scutellaria baicalensis extracellular vesicle freeze-dried powder.

2. The method for preparing extracellular vesicles derived from Scutellaria baicalensis according to claim 1, characterized in that, The specific step (3) is as follows: the supernatant is filtered sequentially through 0.45 μm and 0.22 μm microporous membranes, and the filtrate is collected.

3. The method for preparing extracellular vesicles derived from Scutellaria baicalensis according to claim 1, characterized in that, The volume ratio of the concentrate to the dextran sulfate solution is (2-4):1; the mass concentration of the dextran sulfate solution is 1-3 wt%.

4. The method for preparing extracellular vesicles derived from Scutellaria baicalensis according to claim 1, characterized in that, In step (1), the Scutellaria baicalensis raw material is pre-cooled at -20°C to -10°C before homogenization.

5. The method for preparing extracellular vesicles derived from Scutellaria baicalensis according to claim 1, characterized in that, In step (1), the homogenization is carried out at 36.5-37.5℃.

6. The method for preparing extracellular vesicles derived from Scutellaria baicalensis according to claim 1, characterized in that, In step (5), the conditions for vacuum freeze drying are: temperature -30℃, vacuum degree 5 Pa, and time 36 h.

7. A method for preparing a water-soluble inclusion complex of extracellular vesicles from Scutellaria baicalensis, characterized in that, Includes the following steps: S1: Dissolve cyclodextrin in pure water by heating at 37-45℃ to obtain solution A; S2: Add the freeze-dried powder of Scutellaria baicalensis extracellular vesicles prepared by any one of claims 1-6 to the solution A, and stir the reaction at 37-45℃ for 1-3 h to obtain the inclusion solution; S3: The inclusion solution is freeze-dried under vacuum to obtain a water-soluble inclusion complex of Scutellaria baicalensis extracellular vesicles.

8. The preparation method according to claim 7, characterized in that, The cyclodextrin is selected from one or a combination of two of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin; the mass ratio of the cyclodextrin to the lyophilized powder of Scutellaria baicalensis extracellular vesicles is (1-3):

1.

9. A product for inhibiting epithelial cell ferroptosis, characterized in that, The product is a drug, health food, or functional food, and the product comprises the components described in claim 1. The lyophilized powder of Scutellaria baicalensis extracellular vesicles prepared by any one of the preparation methods described in item 6.

10. The use of the lyophilized extracellular vesicle powder of Scutellaria baicalensis prepared by any one of claims 1-6 in the preparation of products that inhibit epithelial cell ferroptosis.

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