Separation and extraction method and application of extracellular vesicles from brucea javanica

Abstract

The invention relates to the technical field of molecular biology and biomedicine, in particular to a separation and extraction method and application of extracellular vesicles derived from brucea javanica. The separation and extraction method comprises the following steps: S1, preparing supernate containing active ingredients of brucea javanica; s2, sequentially performing micron-scale filtration and nano-scale filtration on the supernate to obtain an extracellular vesicle concentrated solution; and S3, mixing the extracellular vesicle concentrated solution with a glucosamine sulfate solution, and performing freeze-drying to obtain freeze-dried powder of the extracellular vesicles derived from brucea javanica. The separation and extraction method is simple in operation process, mechanical damage to the vesicle structure caused by a traditional ultracentrifugation method is effectively avoided, and the extracted vesicles are uniform and good in form, uniform and concentrated in particle size distribution, good in stability and high in purity.

Description

A method for isolating and extracting extracellular vesicles from Brucea javanica and its application Technical Field

[0001] This invention relates to the fields of molecular biology and biomedical technology, and in particular to a method for isolating and extracting extracellular vesicles derived from Brucea javanica and its application. Background Technology

[0002] Brucea javanica (L.) Merr., commonly known as crow's gall, is the dried, ripe fruit of the plant *Brucea javanica*, belonging to the Simaroubaceae family. It is harvested in autumn when the fruit is ripe, impurities are removed, and it is sun-dried. Other names include "old crow's gall" and "duck egg fruit." It is mainly produced in Guangdong, Guangxi, Taiwan, and Fujian provinces. It is cold in nature, bitter in taste, and slightly toxic. It enters the large intestine and liver meridians, and its functions include clearing heat and drying dampness, stopping dysentery, treating malaria, and killing parasites and detoxifying. Records in *Medical Records of Integrating Chinese and Western Medicine*, *Guangxi Traditional Chinese Medicine*, *Compendium of Materia Medica*, and *Medical Records of Integrating Chinese and Western Medicine* indicate that Brucea javanica is used to treat dysentery and malaria, and externally to treat warts, corns, and hemorrhoids. Modern pharmacological and clinical studies have shown that Brucea javanica mainly contains various active ingredients such as quassinolides, alkaloids, triterpenoids, steroids, phenylpropanoids, and flavonoids, exhibiting anti-tumor, anti-inflammatory, antiviral, and lipid-lowering pharmacological effects. In recent years, the pharmacological effects of the active ingredients in Brucea javanica have been continuously explored, revealing its potential medicinal value.

[0003] Ulcerative colitis is a chronic, nonspecific inflammatory bowel disease of unknown etiology, belonging to one of the major subtypes of inflammatory bowel disease and falling under the category of "dysentery" in Traditional Chinese Medicine. Typical pathological features of ulcerative colitis include persistent inflammation of the intestinal mucosa, damage to epithelial structures, and immune dysfunction. Clinical manifestations are mainly persistent or recurrent diarrhea, mucus and bloody stools, abdominal pain, and tenesmus. Modern pharmacological studies have shown that the occurrence and development of this disease are closely related to multiple mechanisms, including intestinal inflammatory response, mucosal barrier dysfunction, oxidative stress imbalance, and intestinal flora dysbiosis. In clinical treatment, current methods often involve drug enemas for local symptom relief or systemic therapy with monoclonal antibodies. However, existing therapies still face certain limitations. While drug enemas can provide local relief, patient comfort during the procedure is poor, and the infused solution is easily expelled within a short time, resulting in short drug retention time and insufficient absorption, thus affecting treatment efficacy. Furthermore, monoclonal antibody drugs are expensive, and long-term use may lead to drug resistance.

[0004] Plant-derived extracellular vesicles (PDEVs) are an important class of substances secreted by plant cells, containing various biological information such as carbohydrates, proteins, lipids, RNA, and metabolites, playing a crucial role in intercellular communication. Studies have shown that plant-derived PDEVs possess anti-inflammatory and tissue-repair-promoting biological activities, and can be used to treat ulcerative colitis, photoaging of the skin, and diabetic wounds. Furthermore, plant PDEVs have the advantages of wide availability and high safety, making them suitable for assembling carriers for small molecule drugs to prevent and treat diseases. The efficacy of plant PDEVs is closely related to their source; PDEVs from different sources exhibit significant differences in chemical composition, biological activity, and therapeutic effects. Current research has found that PDEVs from Coptis chinensis, ginger, and Atractylodes macrocephala can treat ulcerative colitis, while those from Platycodon grandiflorus and Artemisia annua can treat triple-negative breast cancer. Currently, there are no reports of PDEVs from Brucea javanica having a therapeutic effect on ulcerative colitis.

[0005] The isolation of extracellular vesicles from *Brucea javanica* cells has been reported primarily using the classic multi-step ultracentrifugation method. However, this method is not only time-consuming but also generally has low recovery efficiency. More importantly, the shearing forces from repeated centrifugation may cause mechanical damage to the vesicle structure, which contradicts the requirement to maintain its biological activity and limits the reliability of subsequent studies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a simple method for extracting and separating extracellular vesicles from Brucea javanica, which can be applied to the treatment of ulcerative colitis.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a method for separating and extracting extracellular vesicles derived from Brucea javanica, comprising the following steps: S1: preparing a supernatant containing the effective components of Brucea javanica; S2: subjecting the supernatant to micron-level filtration and nano-level filtration in sequence to obtain a concentrated extracellular vesicle solution; S3: mixing the concentrated extracellular vesicle solution with a glucosamine sulfate solution and freeze-drying to obtain a freeze-dried powder of extracellular vesicles derived from Brucea javanica.

[0008] Another technical solution adopted in this invention is the application of extracellular vesicles derived from Brucea javanica in the preparation of drugs for treating ulcerative colitis.

[0009] The beneficial effects of this invention are as follows: the separation and extraction method of this invention has a simple operation process, and the extracted vesicles have uniform morphology, uniform and concentrated particle size distribution, good stability, and high purity. Specifically, nanoscale filtration is used to extract and purify extracellular vesicles of *Brucea javanica*, achieving efficient enrichment of vesicles through gentle filtration and concentration steps, effectively avoiding the mechanical damage to the vesicle structure caused by traditional ultracentrifugation. Using glucosamine sulfate solution instead of traditional PBS (phosphate buffered saline) as the storage medium for extracellular vesicles and mixing it with the extracellular vesicle concentrate for freeze-drying better maintains the integrity of the vesicle structure, thereby ensuring the long-term stability of its internal bioactive substances. Attached Figure Description

[0010] Figure 1 is a schematic diagram of the morphology of extracellular vesicles derived from *Brucea javanica* under transmission electron microscopy in an embodiment of the present invention; Figure 2 is a particle size diagram of extracellular vesicles derived from *Brucea javanica* detected in an embodiment of the present invention; Figure 3 is a potential diagram of extracellular vesicles derived from *Brucea javanica* detected in an embodiment of the present invention; Figure 4 shows the effect of extracellular vesicles derived from *Brucea javanica* on the body weight of mice with ulcerative colitis in an embodiment of the present invention; Figure 5 shows the effect of extracellular vesicles derived from *Brucea javanica* on the colon length of mice with ulcerative colitis in an embodiment of the present invention; Figure 6... Figure 7 shows the effect of extracellular vesicles derived from *Brucea javanica* on the Disease Activity Index (DAI) of mice with ulcerative colitis in this embodiment of the invention; Figure 8 shows the effect of extracellular vesicles derived from *Brucea javanica* on the H&E (hematoxylin-eosin) staining results of colon tissue of mice with ulcerative colitis in this embodiment of the invention; Figure 9 shows the effect of extracellular vesicles derived from *Brucea javanica* on the barrier proteins ZO-1 (closed band protein 1) and Occludin (closure protein) in colon tissue of mice with ulcerative colitis in this embodiment of the invention; Figure 10 shows the effect of extracellular vesicles derived from *Brucea javanica* on the levels of inflammatory cytokines TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), and IL-6 (interleukin-6) in mice with ulcerative colitis in this embodiment of the invention; Figure 11 shows the effect of extracellular vesicles derived from *Brucea javanica* on the levels of iNOS (inducible nitric oxide synthase) and TNF-α in the RAW264.7 macrophage M1 polarization model induced by LPS (lipopolysaccharide) and IFN-γ (interferon-γ) in this embodiment of the invention. The effect of mRNA levels; Figure 11 shows the effect of extracellular vesicles derived from Brucea javanica on cell viability in the IEC-6 intestinal epithelial cell injury model induced by 3wt% DSS (sodium dextran sulfate) in the embodiments of the present invention. Detailed Implementation

[0011] 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.

[0012] A method for separating and extracting extracellular vesicles from Brucea javanica includes the following steps: S1: preparing a supernatant containing the effective components of Brucea javanica; S2: subjecting the supernatant to micron-level filtration and nano-level filtration in sequence to obtain a concentrated extracellular vesicle solution; S3: mixing the concentrated extracellular vesicle solution with glucosamine sulfate solution and freeze-drying to obtain a freeze-dried powder of extracellular vesicles from Brucea javanica.

[0013] As described above, the beneficial effects of this invention are as follows: The separation and extraction method of this invention first performs micron-level filtration on the supernatant to remove micron-level residual substances; then, it performs nano-level filtration to obtain a concentrated extracellular vesicle solution. Using nano-level filtration to extract and purify the extracellular vesicles of *Brucea javanica* effectively avoids the mechanical damage to the vesicle structure caused by traditional ultracentrifugation. Through gentle filtration and concentration steps, efficient enrichment of vesicles can be achieved under low-speed conditions. The concentrated extracellular vesicle solution is mixed with glucosamine sulfate solution and freeze-dried. Using glucosamine sulfate solution instead of traditional PBS as the storage solution for extracellular vesicles effectively delays the structural denaturation of vesicle membrane proteins during freezing, better maintaining the integrity of the vesicle structure, thereby ensuring the long-term stability of its internal bioactive substances.

[0014] The separation and extraction method of the present invention has a simple operation process, and the extracted vesicles have good morphology, uniform and concentrated particle size distribution, good stability, and high purity, and have good application prospects.

[0015] Further, S1 specifically involves: soaking Brucea javanica in a pre-cooled phosphate buffer solution, then sequentially crushing, juicing, and centrifuging to obtain a supernatant containing the effective components of Brucea javanica.

[0016] Furthermore, the pre-cooling temperature of the phosphate buffer solution is -25 to -15°C. As described above, the fruit of *Brucea javanica* is soaked in a low-temperature phosphate buffer solution, followed by direct crushing, juicing, and centrifugation to gently release the vesicles. This step uses an inexpensive and readily available phosphate buffer solution instead of traditional expensive reagents such as iodixanol, which better maintains the biological activity and stability of extracellular vesicles during the extraction process.

[0017] Furthermore, the centrifugation temperature is 3~5℃, and the relative centrifugal force is 11000~13000g.

[0018] As described above, low-temperature centrifugation can inhibit enzyme activity, prevent vesicles from being degraded by proteases during extraction, and maintain their integrity. A relative centrifugal force of 11,000~13,000g can effectively precipitate plant tissue fragments, large particulate impurities, and extracellular vesicles of the target size, while avoiding excessive mechanical compression and aggregation of vesicles caused by excessive centrifugal force.

[0019] Furthermore, micron-level filtration is achieved by sequentially setting a first micron-level microporous filter membrane and a second micron-level microporous filter membrane, wherein the pore size of the first micron-level microporous filter membrane is larger than that of the second micron-level microporous filter membrane.

[0020] Furthermore, nanoscale filtration is achieved by sequentially setting a first nanoporous membrane and a second nanoporous membrane, wherein the pore size of the first nanoporous membrane is larger than that of the second nanoporous membrane.

[0021] As can be seen from the above description, the pore size of the first micron-sized microporous filter membrane is larger than that of the second micron-sized microporous filter membrane, and the pore size of the first nanoporous membrane is larger than that of the second nanoporous membrane, which can effectively filter while avoiding the influence of flow rate.

[0022] Furthermore, the first micron-sized microporous filter membrane has a pore size of 0.45 μm, and the second micron-sized microporous filter membrane has a pore size of 0.22 μm.

[0023] As described above, a 0.45μm filter membrane is first used to remove most of the micron-sized residues, cell debris, and some bacteria; then a 0.22μm filter membrane is used for final sterilization to remove all bacteria. This sequence avoids large particles directly clogging the more precise and expensive 0.22μm filter membrane, improves filtration efficiency, extends filter membrane life, and ensures the sterility of the final vesicle concentrate.

[0024] Furthermore, the pore size of the first nanoporous membrane is 180~220nm.

[0025] Furthermore, the pore size of the second nanoporous membrane is 25~35nm.

[0026] As described above, the first nanoporous membrane allows most small extracellular vesicles (such as exosomes, typically 30-150 nm in size) and even smaller soluble proteins to pass through, while retaining larger vesicles (such as microvesicles) with a particle size greater than 220 nm, organelle fragments, and residual microscopic plant debris, thus completing preliminary size screening. The second nanoporous membrane can retain vesicles within the target exosome size range (>30 nm), while allowing smaller non-vesicle impurities such as proteins and nucleic acids to pass through and be filtered out, thereby significantly improving the purity of vesicle products. The two form a continuous "filtration window," realizing a refined purification process from "removing large particles" to "retaining target vesicles and removing small impurities," which is a key step in obtaining high-purity, highly uniform extracellular vesicles.

[0027] Furthermore, the freeze-drying temperature is -35 to -25°C, and the vacuum degree is 4 to 6 Pa.

[0028] Furthermore, the volume ratio of the extracellular vesicle concentrate to the glucosamine sulfate solution was 1.8~2.2:1.

[0029] As can be seen from the above description, if the concentration of glucosamine sulfate solution is too low, precipitation will be incomplete, and if it is too high, non-specific co-precipitation of impurities may occur.

[0030] Another technical solution adopted in this invention is the application of extracellular vesicles derived from Brucea javanica in the preparation of drugs for treating ulcerative colitis.

[0031] As described above, existing research on *Brucea javanica* mainly focuses on its crude extracts such as decoctions and alcoholic extracts, oil emulsions, or quassinolide components (such as crocin A, crocin D, and crocinol). However, the extracellular vesicles of *Brucea javanica* are nanoscale lipid bilayer vesicles naturally secreted by plant cells. They encapsulate specific bioactive components such as proteins, lipids, and nucleic acids, and are fundamentally different from traditional extracts in terms of structure, composition, and functional mechanism.

[0032] This invention, through verification, revealed that extracellular vesicles derived from *Brucea javanica* exhibit excellent anti-inflammatory and intestinal barrier repair activities. Extracellular vesicles of *Brucea javanica* at concentrations of 5 and 10 mg / mL inhibited the M1 polarization phenotype of Raw264.7 macrophages, reduced the levels of inflammatory cytokines iNOS and TNF-α mRNA, and improved IEC-6 intestinal epithelial cell damage, demonstrating good anti-inflammatory and intestinal barrier protective activities. Therefore, extracellular vesicles derived from *Brucea javanica* can be used to prepare drugs with anti-inflammatory and intestinal barrier repair activities, namely, drugs for treating ulcerative colitis.

[0033] Another technical solution adopted in this invention is: a drug for treating ulcerative colitis, comprising excipients and extracellular vesicles of Brucea javanica.

[0034] Furthermore, the extracellular vesicles of Brucea javanica are Brucea javanica extracellular vesicle solution or Brucea javanica extracellular vesicle lyophilized powder.

[0035] Furthermore, the excipient is cyclodextrin, which encapsulates the extracellular vesicles of *Brucea javanica* to form inclusion complexes.

[0036] Furthermore, cyclodextrins include at least one of α-cyclodextrin, β-cyclodextrin, hydroxyethyl-β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.

[0037] Preferably, the cyclodextrin is hydroxypropyl-β-cyclodextrin.

[0038] Another technical solution adopted in this invention is: a method for preparing a drug for treating ulcerative colitis, comprising the following steps: Step 1: adding pure water to cyclodextrin and heating to dissolve it to obtain solution A; Step 2: stirring solution A, adding Brucea javanica extracellular vesicle solution or Brucea javanica extracellular vesicle lyophilized powder, and carrying out an inclusion reaction to obtain an inclusion solution; Step 3: freeze-drying the inclusion solution to obtain an inclusion complex.

[0039] Furthermore, the heating temperature in step 1 is 37~45℃.

[0040] Furthermore, in step 2, solution A is stirred at 37~45℃ for 1~3 h.

[0041] Furthermore, the mass ratio of cyclodextrin to the extracellular vesicles of *Brucea javanica* in the solution or lyophilized powder of *Brucea javanica* extracellular vesicles is 1 to 3:1.

[0042] Preferably, the mass ratio of cyclodextrin to the extracellular vesicles of *Brucea javanica* in the solution or lyophilized powder is 1:1.

[0043] Example 1 of the present invention is: a method for isolating and extracting extracellular vesicles from Brucea javanica, the specific steps of which are as follows: S1: Take Brucea javanica fruit, soak it in a phosphate buffer solution pre-cooled at -20℃ for 2 hours, then crush and juice it using a high-speed blender, filter it with gauze, and collect the filtrate; S2: Centrifuge the filtrate at 12000g for 10 minutes at 4℃, discard the precipitate, and obtain the supernatant containing the effective components of Brucea javanica; S3: Pass the supernatant sequentially through a microporous membrane with a pore size of 0.45μm, Microporous membranes with a pore size of 0.22 μm were used to filter out micron-sized residual substances, and the filtrate was collected. S4: The filtrate collected in S3 was passed sequentially through nanoporous membranes with a pore size of 200 nm and 30 nm to obtain extracellular vesicle concentrate. The extracellular vesicle concentrate was stored at -80℃ and had a shelf life of 90 days. S5: The extracellular vesicle concentrate was mixed with glucosamine sulfate solution (concentration of 2 wt%) at a volume ratio of 2:1 and freeze-dried at a temperature of -30℃, a vacuum degree of 5 Pa, and a time of 36 h to obtain freeze-dried powder of extracellular vesicles derived from Brucea javanica. The freeze-dried powder was stored at -80℃.

[0044] Example 2 of the present invention is a method for isolating and extracting extracellular vesicles from Brucea javanica, the specific steps of which are as follows: S1: Take Brucea javanica fruit, soak it in a phosphate buffer solution pre-cooled at -25℃ for 2 hours, then crush and juice it using a high-speed blender, filter it with gauze, and collect the filtrate; S2: Centrifuge the filtrate at 11000g for 10 minutes at 3℃, discard the precipitate, and obtain the supernatant containing the effective components of Brucea javanica; S3: Pass the supernatant sequentially through a microporous membrane with a pore size of 0.45μm and a pore size of... Microporous membranes with a diameter of 0.22 μm were used to filter out micron-sized residual substances, and the filtrate was collected. S4: The filtrate collected in S3 was passed sequentially through nanoporous membranes with a pore size of 180 nm and 25 nm to obtain extracellular vesicle concentrate. The extracellular vesicle concentrate was stored at -80℃ and had a shelf life of 90 days. S5: The extracellular vesicle concentrate was mixed with glucosamine sulfate solution (concentration of 2 wt%) at a volume ratio of 1.8:1 and freeze-dried at a temperature of -35℃, a vacuum degree of 4 Pa, and a time of 36 h to obtain freeze-dried powder of extracellular vesicles derived from Brucea javanica. The freeze-dried powder was stored at -80℃.

[0045] Example 3 of the present invention is: a method for isolating and extracting extracellular vesicles from Brucea javanica, the specific steps of which are as follows: S1: Take Brucea javanica fruit, soak it in a phosphate buffer solution pre-cooled at -15℃ for 2 hours, then crush and juice it using a high-speed blender, filter it with gauze, and collect the filtrate; S2: Centrifuge the filtrate at 13000g for 10 minutes at 5℃, discard the precipitate, and obtain the supernatant containing the effective components of Brucea javanica; S3: Pass the supernatant sequentially through a microporous membrane with a pore size of 0.45μm and a pore size of... The micron-sized residues were removed by filtration through a 0.22 μm microporous membrane, and the filtrate was collected. S4: The filtrate collected in S3 was sequentially passed through a nanoporous membrane with a pore size of 220 nm and a nanoporous membrane with a pore size of 25~35 nm to obtain an extracellular vesicle concentrate. The extracellular vesicle concentrate was stored at -80℃ and had a shelf life of 90 days. S5: The extracellular vesicle concentrate was mixed with glucosamine sulfate solution (concentration of 2 wt%) at a volume ratio of 2.2:1 and freeze-dried at a temperature of -25℃, a vacuum degree of 6 Pa, and a time of 36 h to obtain a freeze-dried powder of extracellular vesicles derived from Brucea javanica. The freeze-dried powder was stored at -80℃.

[0046] Example 4 of the present invention is to verify the effect of extracellular vesicles derived from Brucea javanica on mice with ulcerative colitis induced by 3% sodium dextran sulfate. The extracellular vesicles derived from Brucea javanica in the following example are lyophilized powders from Example 1.

[0047] 1. Experimental Methods: Fifty male SPF-grade C57BL / 6J mice (22-24 g) were randomly divided into five groups: Normal, Model Group (DSS), Mesalazine Group (5-ASA, 200 mg / kg), Low-dose BDEVs-L (10 mg / kg), and High-dose BDEVs-H (20 mg / kg), with ten mice in each group. Except for the Normal group, the other groups were allowed free access to 3wt% sodium dextran sulfate to establish an ulcerative colitis mouse model, while simultaneously receiving an equal volume of physiological saline or a corresponding volume of the drug via gavage for 7 days. Mouse weight was recorded daily during the experiment, and the percentage of weight loss, fecal morphology, and bloody stool were scored according to the Disease Activity Index (DAI) criteria.

[0048] Observation indicators: Macroscopic indicators: mouse body weight, DAI index (disease activity index), serum, colon length; Histopathological examination: H&E staining and PCNA (proliferating cell nuclear antigen) staining of colon tissue; Expression of colon tissue barrier-related proteins ZO-1 and Occludin; Levels of colon tissue inflammatory cytokines TNF-α, IL-1β and IL-6.

[0049] 2. Experimental Results 2.1 Identification of Extracellular Vesicles Derived from Brucea javanica Extracellular vesicles were identified using transmission electron microscopy. It was found that the extracellular vesicles derived from Brucea javanica were round or oval in shape and had a complete membrane structure, as shown in Figure 1.

[0050] The size and potential of extracellular vesicles derived from Brucea javanica were detected. The results showed that most of the extracted extracellular vesicles were between 120 and 130 nm in size and had a potential of around -40 mV, as shown in Figures 2 and 3.

[0051] The above results indicate that the extracellular vesicles isolated from Brucea javanica in this invention meet the standard characteristics after identification, laying a solid foundation for subsequent pharmacodynamic studies and ensuring the quality control of experimental samples.

[0052] 2.2 Pharmacological Study on Mice with Ulcerative Colitis Figure 4 shows the effect of extracellular vesicles derived from *Brucea javanica* on the body weight of mice with ulcerative colitis in this embodiment; Figure 5 shows the effect of extracellular vesicles derived from *Brucea javanica* on the colon length of mice with ulcerative colitis in this embodiment; Figure 6 shows the effect of extracellular vesicles derived from *Brucea javanica* on the disease activity index (DAI) of mice with ulcerative colitis in this embodiment. As shown in Figures 4-6, compared with the normal group, the model group mice showed significantly reduced body weight and exhibited typical pathological symptoms such as hematochezia and loose stools. The disease activity index was significantly increased, and the colon length was significantly shortened, indicating that the ulcerative colitis model was successfully established. Compared with the model group, no hematochezia or loose stools were observed in the 5-ASA group and the low- and high-dose *Brucea javanica* extracellular vesicle groups during treatment, and all significantly improved the weight loss of mice, especially effectively curbing the weight loss by the seventh day. Furthermore, *Brucea javanica* extracellular vesicle intervention significantly reduced the DAI index of the model mice and effectively alleviated the colon shortening phenomenon. The above results indicate that the outer vesicles of Brucea javanica have a potential alleviating effect on ulcerative colitis in mice.

[0053] 2.2 Effects on the histopathology of mouse colon tissue. The H&E staining results are shown in Figure 7. Compared with the normal group, the colon tissue of the model group mice exhibited typical pathological features of colitis, with severe damage to the mucosal structure, disappearance of numerous crypts, a significant reduction in goblet cells, and extensive infiltration of inflammatory cells into the mucosa and submucosa. After intervention with 5-ASA and *Brucea javanica* external vesicles, the pathological damage to the colon tissue was significantly improved. The colon structure became clearer and more intact, the mucosal structure became more continuous, the crypt structure was repaired, and the extent and severity of inflammatory cell infiltration decreased. These results indicate that *Brucea javanica* external vesicles have a significant repairing effect on colon tissue damage in mice with ulcerative colitis.

[0054] 2.3 Expression of ZO-1 and Occludin, Barrier-Related Proteins in Mouse Colon: To assess the integrity of the colonic mucosal physical barrier, the expression and distribution of the tight junction core proteins ZO-1 and Occludin were detected by immunofluorescence staining. Figure 8 shows that in the normal group, the colonic epithelial cells of mice had clear outlines, and ZO-1 and Occludin were continuously and completely expressed along the cell membrane, forming a dense network structure, indicating that the tight junction structure was intact. In the model group, the fluorescence signal intensity of these two proteins was significantly weakened, and the normal continuous distribution was severely disrupted, appearing discontinuous, blurred, or even completely absent. Compared with the model group, the fluorescence signal of ZO-1 and Occludin in the colonic mucosa was significantly enhanced after intervention with *Brucea javanica* exovesicles, and their continuity along the cell boundary was significantly restored. This indicates that *Brucea javanica* exovesicles can effectively promote the reorganization and expression of tight junction proteins, thereby repairing the intestinal epithelial barrier function in mice with ulcerative colitis.

[0055] 2.4 Effects on the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in mouse colonic tissue. To assess the inflammatory status of colonic tissue, the levels of key pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were detected using ELISA. As shown in Figure 9, A represents TNF-α level, B represents IL-1β level, and C represents IL-6 level. Compared with the normal group, the concentrations of all three cytokines in the colonic tissue of the model group mice were significantly upregulated, indicating that the colon of the model mice was in an inflammatory response. Compared with the model group, the levels of TNF-α, IL-1β, and IL-6 in mice treated with *Brucea javanica* exovesicles were significantly decreased. These results collectively indicate that *Brucea javanica* exovesicles can effectively alleviate the inflammatory response of ulcerative colitis.

[0056] Example 5 of the present invention is to verify the effect of extracellular vesicles derived from Brucea javanica on M1 macrophages. The extracellular vesicles derived from Brucea javanica in the following example are lyophilized powders from Example 1.

[0057] 1. Experimental Methods: RAW264.7 cells in logarithmic growth phase were collected at a concentration of 5 × 10⁻⁶ cells / year. 5 Macrophages were seeded at a density of 1 / mL in 6-well plates, and phorbol-12-myristate-13-acetate (PMA, 50 nM) was added to each well to induce maturation for 24 h. Except for the blank wells, LPS (100 ng / mL) + IFN-γ (20 ng / mL) were added to the other wells to induce macrophage polarization towards the M1 phenotype. Treatment wells were treated with *Brucea javanica* exovesicles (0.5, 1, 5, and 10 mg / kg, respectively) for 48 h. After the experiment, cells were collected, and the levels of iNOS and TNF-α mRNA were detected using qPCR (real-time quantitative polymerase chain reaction).

[0058] 2. Experimental Results The qPCR results are shown in Figure 10, where A represents the mRNA level of iNOS and B represents the mRNA level of TNF-α. Compared with the control group, LPS and IFN-γ stimulation significantly upregulated the mRNA levels of the M1 markers iNOS and TNF-α in macrophages, indicating that the macrophage M1 polarization model was successfully constructed. Compared with the model group, 0.5 and 1 mg / kg of *Brucea javanica* extracellular vesicles had no significant inhibitory effect on the mRNA levels of iNOS and TNF-α; when the dose was increased to 5 and 10 mg / kg, *Brucea javanica* extracellular vesicles significantly downregulated the mRNA expression of iNOS and TNF-α. The results confirm that *Brucea javanica* extracellular vesicles can inhibit macrophage M1 polarization.

[0059] Example 6 of the present invention is: the effect of extracellular vesicles derived from Brucea javanica on a model of intestinal epithelial cell damage induced by sodium dextran sulfate. The extracellular vesicles derived from Brucea javanica in the following example are lyophilized powders from Example 1.

[0060] 1. Experimental Methods: IEC-6 cells in logarithmic growth phase were collected at a concentration of 2 × 10⁻⁶. 4 Cells were seeded at a density of 1 / mL in 96-well plates. Except for the blank wells, each well was incubated with 3wt% sodium dextran sulfate for 4 hours. The supernatant was then discarded, and extracellular vesicles of *Brucea javanica* (0.5, 1, 5, and 10 mg / kg, respectively) were added for 24 hours of intervention. Cell viability was assessed using the CCK-8 assay after the experiment.

[0061] 2. Experimental Results The results are shown in Figure 11. Compared with the control group, the viability of IEC-6 cells treated with 3% DSS was significantly reduced. Compared with the model group, 0.5 and 1 mg / kg of *Brucea javanica* extracellular vesicles had no significant effect on IEC-6 cell viability; however, when the dose was increased to 5 and 10 mg / kg, *Brucea javanica* extracellular vesicles significantly upregulated IEC-6 cell viability. These results confirm that *Brucea javanica* extracellular vesicles can improve intestinal epithelial cell damage.

[0062] Example 7 of the present invention is: a drug for treating ulcerative colitis, comprising hydroxypropyl-β-cyclodextrin and a solution of croton tigrin extracellular vesicles, wherein the hydroxypropyl-β-cyclodextrin encapsulates the croton tigrin extracellular vesicles to form an inclusion complex.

[0063] Example 8 of the present invention is: a drug for treating ulcerative colitis, comprising α-cyclodextrin, β-cyclodextrin and a solution of croton tigrin extracellular vesicles, wherein α-cyclodextrin and β-cyclodextrin encapsulate croton tigrin extracellular vesicles to form an inclusion complex.

[0064] Example 9 of the present invention is: a drug for treating ulcerative colitis, comprising hydroxyethyl-β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin and a solution of croton tigrin extracellular vesicles, wherein hydroxyethyl-β-cyclodextrin, methyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin encapsulate croton tigrin extracellular vesicles to form an inclusion complex.

[0065] Example 10 of the present invention is a method for preparing a drug for treating ulcerative colitis, the specific steps of which are as follows: Step 1: Add pure water to hydroxypropyl-β-cyclodextrin and heat at 37°C to dissolve it to obtain solution A; Step 2: Place solution A at 37°C and stir magnetically for 3 h, add Brucea javanica extracellular vesicle solution, and carry out inclusion reaction at 37°C for 2 h; the mass ratio of cyclodextrin to Brucea javanica extracellular vesicle solution or Brucea javanica extracellular vesicle freeze-dried powder is 1:1 to obtain inclusion solution; Step 3: Freeze-dry the inclusion solution to obtain inclusion complex.

[0066] Example 11 of the present invention is a method for preparing a drug for treating ulcerative colitis, the specific steps of which are as follows: Step 1: Add pure water to hydroxypropyl-β-cyclodextrin and heat at 45°C to dissolve it to obtain solution A; Step 2: Place solution A at 45°C and stir magnetically for 1 h, add Brucea javanica extracellular vesicle solution or Brucea javanica extracellular vesicle lyophilized powder, and carry out an inclusion reaction at 37°C for 2 h; the mass ratio of cyclodextrin to Brucea javanica extracellular vesicle solution or Brucea javanica extracellular vesicle lyophilized powder is 3:1, to obtain an inclusion solution; Step 3: Freeze-dry the inclusion solution to obtain the inclusion compound.

[0067] In summary, the method and application for the separation and extraction of extracellular vesicles from Brucea javanica provided by this invention have the following advantages: 1. The Brucea javanica fruit is soaked in a low-temperature phosphate buffer solution, and then directly crushed, juiced and centrifuged to gently release the vesicles, which can better maintain the biological activity and stability of the extracellular vesicles.

[0068] 2. Nanoscale filtration is used to extract and purify extracellular vesicles of Brucea javanica, effectively avoiding the mechanical damage to the vesicle structure caused by traditional ultracentrifugation. 3. The separation and extraction method has a simple operation process, and the extracted vesicles have good morphology, uniform and concentrated particle size distribution, good stability, and high purity, showing good application prospects. 4. Glucosamine sulfate solution is used instead of traditional PBS as the storage medium for extracellular vesicles during freeze-drying, effectively delaying the structural denaturation of vesicle membrane proteins during freezing, thereby ensuring the long-term stability of the internal bioactive substances.

[0069] 5. Extracellular vesicles derived from Brucea javanica exhibit good anti-inflammatory and intestinal barrier repair activities, can reduce the levels of inflammatory cytokines iNOS and TNF-α mRNA, and improve IEC-6 intestinal epithelial cell damage.

[0070] 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 isolating and extracting extracellular vesicles derived from Brucea javanica, characterized in that, Includes the following steps: S1: Prepare a supernatant containing the effective components of Brucea javanica; S2 S3: The supernatant is subjected to micron-level filtration and nano-level filtration in sequence to obtain extracellular vesicle concentrate; S4: The extracellular vesicle concentrate is mixed with glucosamine sulfate solution and freeze-dried to obtain freeze-dried powder derived from extracellular vesicles of Brucea javanica.

2. The method for isolating and extracting extracellular vesicles from Brucea javanica according to claim 1, characterized in that, S1 specifically involves soaking Brucea javanica in a pre-cooled phosphate buffer solution, then sequentially crushing, juicing, and centrifuging to obtain a supernatant containing the effective components of Brucea javanica.

3. The method for isolating and extracting extracellular vesicles from Brucea javanica according to claim 2, characterized in that, The centrifugation temperature is 3~5℃, and the relative centrifugal force is 11000~13000g.

4. The method for isolating and extracting extracellular vesicles from Brucea javanica according to claim 1, characterized in that, The micron-level filtration is achieved by sequentially setting a first micron-level microporous filter membrane and a second micron-level microporous filter membrane, wherein the pore size of the first micron-level microporous filter membrane is larger than that of the second micron-level microporous filter membrane.

5. The method for isolating and extracting extracellular vesicles from Brucea javanica according to claim 1, characterized in that, The nanoscale filtration is achieved by sequentially setting a first nanoporous membrane and a second nanoporous membrane, wherein the pore size of the first nanoporous membrane is larger than that of the second nanoporous membrane.

6. The method for isolating and extracting extracellular vesicles from Brucea javanica according to claim 5, characterized in that, The pore size of the first nanoporous membrane is 180~220nm.

7. The method for isolating and extracting extracellular vesicles from Brucea javanica according to claim 5, characterized in that, The pore size of the second nanoporous membrane is 25~35nm.

8. The method for isolating and extracting extracellular vesicles from Brucea javanica according to claim 1, characterized in that, The freeze-drying temperature is -35 to -25°C, and the vacuum degree is 4 to 6 Pa.

9. The method for isolating and extracting extracellular vesicles from Brucea javanica according to claim 1, characterized in that, The volume ratio of the extracellular vesicle concentrate to the glucosamine sulfate solution is 1.8~2.2:

1.

10. Application of extracellular vesicles derived from Brucea javanica in the preparation of drugs for treating ulcerative colitis.

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