Application of Chinese torreya exosome nano-vesicles in preparation of medicine for treating or improving ulcerative colitis

Abstract

The invention belongs to the field of medicines, and particularly relates to application of Chinese torreya exosome nano-vesicles in preparation of medicines for treating or improving ulcerative colitis. The invention discloses a traditional medicine which discovers that XFEV not only can obviously reduce proinflammatory factors TNF-alpha and IL-6 and improve an anti-inflammatory factor IL-10, but also can repair a colonic mucosa crypt structure and enhance an intestinal barrier function, and is inferred to indirectly resist inflammation by regulating intestinal flora and is different from a single-mechanism traditional medicine. A plant exosome preparation process is optimized, uniform particles are obtained by combining differential centrifugation with sucrose gradient purification, the curative effect of XFEV delivered by an embedding method is proved to be remarkably superior to that of simple mixing, the targeting and protective advantages of the exosome as a natural carrier are highlighted, and a low-toxicity and high-efficiency new strategy is provided for UC treatment.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and in particular relates to the application of Torreya grandis exosome nanovesicles in the preparation of drugs for treating or improving ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a type of chronic inflammatory bowel disease, clinically characterized by mucus and bloody stools, abdominal pain, diarrhea, and weight loss. Endoscopic findings often reveal continuous, diffuse erosions, frequently accompanied by ulcers, edema, congestion, and inflammatory polyp formation. The disease course is protracted and prone to relapse. Maintenance therapy includes 5-aminosalicylic acid, thiopurines, biologics, and small molecule drugs. However, relapse is common after discontinuation of medication, either due to increased drug tolerance from long-term use or the combined side effects of high-dose medication.

[0003] Torreya grandis ( Torreyagrandis Torreya seeds contain many bioactive components, including polyphenols, tocopherols, sterols, squalene, and unsaturated fatty acids (such as oleic acid, linoleic acid, and pinocembryonic acid). These components give Torreya seeds excellent health benefits. Torreya seed extract (EST) and Torreya kernel oil (TKO) can significantly regulate the composition of the gut microbiota. After TKO treatment, beneficial bacteria (such as Firmicutes) in the mouse gut significantly increased, while harmful bacteria (such as...)... Bacteroidetes, Desulfobacteria, Fusobacteria () decreased. Furthermore, TKO also significantly increased. Akkermansia The abundance of this microbial community is negatively correlated with inflammation, obesity, diabetes, and colon cancer. Torreya grandis can enrich the diversity of the gut microbiota and regulate the types of gut microbial metabolites, such as short-chain fatty acids (SCFAs). These microbial metabolites have positive immunomodulatory and anti-inflammatory effects.

[0004] With the increasing application of nanotechnology in the medical field, exosomes and their analogues have become a new research hotspot. Exosomes are nanoscale vesicles secreted by cells that can carry many bioactive components and participate extensively in physiological and pathological processes such as immune regulation and angiogenesis. They are relatively abundant in origin, structurally stable, biocompatible, and possess natural homing properties, allowing them to penetrate biological barriers, thus making them considered ideal drug delivery carriers. Plant-derived exosome-like nanoparticles (such as ginger, grape, and Dendrobium officinale) have been shown to have significant alleviating effects on ulcerative colitis, but there are currently no reports on exosomes derived from Torreya grandis. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of Torreya grandis exosome nanovesicles in the preparation of drugs for treating or improving ulcerative colitis.

[0006] Application of Torreya grandis exosome nanovesicles in the preparation of drugs for treating or improving ulcerative colitis.

[0007] As a further aspect of the present invention, the preparation process of the Torreya grandis exosome nanovesicles involves sequentially purifying Torreya grandis juice through differential centrifugation, ultracentrifugation, ultrafiltration, and sucrose gradient.

[0008] As a further aspect of the present invention, the preparation of the Torreya grandis exosome nanovesicles specifically includes the following steps: (1) After the viscous Torreya grandis juice was centrifuged four times at different speeds to collect the supernatant, the supernatant was filtered through a 0.22 μm filter membrane and collected. (2) The supernatant after filtration by the filter membrane is subjected to ultrafiltration, and the upper solution is taken and filtered with a 0.22μm aqueous filter head for sterilization; (3) Centrifuge the filtered sterilized solution, resuspend it with PBS buffer, purify it in a sucrose gradient, take 45-60% layer solution, centrifuge, wash the precipitate with an appropriate amount of PBS buffer, centrifuge again, and precipitate.

[0009] As a further embodiment of the present invention, in step (1), the Torreya grandis juice is obtained by juicing Torreya grandis fruit after removing the shell.

[0010] In step (1), the first centrifugation is 1×10⁻⁶. 3 g, centrifuge horizontally at 4℃ for 10 min, then centrifuge a second time at 2×10⁻⁶. 3 g, centrifuge horizontally at 4℃ for 20 min, then centrifuge a third time at 4×10⁻⁶. 3 g, centrifuge horizontally at 4℃ for 30 min, the fourth centrifugation is 1×10. 4 g, centrifuge horizontally at 4℃ for 1 hour.

[0011] As a further aspect of the present invention, in step (2), the ultrafiltration parameter is 5×10 3 g, centrifuge horizontally at 4℃ for 30 minutes.

[0012] As a further embodiment of the present invention, in step (3), sucrose gradients of 8%, 30%, 45% and 60% are dissolved in 20mM Tri-HCl at pH=7.2.

[0013] As a further embodiment of the present invention, in step (3), after the precipitate is weighed, it is resuspended in PBS buffer to form Torreya grandis exosome nanovesicle solution XFEV and stored in an environment of -80°C.

[0014] As a further aspect of the present invention, the preparation of the Torreya grandis exosome nanovesicles further includes: incubating 1 mg / kg Torreya grandis exosome nanovesicles with 50 mg / kg 5-ASA aqueous solution at 37°C and pH=7.4 for 24 h to obtain encapsulated Torreya grandis exosome nanovesicles.

[0015] As a further aspect of the present invention, the drug comprises Torreya grandis exosome nanovesicles and a pharmaceutically acceptable carrier.

[0016] As a further aspect of the present invention, the dosage of the Torreya grandis exosome nanovesicles is 1 mg / kg / day per dose.

[0017] The beneficial effects of this invention are as follows: This invention discovers that XFEV can not only significantly reduce pro-inflammatory factors TNF-α and IL-6 and increase anti-inflammatory factor IL-10, but also repair colonic mucosal crypt structure and enhance intestinal barrier function. It is speculated that XFEV indirectly combats inflammation by regulating the gut microbiota, which differs from traditional drugs with a single mechanism. The plant exosome preparation process was optimized, using differential centrifugation combined with sucrose gradient purification to obtain uniform particles of 144.4 nm. Furthermore, it was confirmed that the efficacy of XFEV delivery via encapsulation is significantly superior to simple mixing, highlighting the targeting and protective advantages of exosomes as natural carriers, and providing a low-toxicity, high-efficiency new strategy for UC treatment. Attached Figure Description

[0018] Figure 1 Changes in body weight in mice with colitis; Figure 2 Changes in the DAI index in colitis mice; Figure 3 Gross image of the colon in a mouse with colitis; Figure 4 The effects of Torreya grandis exosome nanovesicles (XFEV) on colonic tissue of mice with DSS-induced colitis: A: H&E staining blank group; B: H&E staining model group; C: H&E staining 5-ASA group; D: H&E staining XFEV (packaged) group; E: H&E staining XFEV-ASA group; F: H&E staining XFEV group. Figure 5 Effects of Torreya grandis exosome nanovesicles (XFEV) on serum inflammatory factor expression levels in mice with DSS-induced colitis; G: IL-1β expression level; H: TNF-α expression level; I: IL-6 expression level; J: IL-10 expression level; Note: Pro-inflammatory factors in the model group were significantly different from those in the control group (P < 0.001); compared with the drug-treated group (P > 0.05); anti-inflammatory factors in the model group were significantly different from those in the drug-treated group (P < 0.001). Figure 6Effects of Torreya grandis exosome nanovesicles (XFEV) on complete blood counts in mice with DSS-induced colitis; A: WBC concentration; B: RBC concentration; C: MCV concentration; D: LYM concentration; E: MPV concentration; F: PLT concentration; G: NEU concentration; Figure 7 This is an electron microscope image of Torreya grandis exosome nanovesicles (XFEV). Detailed Implementation

[0019] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0020] Unless otherwise specified, the preparation methods used in this embodiment are conventional methods known to those skilled in the art, and the materials or reagents used are commercially available products unless otherwise specified.

[0021] 1. Materials and Methods 1.1 Preparation of Torreya grandis exosome nanovesicles (XFEV) The main methods for isolating PELNs (plant-derived exosome-like nanovesicles) include differential ultracentrifugation, size exclusion chromatography, polymer precipitation, ultrafiltration, immunoaffinity capture, and dialysis electrophoresis. The preparation of *Torreya grandis* exosome nanovesicles includes the following steps: After removing the shells from the Torreya nuts, add them to a juicer along with a small amount of deionized water. First, extract 1 x 10 ml of the thick Torreya nut juice. 3 g. After centrifuging horizontally at 4℃ for 10 min, collect the supernatant, then aliquot the supernatant into centrifuge tubes and centrifuge for another 2 × 10⁻⁶ min. 3 g. After centrifuging horizontally at 4℃ for 20 min, collect the supernatant and then centrifuge 4×10⁻⁶ times. 3 After centrifuging horizontally at 4℃ for 30 min, collect the supernatant and finally 1×10⁻⁶ g. 4 g. After centrifugation at 4℃ for 1 hour, the supernatant was collected and filtered through a 0.22μm filter membrane and collected into a centrifuge tube.

[0022] Pour the supernatant from the previous step into the ultrafiltration tube, 5×10 3 After centrifugation at 4℃ for 30 min, the solution in the upper ultrafiltration tube is collected, mixed, and then filtered through a 0.22 μm aqueous filter for sterilization. The sterilized solution is then subjected to a 1×10⁻⁶ filter. 4 Centrifuge at 4℃ for 2 hours, resuspend in PBS buffer, and combine to 500 μL. Further purify using a sucrose gradient (8%, 30%, 45%, and 60% sucrose dissolved in 20 mM Tri-HCl, pH 7.2). Take 1 × 10⁻⁶ g of the 45-60% layer solution. 4Centrifuge at 4℃ for 2 hours, wash the precipitate with an appropriate amount of PBS buffer, and then centrifuge at 1×10⁻⁶. 4 Centrifuge at 4℃ for 2 hours, weigh the precipitate, and resuspend it in PBS buffer to prepare a Torreya grandis exosome nanovesicle solution (XFEV 20 mg / ml). Store at -80℃, avoiding repeated freeze-thaw cycles during use. Figure 7 As shown, it can be seen that uniform Torreya grandis exosome nanovesicles with a diameter of 144.4 nm can be obtained by differential centrifugation combined with sucrose gradient purification.

[0023] Furthermore, *Torreya grandis* exosome nanovesicles (XFEV) and pharmaceutically acceptable carriers can be formulated into drugs, with dosage forms including tablets, capsules, lozenges, injections, suspensions, suppositories, ointments, bladder instillations, oral preparations, suppositories, or sustained-release preparations. The carrier can be any of liposomes, nanoparticles, hydrogels, or microspheres.

[0024] 1.2 Laboratory animals and drugs UC mouse model: 48 male SPF-grade C57BL / 6J mice, 6-8 weeks old, weighing (18-22g), were used to establish a colitis model by administering DSS (3.5%, w / v). 50 mice from the same batch were divided into 6 groups (n=8).

[0025] 1) Blank control group (deionized water 0.3 mg / 20 g / d); 2) DSS group (3.5% DSS); 3) 5-ASA group (50 mg / kg); 4) XFEV (package) group (purified XFEV (1 mg / kg) and 5-ASA (50 mg / kg) aqueous solution were co-incubated at 37℃ and pH 7.4 for 24 h, and 5-ASA was allowed to spontaneously enter the exosome by utilizing the concentration gradient of drug molecules and membrane permeability). 5) XFEV group (XFEV 1mg / kg); 6) XFEV-ASA group (5-ASA 50mg / kg, XFEV 0.5mg / kg directly mixed).

[0026] The dosage was initially determined through experiments and administered orally daily for 7 days. The mice were placed in an animal room with simulated daily lighting (12:12h light-dark cycle) and the temperature was adjusted to (23±0.5℃) to allow them to adapt for one week. The control group mice were fed normally without any treatment. On the first day of the experiment, the mice in the remaining groups were given freshly prepared DSS (3.5%, w / v) to induce the UC mouse model, with a cycle of 7 days (3; 4; 5; 6). The corresponding dosage of the drug was administered to each group starting on the second day of the experiment, with a cycle of 7 days.

[0027] The specific dosing regimen for constructing the colitis model is shown in the table below (dosage volume is 0.1 ml, total required 16.8 ml).

[0028]

[0029] Mice were induced to develop ulcerative colitis using 3.5% DSS (diuretic saline). After the adaptation period, the mice were observed daily. During the modeling period, the DAI (diarrhea-associated acute inflammatory response) score of the DSS-induced mice increased from 0 to 4. The main symptoms were diarrhea, mucus-like stools, fecal occult blood, gross bloody stools, and weight loss. Upon dissection, the mice's intestines were blackened, consistent with Kimura et al.'s description of a DSS-induced ulcerative colitis model, indicating successful modeling. After gavage administration of XFEV (xyphenidate-free vegetative-exposure progesterone), the mice's DAI score decreased from 1 to 0, and the diarrhea significantly improved. No intestinal redness, swelling, or congestion was observed.

[0030] 2. Effects of Torreya grandis exosome nanovesicles (XFEV) on UC mice 2.1 Weight Mice were weighed starting from day 1 after the start of the experiment. DAI (Diagnosis Intensity) in UC mice was assessed based on fecal consistency (score 0-3), fecal bleeding (score 0-3), and weight loss (score 0-4). When the DSS group mice lost approximately 15% of their body weight, administration was discontinued in all treatment groups, mice were sacrificed, and serum was collected. A portion was used for subsequent complete blood counts, and the remainder was used to measure serum cytokines such as TNF-α, IL-6, and IFN-γ using kits and an ELISA reader. Colons were extracted, their length measured, and photographs recorded. A small portion of the colon tissue was then fixed in 4% paraformaldehyde solution, dehydrated with ethanol, embedded in paraffin, and sectioned (5 μm) for H&E staining to assess crypt damage, the severity of inflammation, and ulceration, and histological scoring was performed.

[0031] The mouse weight recording results are as follows Figure 1 As shown in the figure, the weight of the control group gradually increased over 7 days, indicating a stable experimental environment and minimal external interference. Compared with the modeling group, the 5-ASA group showed no significant change before day 4, but the decrease was significantly smaller after day 4. Compared with the XFEV (packed) group, the weight was significantly lower on day 7. This may be because the high dose of 5-ASA reagent induced inflammation, thus causing a slow weight loss. The XFEV (packed) group showed a significantly faster weight recovery rate after day 2 than other treatment groups. There was no significant difference in weight between the control group and the control group on day 7. XFEV may reduce the release of pro-inflammatory factors and promote colonic epithelial cell proliferation or mucus secretion by inhibiting pathways such as NF-κB. The weight of the XFEV-ASA group and the XFEV group tended to increase steadily, but it was significantly lower than that of the XFEV (packed) group, which may indicate that XFEV is more suitable for embedding administration.

[0032] 2.2 Disease Activity Index (DAI) DAI calculation formula: DAI = (weight loss score + fecal characteristics score + bloody stool score) / 3.

[0033] Experimental method: On the first day of the experiment, the weight of mice in each group was weighed and recorded, feces were observed and collected, and scores were recorded.

[0034]

[0035] From the DAI indicator ( Figure 2 According to the DSS model, high doses of 5-ASA (>30 mg / kg) may aggravate diarrhea by damaging the mucus layer, but because it still has an anti-inflammatory mechanism, its DAI index remains stable at around 2. Mice in the drug-treated group may have their inflammatory environment improved and their barrier repaired due to the drug's effect, thus effectively reducing their DAI index. However, different routes of administration may lead to different final effects.

[0036] 2.3 Weighing of spleen and colon, and measurement of colon length Spray the mouse abdomen with 70% alcohol, then open the mouse's abdomen through a midline incision and remove the spleen. Increased spleen weight is generally associated with the degree of inflammation and anemia. Lift the colon with forceps and carefully and slowly pull until the cecum is visible. Separate the colon and cecum from the anus. Select one representative from each group to take a gross intestinal photograph from the cecum to the rectum. Record the data after measuring its length. After straightening the colon, it can be directly separated from the cecum and quickly rinsed with cold PBS to remove feces and blood. The cecum can be discarded because DSS induces little or no inflammation in this area.

[0037] like Figure 3 As shown in the figure below, after euthanizing the mice, their colon lengths were measured. The colons of the control group were smooth and intact, with a length of approximately 7 cm. The colons of the model group showed a distinctly reddish-brown color and bloody stools, with a length of approximately 4 cm, significantly shorter than the control group. The 5-ASA colons were smooth and intact, with a length of approximately 6 cm. The colons of the other three treatment groups were bright and smooth, with relatively normal stools, and a length of approximately 6 cm.

[0038] 2.4 Spleen H&E staining (colon tissue) and histological scoring (1) Dewax paraffin sections to water. After washing with a gradient of alcohols (100%-95%-90%-85%-75%), rinse with deionized water; (2) Stain the cell nuclei with hematoxylin staining solution. After cleaning, place the slides in hematoxylin staining solution for 8 minutes, wash with deionized water, let stand in 1% hydrochloric acid alcohol for a few seconds, then use 0.6% ammonia water to turn blue, and finally rinse with running deionized water; (3) Stain the cytoplasm with eosin staining solution. Immerse the slide in eosin staining solution for 3 minutes; (4) Dehydration and mounting: The sections are dehydrated by alcohol and xylene in turn to make them transparent, and then mounted with neutral resin.

[0039] Blank control group: H&E staining showed that the mouse colon tissue structure was intact; the mucosal layer was neatly arranged; and the crypt structure was clearly visible. Figure 4 No obvious inflammatory cell infiltration was observed in the lamina propria, and the submucosal blood vessels were normally distributed, consistent with the histological characteristics of a healthy colon.

[0040] The DSS model group exhibited typical pathological changes of colitis: large-area shedding of the mucosal layer, destruction of crypt structure, infiltration of a large number of neutrophils in the lamina propria, increased inflammatory cell density compared with the control group, submucosal edema accompanied by vasodilation.

[0041] 5-ASA treatment group: Mucosal repair, partial reconstruction of crypt structure, reduced incidence of crypt abscess, decreased neutrophil density, reduced edema area, increased goblet cell count, and partial recovery of mucus secretion function.

[0042] XFEV (packet) group and other drug-treated groups showed significant tissue repair: mucosal repair, intact crypt regeneration structure, restoration of the number of newly formed goblet cells, and reduced inflammatory cell infiltration. Figure 4 This suggests that XFEV may have a role in relieving intestinal inflammation.

[0043] 2.5 Measurement of serum cytokines and routine blood tests Blood was collected through the eyeball into sterile, enzyme-free centrifuge tubes. After standing at room temperature for half an hour, the blood was centrifuged (3500 rpm, 10 min, 4℃). The yellow, clear supernatant was collected as serum and aliquoted into multiple 1.5 mL centrifuge tubes. The levels of cytokines such as TNF-α, IL-6, and IFN-γ in the serum were measured using an ELISA reader and a kit. Simultaneously, blood samples were collected for subsequent routine blood tests to assess the levels of WBC, LYMPH, MON, and other parameters.

[0044] The levels of inflammatory cytokines TNF-α and IL-10 in mouse serum were determined using enzyme-linked immunosorbent assay (ELISA). The procedure is as follows: Coating: Add 100 μL of antibody dilution solution (1-10 μg / mL) to each well of the microplate and incubate at 4°C for 12-16 h; the next day, discard the liquid and wash with washing buffer 5 times for 5 min each time.

[0045] Blocking: Add 200 μL of blocking solution to each well and incubate at 37°C for 2 hours.

[0046] Washing: Remove the sealing film and wash with washing solution 5 times, 5 minutes each time.

[0047] Sample addition: Add 100 μL of diluted sample to each well, incubate at 37°C for 2 h, and then repeat step 3 for washing.

[0048] Add antibody and incubate: Add 100 μL of diluted antibody (according to the instructions) to each well, incubate for 1 hour, and then repeat step 3 for washing.

[0049] Add enzyme conjugate and incubate: Add 100 μL of diluted enzyme conjugate to each well, incubate in the dark for half an hour, and then repeat step 3 for washing.

[0050] Developing and stopping: Add 100 μL of LTMB colorimetric solution to each well and incubate at 37°C in the dark for half an hour until a clear color gradient appears. Add 100 μL of 2M sulfuric acid solution to stop the color, and the solution will change from blue to yellow.

[0051] Results determination: Immediately after adding the stop solution, the optical density at 450 nm was measured using an ELISA reader, and the sample concentration was calculated based on the concentration gradient of the standard.

[0052] Expression of inflammatory factors in mice, such as Figure 5 As shown in the experimental data, the expression of the pro-inflammatory factor IL-1β in mice was significantly increased after DSS induction (P < 0.001), while the content of pro-inflammatory factors in mice treated with XFEV decreased but not significantly (P > 0.05).

[0053] The expression levels of anti-inflammatory factors in the model group were significantly decreased (P < 0.001). Although the IL-10 expression level in the 5-ASA group was higher than that in the model group, it was still lower than that in the blank group, possibly because IL-10 was partially restored through the classical anti-inflammatory pathway, but the immunosuppressive deficiency was not completely reversed. The significantly increased IL-10 expression levels in all three treatment groups compared to the model group (P < 0.001) indicate that XFEV has a certain therapeutic effect on UC. However, the difference between the treatment groups was not significant (P > 0.05), suggesting that the combined treatment effect was not obvious. Therefore, different administration methods may affect its efficacy.

[0054] Combination Figure 6 Blood routine data showed that the colitis model successfully induced inflammation (WBC↑), and XFEV and 5-ASA could suppress inflammation but were accompanied by the risk of thrombocytopenia.

[0055] 3. Conclusion This invention successfully extracted Torreya grandis exosome-like nanovesicles with a particle size of 144.4 nm from fresh Torreya grandis fruit by combining differential centrifugation, ultracentrifugation, ultrafiltration and sucrose gradient purification.

[0056] This invention established a mouse model of ulcerative colitis induced by dextran sulfate sodium DSS and investigated the alleviating effect of XFEV on dextran sulfate sodium DSS-induced ulcerative colitis using methods such as XFEV intervention, serum inflammatory factor detection, morphological observation of colonic tissue, and immunohistochemical analysis of the colon. The results showed that XFEV could alleviate the apparent symptoms of weight loss and colonic shortening UC mice induced by dextran sulfate sodium DSS, reduce colonic tissue damage, maintain the integrity of the colonic structure, and promote the relief of inflammation.

[0057] This invention analyzes the results of three different administration methods to show that different administration methods lead to different drug efficacy of Torreya grandis nanoliposome vesicles (XFEV (packet) > XFEV-ASA > XFEV), proving that Torreya grandis exosome nanovesicles can be used as an excellent delivery carrier.

[0058] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various improvements without departing from the concept of the present invention, and these improvements all fall within the scope of protection of the present invention.

Claims

1. Application of Torreya grandis exosome nanovesicles in the preparation of drugs for treating or improving ulcerative colitis.

2. The application according to claim 1, characterized in that, The preparation process of the Torreya grandis exosome nanovesicles involves sequentially purifying Torreya grandis juice through differential centrifugation, ultracentrifugation, ultrafiltration, and sucrose gradient.

3. The application according to claim 2, characterized in that, The preparation of the Torreya grandis exosome nanovesicles specifically includes the following steps: (1) After the viscous Torreya grandis juice was centrifuged four times at different speeds to collect the supernatant, the supernatant was filtered through a 0.22 μm filter membrane and collected. (2) The supernatant after filtration by the filter membrane is subjected to ultrafiltration, and the upper layer solution is taken and filtered with a 0.22μm aqueous filter head for sterilization; (3) Centrifuge the filtered sterilized solution, resuspend it with PBS buffer, purify it in a sucrose gradient, take 45-60% layer solution, centrifuge, wash the precipitate with an appropriate amount of PBS buffer and centrifuge again to obtain Torreya grandis exosome nanovesicles.

4. The application according to claim 3, characterized in that, In step (1), the Torreya grandis juice is obtained by juicing Torreya grandis fruit after removing the shells; In step (1), the first centrifugation is 1×10⁻⁶. 3 g, centrifuge horizontally at 4℃ for 10 min, then centrifuge a second time at 2×10⁻⁶. 3 g, centrifuge horizontally at 4℃ for 20 min, then centrifuge a third time at 4×10⁻⁶. 3 g, centrifuge horizontally at 4℃ for 30 min, the fourth centrifugation is 1×10. 4 g, centrifuge horizontally at 4℃ for 1 hour.

5. The application according to claim 3, characterized in that, In step (2), the ultrafiltration parameter is 5 × 10⁻⁶. 3 g, centrifuge horizontally at 4℃ for 30 minutes.

6. The application according to claim 3, characterized in that, In step (3), sucrose gradients of 8%, 30%, 45% and 60% are dissolved in 20 mM Tri-HCl at pH 7.

2.

7. The application according to claim 3, characterized in that, In step (3), after weighing the precipitate, it is resuspended in PBS buffer to form Torreya grandis exosome nanovesicle solution XFEV and stored at -80°C.

8. The application according to claim 3, characterized in that, The preparation of the Torreya grandis exosome nanovesicles further includes: incubating 1 mg / kg Torreya grandis exosome nanovesicles with 50 mg / kg 5-ASA aqueous solution at 37°C and pH=7.4 for 24 h to obtain encapsulated Torreya grandis exosome nanovesicles.

9. The application according to claim 1, characterized in that, The drug comprises Torreya grandis exosome nanovesicles and a pharmaceutically acceptable carrier.

10. The application according to claim 1, characterized in that, The dosage of the Torreya grandis exosome nanovesicles is 1 mg / kg / day per dose.

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