Preparation method and application of tartary buckwheat tea-derived nanovesicles

Abstract

The present application relates to a kind of tartary buckwheat tea source nanovesicle preparation method and its application, belong to biological nanometer material technical field, solve the technical problem of high yield, high purity extraction plant nanovesicle from tartary buckwheat tea, its preparation method includes the following steps: S1, weighing, soaking;S2, break, filter;S3, differential centrifugation;S4, extrusion, prepare tartary buckwheat tea source nanovesicle, subpackaging in EP tube and stored at-80 ℃ keep ready for use;Tartary buckwheat tea source nanovesicle prepared using this method is applied to the preparation of the drug for treating ulcerative colitis.The BVs obtained by differential centrifugation method combined with extrusion method, compared with traditional ultracentrifugation method, the extraction step is simpler, the yield is higher, easy to scale production, and the obtained BVs have more abundant metabolic products and lipid components, can effectively exert antioxidant property, repair intestinal barrier and regulate intestinal flora, thereby improving ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of bio-nanomaterials technology, specifically relating to a method for preparing tartary buckwheat tea-derived nanovesicles (BVs) and their application in the preparation of drugs for treating ulcerative colitis. Background Technology

[0002] Ulcerative colitis is a type of inflammatory bowel disease, characterized by a prolonged course and frequent relapses. Long-term illness increases the risk of colon cancer (Li Y. Gut microbiota in the pathogenesis and treatment of inflammatory bowel disease: a critical review of mechanisms and therapeutic advances. Front. Med. 13:1738-292.). Currently, commonly used drugs in clinical practice mainly include aminosalicylic acids, glucocorticoids, and immunosuppressants, but many of them have a series of adverse reactions, which limits their clinical use (Cao Xiaoying, Wei Zhifeng. Research progress on drugs for the treatment of ulcerative colitis [J]. Drug Information, 2024, 13(5): 429-437.).

[0003] In recent years, extracellular nanovesicles of plants and their processed products have attracted much attention due to their advantages such as abundant and readily available resources, low immune risk, and good biosafety (Peng J, Liu R, Xu J, Yao Y, Li B, Chen D, Chang Z, Zhao R, Feng Y, Hou R, Lee M, Zhang X. Acid-responsive aggregatedcarrot-derived nanoantioxidants aLLeviate oxidative stress and restoreosteobLast activity. J. NanobiotechnoL. 2025; 23: 206.).

[0004] Buckwheat is a representative crop of miscellaneous grains, and its grains are often consumed in the form of buckwheat tea in daily life. Buckwheat tea processed by low-temperature roasting has higher content of flavonoids and free polyphenols compared to plain buckwheat grains (Zhu F. Buckwheat grain phenolics: Composition, bioactivity, and processing. Food Chem. 2026 Jul 14;525(Pt 2):150412.). Furthermore, existing studies have confirmed that buckwheat plant nanovesicles are rich in regulatory miRNAs, which can regulate gut microbiota (Liu Y, Tan ML, Zhu WJ, Cao YN, Peng LX, Yan ZY, Zhao G. In Vitro Effects of Tartary Buckwheat-Derived NanovesicLes on GutMicrobiota. J Agric Food Chem. 2022 Mar 2;70(8):2616-2629.). Ulcerative colitis causes significant dysbiosis and inflammatory response. Based on existing research, it is speculated that nanovesicles derived from buckwheat tea may have the potential to intervene in ulcerative colitis. Currently, the mainstream preparation method for nanovesicles is ultracentrifugation combined with sucrose density gradient purification (Zhang J, Zhou C, Tan M, Cao Y, Ren Y, Peng L. Optimization and Characterization of PEG Extraction Process for Tartary Buckwheat-Derived NanoparticLes. Foods. 2024 Aug 21;13(16):2624.), but there are significant shortcomings in its application research: Firstly, the ultra-high centrifugal shear force easily damages the vesicle phospholipid membrane structure, causing particle aggregation, loss of flavonoids and miRNA active components, and insufficient vesicle recovery rate. Secondly, buckwheat tea is rich in starch, polysaccharide, and protein impurities, which are difficult to completely remove through the above purification process. These impurities can interfere with the in vivo efficacy evaluation of colitis and aggravate intestinal irritation (Liu Y, Tan ML, Zhu WJ, Cao YN, Peng LX, Yan ZY, Zhao G. In Vitro Effects of Tartary Buckwheat-Derived NanovesicLes on GutMicrobiota. J Agric Food Chem. 2022 Mar 2;70(8):2616-2629.).

[0005] Therefore, there is an urgent need to extract plant nanovesicles from buckwheat tea with high yield and high purity, and to explore their translational applications in ulcerative colitis. Summary of the Invention

[0006] The main objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing buckwheat tea-derived nanovesicles and their applications. The method involves separating BVs using differential centrifugation combined with extrusion, and then using the BVs prepared by this method to prepare drugs for ulcerative colitis. Compared with the traditional ultracentrifugation method, the extraction steps are simpler, the yield is higher, and it is easier to scale up production. Furthermore, the obtained BVs have richer metabolites and lipid components, which can effectively exert antioxidant properties, repair the intestinal barrier, and regulate the intestinal flora, thereby improving ulcerative colitis.

[0007] This invention is achieved through the following technical solution: A method for preparing buckwheat tea-derived nanovesicles includes the following steps: S1. Weighing and soaking: Weigh 20-100g of buckwheat tea (dry weight), add it to a blender, and soak it in 120-600mL of phosphate-buffered saline (PBS) pre-cooled to 4℃ for 1-3 minutes. S2. Crushing and Filtration: Immediately after the phosphate buffer soaking is completed, start the high-speed blender to crush the phosphates, ensuring low-temperature extraction, with a crushing time of 1-3 minutes; then, filter the filtrate using a filter bag and collect it for later use. S3. Differential centrifugation: Transfer the filtrate prepared in step S2 to a centrifuge tube and perform differential centrifugation at 4°C: The acceleration of the first centrifugation is 1000g~2000g, and the centrifugation time is 10min~20min, to obtain supernatant a; Then, supernatant a was centrifuged a second time at an acceleration of 3000g to 5000g for 20 to 45 minutes to obtain supernatant b. Finally, the supernatant b was subjected to a third centrifugation treatment at an acceleration of 10000g~13000g for 45min~60min to obtain supernatant c. S4. Extrusion: The supernatant c obtained from the last centrifugation in step S3 is extruded sequentially through a coarse sieve microporous membrane with a pore size of 0.45 μm and a fine sieve microporous membrane with a pore size of 0.2 μm or 0.22 μm to obtain buckwheat tea-derived nanovesicles, which are then packaged in EP tubes and stored at -80℃ for later use.

[0008] Further, in step S1, the concentration of the phosphate buffer is 0.01M and the pH value is 7.2~7.4.

[0009] Furthermore, in step S4, the obtained buckwheat tea-derived nanovesicles have a characteristic lipid bilayer (similar to extracellular vesicles), a particle size of 100nm~200nm, a zeta potential of -5mV~-15mV, are rich in flavonoids, benzene and its substituted derivatives, have antioxidant potential, and glycerophospholipids and steroids and their derivatives have barrier repair potential.

[0010] The application of buckwheat tea-derived nanovesicles prepared by the method described above in the preparation of a drug for treating ulcerative colitis. The buckwheat tea-derived nanovesicles provided by this invention are rich in various bioactive components such as lipids and flavonoids, and can be targeted to the colon, achieving a significant increase in antioxidant properties in the intestine, effectively clearing inflammatory factors, and improving ulcerative colitis by repairing the intestinal barrier and regulating the intestinal flora.

[0011] The beneficial effects of this invention are as follows: 1) The present invention uses differential centrifugation combined with extrusion to prepare buckwheat tea-derived nanovesicles. The raw materials are close to daily life and dietary habits, have wide applications, are easy to obtain, have simple extraction processes, are easy to scale up, and have good biosafety. 2) The buckwheat tea-derived nanovesicles provided in this invention were identified through metabolomics and lipidomics, revealing that they contain diazacyclohexane heterocyclic derivatives and phenylpropanoid-polyketide metabolites. Diazacyclohexane derivatives can regulate inflammatory signaling pathways; phenylpropanoids and polyketides contain a large number of phenolic secondary metabolites, exhibiting strong antioxidant effects; simultaneously, the abundant glycerophospholipids and steroids possess potential for barrier repair, potentially providing a treatment for ulcerative colitis with good patient compliance and biosafety. Attached Figure Description

[0012] Figure 1 Transmission electron microscope image of the BVs nanovesicles prepared in Example 1; Figure 2 The particle size distribution diagram of the BVs nanovesicles prepared in Example 1; Figure 3 Potentiometric diagram of BVs nanovesicles prepared in Example 1; Figure 4 A comparison of protein concentrations in different batches of BVs nanovesicles prepared in Example 1; Figure 5 Comparison of ABTS scavenging rates of BVs nanovesicles prepared in Example 1; Figure 6 Comparison of DPPH scavenging rates of BVs nanovesicles prepared in Example 1; Figure 7 A comparison diagram of the biocompatibility of BVs nanovesicles; Figure 8 A comparison of the uptake efficiency of BVs nanovesicles in colon cells; Figure 9 This is a graph showing the change in body weight in mice after intervention with BVs nanovesicles. Figure 10 Comparison of blood routine results under BVs nanovesicle intervention; Figure 11 Comparison of liver and kidney function under BVs nanovesicle intervention; Figure 12 A comparative diagram showing the safety changes of major organs in mice after BVs nanovesicle intervention; Figure 13 A graph showing the relationship between body weight and DAI score after BVs nanovesicle preventive treatment; Figure 14 Image showing colon length after BVs nanovesicle treatment; Figure 15 HE staining image of BVs nanovesicles for the treatment of ulcerative colitis; Figure 16 AB-PAS diagram for BVs nanovesicles in the treatment of ulcerative colitis; Figure 17 A comparative diagram showing the effects of BVs nanovesicles on the gut microbiota of mice with colitis. Detailed Implementation

[0013] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Example 1

[0014] A method for preparing buckwheat tea-derived nanovesicles includes the following steps: S1. Weighing and soaking: Weigh 20g of dry buckwheat tea (originating from Datong City, Shanxi Province), add it to a blender, and soak it for 2 minutes in 120mL of PBS pre-cooled to 4℃; the concentration of PBS is 0.01M and the pH value is 7.4. S2. Crushing and Filtration: Immediately after the PBS soaking is completed, start the blender (process parameters: rated voltage 220V, rated power 2200W, rated frequency 50Hz, rated speed 35000~48000 rpm; including levels 1~9, level 1 speed is 35000 rpm, level 9 speed is 45000 rpm). Gradually increase the speed from level 1 to level 9 for crushing, and the crushing time is 2 minutes. Then, filter the mixture using a 10cm×15cm herbal filter bag and collect the filtrate in a 250mL beaker for later use. S3. Differential centrifugation: Transfer the filtrate prepared in step S2 to a centrifuge tube with a volume of 50 mL and perform differential centrifugation at a temperature of 4℃: The acceleration of the first centrifugation is 1200 g and the centrifugation time is 10 min, and the supernatant a is obtained. Then, supernatant a was centrifuged a second time at an acceleration of 3000g for 20 minutes to obtain supernatant b. Finally, the supernatant b was centrifuged for the third time at an acceleration of 12000g for 60min to obtain supernatant c. S4. Extrusion: The supernatant c obtained from the last centrifugation in step S3 is extruded sequentially through a coarse sieve microporous membrane with a pore size of 0.45 μm and a fine sieve microporous membrane with a pore size of 0.22 μm to obtain buckwheat tea-derived nanovesicles, which are dispensed into 5 mL EP tubes and stored at -80℃ for later use.

[0015] Based on this, a drug for treating ulcerative colitis was prepared using the buckwheat tea-derived nanovesicles obtained by the preparation method described in Example 1.

[0016] The following verifies the excellent performance of the BVs prepared in Example 1 from multiple dimensions.

[0017] I. Characterization of BVs.

[0018] The morphology and structure of BVs were observed using transmission electron microscopy (TEM). Figure 1 As shown, BVs are round and have a characteristic lipid bilayer similar to extracellular vesicles.

[0019] The particle size and potential distribution of BVs were detected by dynamic light scattering (DLS), and the results showed that the average particle size of BVs was approximately 170 nm (e.g., Figure 2As shown), the Zeta potential is approximately -6.57 mV (as shown). Figure 3 (As shown).

[0020] The protein content was detected using a BCA assay kit, and the results showed that the protein concentration was consistently around 6.9 mg / mL (e.g., ...). Figure 4 (As shown).

[0021] II. Evaluation of antioxidant properties.

[0022] Antioxidant performance: The scavenging effects of nanovesicles on ABTS and DPPH were detected using a free radical scavenging kit (e.g., ...). Figure 5 , Figure 6 (As shown). After treatment with different concentrations of buckwheat tea-derived nanovesicles, the free radical scavenging rates of both groups showed a significant dose-dependent effect. The 1000 μg / mL nanovesicles scavenged up to 90% of ABTS free radicals and nearly 80% of DPPH free radicals.

[0023] III. Biocompatibility of BVs.

[0024] The cellular safety of BVs was evaluated using NCM460 normal colonic epithelial cells. Resuscitated NCM460 cells were routinely cultured and passaged, and after adjusting cell density, seeded into 96-well plates and incubated at 37°C in a 5% CO2 incubator for adherent growth. Gradual drug administration groups were administered at six concentration gradients (12.5–500 μg / mL) using complete culture medium, with equal amounts administered to each well and cultured for 24 h. Cell viability at different concentrations was calculated using a CCK-8 assay kit to assess the toxicity of buckwheat tea nanovesicles to normal colonic cells. The experiments demonstrated that the drug had good biocompatibility and could promote cell proliferation (e.g., ...). Figure 7 (As shown).

[0025] IV. Colonic cell uptake efficiency of BVs.

[0026] (1) Fluorescent labeling of BVs: After the centrifuge was cooled to 4℃, 30μL of 1mg / mL DiO stock solution was added to 940μL of 2mg / mL BVs. The mixture was incubated at 37℃ in the dark, shaken at 200rpm for 30min. After that, the mixture was dialyzed with a 1000 kDa dialysis bag for 6h to remove free DiO, and finally DiO-labeled BVs (DiO-BVs) were obtained.

[0027] (2) Uptake efficiency: NCM460 cells were seeded in confocal microplates. When the cell confluence reached 80% or higher, 1.5 mL of cell culture medium containing 250 μg / mL DiO-BVs was added to each well. The cells were then incubated in an incubator. At the preset time points (0, 2, 4, and 8 h), the culture medium was aspirated, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, stained with Hochest 33342 dye at room temperature for 5 min, and the cell uptake was observed using CLSM (e.g., ...). Figure 8 As shown in the figure, after 8 hours of incubation, the cell uptake efficiency was significantly enhanced, indicating that BVs can be effectively taken up by colonic epithelial cells, providing an effective guarantee for the treatment of ulcerative colitis.

[0028] Fifth, BVs have biological safety.

[0029] The in vivo biosafety of BVs was evaluated using a mouse acute short-term gavage model. Mice were randomly divided into a normal control group and a treatment group, with replicates in each group. The treatment group received 30 mg / kg of buckwheat tea nanovesicle solution via gavage for 7 consecutive days. Mouse weight was monitored daily during the experiment (e.g., ...). Figure 9 (As shown).

[0030] After the experiment, the mice were dissected, and whole blood and serum samples were collected for routine blood tests and liver and kidney function tests (such as...). Figure 10 , Figure 11 (As shown); Simultaneously, mouse heart, liver, spleen, lung, kidney, and colon tissues were isolated, fixed, photographed, and observed to systematically evaluate the safety changes in major mouse organs under short-term intervention with high-dose buckwheat tea nanovesicles (e.g., Figure 12 (As shown).

[0031] The preventive and therapeutic effects of BVs on colitis.

[0032] By collecting basic data such as body weight, DAI score, and colon length during the experiment, the therapeutic effect of BVs was preliminarily evaluated. After BVs preventive treatment, the weight loss of colitis mice was alleviated and the DAI score decreased (e.g., Figure 13 As shown), the colon length in the high-dose BVs administration group significantly recovered to near normal (e.g., Figure 14 (As shown).

[0033] As shown in Figure 15, HE staining, the gold standard for detecting colitis, was used to verify the pathological recovery of colitis. After dewaxing paraffin sections to water, hematoxylin staining was performed for 3 min, followed by rinsing with running water; differentiation was carried out in differentiation solution for 3-5 s, followed by rinsing with running water; blueing was performed in blue solution for 3-6 s, followed by rinsing with running water. The sections were then dehydrated in 95% alcohol for 1 min and stained with eosin for 15 s. After dehydration and clearing in anhydrous ethanol, n-butanol, and xylene for 1 min each, the sections were mounted with neutral resin and examined under a microscope. Image analysis showed that BVs had a significantly higher efficacy than the positive drug group, and this efficacy was dose-dependent.

[0034] After dewaxing paraffin sections to water, they were sequentially immersed in AB-PAS staining solution C for 15 min, washed with tap water until colorless; stained with staining solution B for 15 min, washed with tap water, and washed twice with distilled water; stained with staining solution A (at room temperature) in the dark for 1 h, and rinsed with running water for 5 min. The sections were then dehydrated and cleared with anhydrous ethanol and xylene for 5 min each, and mounted with neutral resin. They were then examined under a microscope, and images were acquired and analyzed to assess the treatment effect (e.g., Figure 16 (As shown).

[0035] The impact of BVs on gut microbiota was assessed using 16S rRNA.

[0036] (1) Sample collection; On the day of the autopsy, the cecal contents of the control group, model group, and ESSS group were quickly collected into sterile cryovials and rapidly transferred to liquid nitrogen for freezing, and then transferred to a -80°C freezer for storage.

[0037] (2) Extraction of total DNA from the microbiome; Bacterial DNA was extracted from cecal contents using a standardized procedure. The DNA was quantified using Nanodrop, and the quality of the extracted DNA was detected by 1.2% agarose gel electrophoresis.

[0038] (3) PCR amplification of the target fragment; Using microbial ribosomal RNA or specific gene fragments as target sequences, primers are designed based on conserved regions and sample-specific barcodes are added to amplify variable regions of rRNA genes or specific gene fragments by PCR.

[0039] (4) Purification and recovery of amplification products using magnetic beads; Add 0.8 times the volume of magnetic beads (Vazyme VAHTS™ DNA Clean Beads) to the amplification product, vortex to mix, and then magnetically adsorb for 5 min, discarding the supernatant; add 20 μL of 0.8 times the volume of magnetic bead washing buffer, resuspend, and magnetically adsorb for 5 min, discarding the supernatant; add 200 μL of 80% ethanol, reverse the adsorption, and discard the ethanol; let stand at room temperature for 5 min to allow the ethanol to evaporate; add 25 μL of ELution Buffer to elute, magnetically adsorb for 5 min, and transfer the supernatant to a new centrifuge tube for storage.

[0040] (5) Quantitative fluorescence analysis of amplified products; The PCR amplification products were quantitatively analyzed using fluorescence. The fluorescent reagent was the Quant-iT PicoGreen dsDNAAssay Kit, and the quantification instrument was a MicropLate reader (BioTek, FLx800). Based on the fluorescence quantification results, the samples were mixed in appropriate proportions according to the sequencing volume requirements of each sample.

[0041] (6) Sequencing library preparation; Sequencing libraries were prepared using the TruSeq Nano DNA LT Library Prep Kit from ILLumina.

[0042] (7) Perform high-throughput sequencing on the instrument; (8) Sample data processing and analysis.

[0043] Using 16S rRNA sequencing, compared with the DSS group, the Simpson index, Pilo evenness index, and Shannon index of the gut microbiota in the BVs group were all decreased. This indicates that BVs reduced the richness and evenness of pathological microorganisms induced by DSS. It is speculated that this may be because after BVs intervention, as the relative abundance of dominant BV species (such as Akkermania) increases, the gut microbiota balance is reshaped. This process may inevitably lead to the disappearance of some harmful or beneficial bacteria, thereby reducing the gut diversity index (e.g., Figure 17 (As shown). Example 2

[0044] A method for preparing buckwheat tea-derived nanovesicles includes the following steps: S1. Weighing and soaking: Weigh 20g of dry buckwheat tea (originating from Datong City, Shanxi Province), add it to a blender, and soak it for 2 minutes in 120mL of PBS pre-cooled to 4℃; the concentration of PBS is 0.01M and the pH value is 7.4. S2. Disruption and Filtration: Immediately after the PBS soaking is completed, start the high-speed blender to disrupt the PBS for 2 minutes; then, filter the PBS using a filter bag and collect the filtrate for later use. S3. Differential centrifugation: Transfer the filtrate prepared in step S2 to a centrifuge tube and perform differential centrifugation at 4°C: The acceleration of the first centrifugation is 1200g and the centrifugation time is 10min, to obtain supernatant a; Then, supernatant a was centrifuged a second time at an acceleration of 3000g for 20 minutes to obtain supernatant b. Finally, the supernatant b was centrifuged for the third time at an acceleration of 12000g for 60min to obtain supernatant c. S4. Extrusion: The supernatant c obtained from the final centrifugation in step S3 is extruded sequentially through a coarse microporous membrane with a pore size of 0.45 μm and a fine microporous membrane with a pore size of 0.22 μm to obtain buckwheat tea-derived nanovesicles. These nanovesicles are dispensed into EP tubes and stored at -80℃ for later use. The obtained buckwheat tea-derived nanovesicles have a characteristic lipid bilayer, a particle size of 100 nm to 200 nm, and a zeta potential of -5 mV to -15 mV. Example 3

[0045] A method for preparing buckwheat tea-derived nanovesicles includes the following steps: S1. Weighing and soaking: Weigh 20-100g of buckwheat tea (produced in Datong City, Shanxi Province), add it to a blender, and soak it in 120-600mL of PBS pre-cooled to 4℃ for 1-3 minutes; the concentration of PBS is 0.01M and the pH value is 7.2-7.4. S2. Disruption and Filtration: Immediately after the PBS soaking is completed, start the blender to disrupt the PBS for 1-3 minutes; then, filter the PBS using a filter bag and collect the filtrate for later use. S3. Differential centrifugation: Transfer the filtrate prepared in step S2 to a centrifuge tube and perform differential centrifugation at 4°C: The acceleration of the first centrifugation is 1000g~2000g and the centrifugation time is 10min~20min, to obtain supernatant a; Then, supernatant a was centrifuged a second time at an acceleration of 3000g to 5000g for 20 to 45 minutes to obtain supernatant b. Finally, the supernatant b was subjected to a third centrifugation treatment at an acceleration of 10000g~13000g for 45min~60min to obtain supernatant c. S4. Extrusion: The supernatant c obtained from the final centrifugation in step S3 is extruded sequentially through a coarse microporous membrane with a pore size of 0.45 μm and a fine microporous membrane with a pore size of 0.2 μm or 0.22 μm to obtain buckwheat tea-derived nanovesicles. These nanovesicles are dispensed into EP tubes and stored at -80℃ for later use. The obtained buckwheat tea-derived nanovesicles have a characteristic lipid bilayer, a particle size of 170 nm, and a zeta potential of -6.57 mV.

[0046] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing buckwheat tea-derived nanovesicles, characterized in that, Includes the following steps: S1. Weighing and soaking: Weigh 20-100g of buckwheat tea (dry weight), add it to a blender, and soak it for 1-3 minutes in 120-600mL of phosphate buffer solution pre-cooled to 4℃. S2. Crushing and Filtration: Immediately after the phosphate buffer soaking is completed, start the high-speed blender to crush the phosphate buffer solution for 1-3 minutes. Then, filter the solution using a filter bag and collect the filtrate for later use. S3. Differential centrifugation: Transfer the filtrate prepared in step S2 to a centrifuge tube and perform differential centrifugation at 4°C: The acceleration of the first centrifugation is 1000g~2000g and the centrifugation time is 10min~20min, to obtain supernatant a; Then, supernatant a was centrifuged a second time at an acceleration of 3000g to 5000g for 20 to 45 minutes to obtain supernatant b. Finally, the supernatant b was subjected to a third centrifugation treatment at an acceleration of 10000g~13000g for 45min~60min to obtain supernatant c. S4. Extrusion: The supernatant c obtained from the last centrifugation in step S3 is extruded sequentially through a coarse sieve microporous membrane with a pore size of 0.45 μm and a fine sieve microporous membrane with a pore size of 0.2 μm or 0.22 μm to obtain buckwheat tea-derived nanovesicles, which are then packaged in EP tubes and stored at -80℃ for later use.

2. The method for preparing buckwheat tea-derived nanovesicles according to claim 1, characterized in that, In step S1, the concentration of the phosphate buffer is 0.01M and the pH value is 7.2~7.

4.

3. The method for preparing buckwheat tea-derived nanovesicles according to claim 1, characterized in that, In step S4, the obtained buckwheat tea-derived nanovesicles have a characteristic lipid bilayer, and the particle size of the buckwheat tea-derived nanovesicles is 100nm~200nm, with a Zeta potential of -5mV~-15mV.

4. The use of buckwheat tea-derived nanovesicles prepared by any one of claims 1 to 3 in the preparation of a medicament for treating ulcerative colitis.

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