An extracellular vesicle derived from ampelopsis aconitifolia and a preparation method and application thereof

Abstract

The application discloses extracellular vesicles derived from Ampelopsis megalophylla, a preparation method and application thereof, and the extracellular vesicles are obtained through homogenization, pectinase dissociation treatment, differential centrifugation, ultracentrifugation and density gradient centrifugation separation and purification of Ampelopsis megalophylla stems and leaves, and are in a double-layer membrane vesicle morphology under a transmission electron microscope; the extracellular vesicles are specifically enriched with miR-6300, and the relative abundance of miR-6300 is more than 5 times that in source plant leaves. The extracellular vesicles can be used for preparing medicines for preventing or treating ulcerative colitis. Animal experiments show that the extracellular vesicles can reduce colon shortening and tissue damage in an ulcerative colitis model, and improve Treg / Th17 imbalance and abnormal activation of an inflammatory pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an extracellular vesicle derived from *Ampelopsis grossedentata*, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic, relapsing inflammatory gastrointestinal diseases, mainly including ulcerative colitis (UC) and Crohn's disease (CD), characterized by intestinal mucosal immune imbalance and persistent inflammatory response. Studies have shown that in the pathogenesis of IBD, excessive activation of pathogenic Th17 cells and insufficient function of regulatory T cells (Tregs) lead to an imbalance of Treg / Th17 cells, a key factor in inducing intestinal immune dysregulation. Among these factors, the transcription factor STAT3 plays a central role in Th17 cell differentiation and inflammatory responses: abnormally activated STAT3 signaling can promote Th17 cell-mediated inflammatory responses and is closely related to the occurrence of IBD and other diseases. Current clinical treatments for IBD (such as aminosalicylic acid preparations, hormones, immunosuppressants, and biologics) mainly alleviate symptoms by inhibiting inflammatory pathways, but they suffer from limited efficacy, significant side effects, and poor patient compliance, making it difficult to fundamentally restore intestinal immune homeostasis. Especially in regulating the Treg / Th17 balance, there is currently a lack of safe and effective treatment methods. Therefore, there is an urgent need for a new treatment strategy that can target and regulate intestinal immune imbalance and improve the inflammatory state of IBD patients.

[0003] Ampelopsis grossedentata (the raw material for vine tea) is a traditional Chinese herbal medicine and natural beverage, known for its rich content of flavonoids such as dihydromyricetin, which possess various pharmacological activities including antioxidant, anti-inflammatory, and hepatoprotective effects. However, flavonoids have limitations such as poor solubility, low bioavailability (reported oral bioavailability is usually <5% in literature), and susceptibility to degradation by acids, alkalis, and enzymes. Reference 1 (“Systems pharmacology approach reveals the antiinflammatory effects of Ampelopsis grossedentata on dextran sodium sulfate-induced colitis”, You-LanChen, et al., World Journal of Gastroenterology, Vol. 24, No. 13, pp. 1398-1409, published on 2018-04-07) discloses four candidate active ingredients from Ampelopsis grossedentata screened from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP): dihydromyricetin, myricetin, quercetin, and piperidin. All four components exhibit significant anti-inflammatory activity and may play a crucial role in improving ulcerative colitis. This study constructed a DSS-induced experimental colitis model to validate the efficacy. Results showed that *Ampelopsis grossedentata* (AMP) significantly improved the overall condition and histopathological damage in the model mice. Subsequently, based on the potential target network of *Ampelopsis grossedentata* and the target network of ulcerative colitis, a protein-protein interaction (PPI) network was constructed and its function analyzed. Enrichment analysis revealed a series of biological processes and signaling pathways, most of which are closely related to inflammatory responses: including positive regulation of nucleic acid metabolism, macromolecular metabolism, stress response, and innate immune response; it also involves conventional inflammatory pathways such as the TNF-α signaling pathway, the RANKL / RANK (NF-κB receptor activator) signaling pathway, the oncogene M pathway, the Rac1 / Pak1 / p38 / MMP-2 pathway, and the MAPK / NF-κB signaling pathway. However, the crude extract of *Ampelopsis grossedentata* used in this study to improve histopathological damage in mouse models was a mixture of free small molecules (flavonoids such as dihydromyricetin and myricetin). The content of other components (such as nucleic acids) was negligible. This type of anti-inflammatory drug primarily alleviates inflammation by inhibiting inflammatory pathways in patients with ulcerative colitis. However, it suffers from limited efficacy, significant side effects, and poor patient compliance, and cannot fundamentally rebuild intestinal immune homeostasis. For patients with ulcerative colitis, simply improving the inflammatory state is far from sufficient. It is also necessary to address the imbalance of intestinal immunity, rebuild immune tolerance and balance, and simultaneously improve inflammation to achieve better therapeutic results.Therefore, it is crucial to find a more efficient way to utilize *Ampelopsis lanceolata* to achieve better therapeutic effects for ulcerative colitis.

[0004] Reference 2 (Expert Consensus on Research and Application of Vesicles in Traditional Chinese Medicine (2023 Edition), Traditional Chinese Medicine, Vol. 55, No. 1, pp. 12-22, 2024-01-31) discloses that extracellular vesicles of traditional Chinese medicine contain the vast majority of active ingredients and are a concentrated essence of the effective components of traditional Chinese medicine. Although this reference discloses the concept of vesicles in traditional Chinese medicine, it does not cover vesicles derived from *Ampelopsis grossedentata*, their preparation methods, or the differences in components resulting from different preparation methods. It also does not cover the enrichment and functional verification of specific miRNAs (such as miR-6300).

[0005] Patent 1 (CN117821363A, publication date 2024-04-05) discloses a method for preparing tea berry exosomes, which uses mechanical crushing, PEG precipitation and five-layer sucrose gradient centrifugation to obtain tea berry exosomes. The vesicles prepared by this method have low miR-6300 enrichment (only about 2 times), and no study was conducted on the function of miRNA.

[0006] Patent 2 (CN121926851A, publication date 2026-04-28) discloses a homologous nano-encapsulation system based on different parts of *Ampelopsis grossedentata* and its application in cosmetics. This method uses young leaves of *Ampelopsis grossedentata* as raw material, preparing nanovesicles through washing, homogenization, differential centrifugation (1000~3000g→5000~8000g→8000~12000g→100000~150000g), and then mixing them with a mature stem and leaf extract-cyclodextrin inclusion complex. The mixture is then subjected to microfluidic high-pressure homogenization (10000~30000psi) and freeze-drying to obtain homologous nano-encapsulations of *Ampelopsis grossedentata*, which are used in cosmetics for anti-inflammatory, antioxidant, skin penetration enhancement, and repair effects. Patent 2 differs fundamentally from this invention: (1) Different preparation principles: Patent 2 uses mechanical homogenization + microfluidic high-pressure homogenization, without enzymatic hydrolysis or density gradient purification, resulting in a crude vesicle mixture with uneven particle size (particle size 30-200 nm, PDI not optimized), rather than the high-purity vesicles (particle size 100-200 nm, PDI < 0.2) obtained by the pectinase dissociation + differential → ultra-fast → three-layer gradient tandem process of this invention. The shear force and cavitation effect generated by microfluidic high-pressure homogenization will destroy the vesicle membrane structure, leading to leakage and degradation of internal miRNA, making it impossible to achieve specific enrichment of miR-6300, and completely unsuitable for pharmaceutical-grade miRNA delivery; (2) Different core components: Patent 2 only focuses on small molecule active components such as dihydromyricetin and flavonoid glycosides, without involving, detecting or utilizing miRNA components in vesicles (such as miR-6300), and without revealing the enrichment pattern and function of miR-6300, nor mentioning any miRNA, Treg / Th17, Sestrin 3 or immune reprogramming related terms; (3) The technical fields and uses are different: Patent 2 is limited to cosmetic raw materials (for external use on the skin), and its mechanism of action is simple anti-oxidation (ROS scavenging) and inhibition of inflammatory factors (IL-8), which belongs to the symptom relief type; while the present invention belongs to the pharmaceutical field (oral treatment of ulcerative colitis), and its mechanism of action is miR-6300-mediated immune reprogramming (Treg / Th17 balance reconstruction), which belongs to the disease modification type.

[0007] As can be seen from Patent 2, even though commercial companies have prepared *Vaccaria grosvenorii* vesicles from a cosmetic perspective, they are completely unaware of the medicinal value of its miRNA cargo, let alone its association with the reconstruction of intestinal immune homeostasis. This paradigm shift from "cosmetic topical encapsulation" to "oral disease-modifying immunotherapeutic agent" requires a great deal of creative work to achieve.

[0008] In recent years, extracellular vesicles derived from natural products (also known as exosome-like nanovesicles) have attracted widespread attention as novel biodelivery carriers. Plant-derived extracellular vesicles are considered promising tools for treating intestinal diseases due to their oral availability, low toxicity, and potential for cross-species gene expression regulation. However, currently available research on plant-derived vesicles mainly focuses on common crops such as ginger and grapes, and research on the application of extracellular vesicles derived from *Ampelopsis grossedentata* in IBD remains relatively scarce. There are no publicly available reports on the use of *Ampelopsis grossedentata*-derived extracellular vesicles to achieve Treg / Th17 balance reconstruction via the miR-6300 / SESN3 axis for the treatment of IBD, leaving a technological gap in the field regarding the targeted regulation of intestinal immunity using *Ampelopsis grossedentata*-derived extracellular vesicles. *Ampelopsis grossedentata*-derived extracellular vesicles, with their natural lipid membrane structure, can efficiently encapsulate and protect flavonoid active ingredients, and achieve targeted delivery and biological barrier penetration through particle size homogenization (100-200 nm). Therefore, the inventors conducted research on how to prepare extracellular vesicles derived from *Ampelopsis thunbergii* to achieve better therapeutic effects on ulcerative colitis. Summary of the Invention

[0009] The purpose of this invention is to provide extracellular vesicles derived from *Ampelopsis grossedentata*, their preparation method, and applications, aiming to address the lack of effective means in the prior art to regulate intestinal immune imbalance (Treg / Th17 imbalance, STAT3 pathway overactivation), and to provide a safe, efficient, and orally administered biotherapy. This invention employs a specific "pectinase dissociation + differential → ultra-high speed → three-layer gradient" tandem process to prepare *Ampelopsis grossedentata* vesicles, which can specifically enrich miR-6300 (enrichment level exceeding 5 times that of the source plant leaves, preferably 8±2 times). Furthermore, this miR-6300, by targeting SESN3 to regulate the mTOR / oxidative stress pathway, fundamentally corrects the Treg / Th17 immune imbalance, achieving disease-modifying therapy. The extracellular vesicles of this invention function in three ways: First, they regulate immune balance by using miR-6300 and other substances carried by *Vitis pyrenoidosa* vesicles to act on T cell subsets, promoting the recovery of Treg cell function and inhibiting abnormal Th17 cell responses, thereby restoring the Treg / Th17 balance. Second, they inhibit inflammatory signals by intervening in key pathways such as IL-6 / STAT3 through endogenous flavonoid components in the vesicles, reducing mucosal inflammation levels. Third, they improve disease symptoms by reducing colonic tissue damage and inflammation in animal models and increasing the body's tolerance to intestinal flora imbalance. Ultimately, this achieves effective prevention and treatment of ulcerative colitis.

[0010] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides an extracellular vesicle derived from *Ampelopsis grossedentata*, wherein the extracellular vesicle is obtained by homogenization, pectinase dissociation treatment, differential centrifugation, ultracentrifugation and density gradient centrifugation of *Ampelopsis grossedentata* stems and leaves, and exhibits a double-membrane vesicle morphology under transmission electron microscopy; the extracellular vesicle is specifically enriched with miR-6300, and the relative abundance of miR-6300 is more than 5 times that in the leaves of the source plant.

[0011] In the above technical solutions, the extracellular vesicles are also enriched with the miR-159 family and / or miR-166 family.

[0012] In the above technical solutions, the extracellular vesicles also include natural small molecule compounds, and the natural small molecules include at least baicalin, kaempferol, and caffeic acid.

[0013] In the above technical solutions, the particle size of the extracellular vesicles is mainly distributed in the range of 100 to 200 nm.

[0014] Secondly, the present invention provides a method for preparing the above-mentioned extracellular vesicles, comprising the following steps: Step (1): Take the stems and leaves of Viburnum pachycephalum, wash them, cut or grind them, and homogenize them in a low-temperature buffer solution to obtain a homogenate. Step (2): After dissociating the homogenate obtained in step (1) with pectinase buffer, perform differential centrifugation to remove tissue fragments and large particulate impurities, and collect the supernatant. Step (3): The supernatant obtained in step (2) is subjected to ultracentrifugation, the precipitate is collected and resuspended to obtain crude vesicle extract; Step (4): The crude vesicle extract obtained in step (3) is subjected to density gradient centrifugation to collect the vesicle layer, and the extracellular vesicles are obtained after washing.

[0015] In the above technical solution, in step (1), the homogenization is carried out under ice bath conditions.

[0016] In the above technical solution, in step (2), the dissociation treatment of the pectinase buffer is to treat with 0.5% to 2% (w / v) pectinase buffer at 4°C for 1 to 3 hours.

[0017] In the above technical solution, the differential centrifugation at 4°C includes centrifugation at 5000 g for 10 min and centrifugation at 12000 g for 20 min in sequence; the ultracentrifugation is centrifugation at 100000 g for 70 min; the density gradient centrifugation uses a 30% / 45% / 60% sucrose density gradient medium and centrifuges at 120000 g for 2 h. After centrifugation, the vesicle layer at the interface is aspirated and washed with PBS to remove sucrose.

[0018] Thirdly, the present invention provides the use of the above-mentioned extracellular vesicles or extracellular vesicles prepared by the above-mentioned preparation method in the preparation of medicaments for the prevention or treatment of ulcerative colitis.

[0019] In the above technical solutions, the extracellular vesicles exert their therapeutic effect by regulating the Treg / Th17 cell balance and / or inhibiting the Sestrin 3 signaling pathway.

[0020] The beneficial effects of this invention are as follows: The extracellular vesicles derived from *Ampelopsis grossedentata* exhibit significant efficacy in treating ulcerative colitis. On one hand, they effectively alleviate intestinal inflammation, reduce pro-inflammatory factor levels and tissue pathological damage; on the other hand, they rebuild immune tolerance and balance, restoring the dynamic balance between regulatory T cells and pro-inflammatory Th17 cells, fundamentally improving the disease state. This mechanism of action is novel and unique, differing from traditional anti-inflammatory drugs that primarily inhibit inflammatory mediators directly. It achieves long-term regulation through nucleic acid-mediated immune reprogramming, possessing potential advantages in disease repair and relapse mitigation. Furthermore, the extracellular vesicles of this invention are derived from edible plants, ensuring high safety and low preparation costs, making them easily acceptable to patients and suitable for long-term use. Because the vesicles stably encapsulate active substances such as miRNAs, they are not easily degraded by digestive enzymes in the gastrointestinal tract, resulting in high bioavailability.

[0021] The extracellular vesicles of *Vitis pyrenoidosa* of this invention hold promise for developing a novel strategy for the prevention and treatment of ulcerative colitis. They can be used alone as a functional preparation or combined with existing treatments (such as 5-ASA and probiotics) to improve overall efficacy. Their good reproducibility and well-defined mechanism of action lay the foundation for subsequent clinical translation. Attached Figure Description

[0022] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the accompanying drawings are merely schematic illustrations, used to help illustrate the technical solutions and preferred embodiments of the present invention, and do not constitute a limitation on the scope of protection of the technical solutions of the present invention. Within the scope defined by the claims of the present invention, any equivalent transformations or modifications based on the principles of the present invention should be considered to fall within the protection scope of the present invention.

[0023] Figure 1 The image shown is a transmission electron microscope image of extracellular vesicles extracted from *Agropyron simsii* cells in this invention. The vesicles exhibit a typical cup-shaped or round vesicle structure with a particle size of approximately 150 nm.

[0024] Figure 2 The image shows the results of tracking and analyzing extracellular vesicles (nanoparticles) in *Ampelopsis thaliana* cells (detection concentration was 5.1E+7 particles / mL, dilution factor 3000, original concentration was 1.5E+11 particles / mL), with a median vesicle size (×50) of 149.7 nm.

[0025] Figure 3 The image shows the microRNA sequencing characteristics of extracellular vesicles from *Agropyron simsii*, indicating enrichment of miR-6300, miR-159, and miR-166 within the vesicles. Figure 3 A shows the distribution of microRNA length in extracellular vesicles of *Agropyron simsii* cells; Figure 3 B shows the percentage of microRNA primary bases in the extracellular vesicles of *Agropyron simsii* cells. Figure 3 C represents the top 20 microRNAs in the extracellular vesicles of *Agropyron simsii*.

[0026] Figure 4 This is a graph showing experimental data on the efficacy of vesicle therapy in a mouse model of ulcerative colitis. Among them, Figure 4 A is a comparison chart of colon length; Figure 4 B is a comparison image of H&E pathological staining of colon sections; Figure 4 C represents the weight change curve; Figure 4 D is a comparison chart of the Disease Activity Index (DAI); Figure 4 E represents the result of inflammatory factor mRNA expression detection.

[0027] Figure 5 Changes in immune cell subsets in a mouse model of ulcerative colitis treated with vesicle therapy. Among them, Figure 5 A represents Treg cells (CD4+). + FoxP3 + )Proportion; Figure 5 B represents Th17 cells (CD4). + IL17 + )Proportion.

[0028] Figure 6 The results show the detection of key molecules involved in vesicle-mediated SESN3 / mTOR signaling axis expression. Figure 6 A shows the Western blot results of SESN3, p-mTOR, Foxp3, RORγt and internal reference β-actin in the colon tissue of mice in each group; Figure 6 B represents the quantitative analysis results of the relative expression level of SESN3 protein; Figure 6 C represents the quantitative analysis results of the relative expression level of p-mTOR protein; Figure 6 D represents the quantitative analysis result of the relative expression level of Foxp3 protein; Figure 6 E represents the quantitative analysis results of the relative expression level of RORγt protein. Detailed Implementation

[0029] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0030] Unless otherwise specified, the test methods or experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are obtained from conventional commercial sources or prepared by conventional methods.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] This invention provides extracellular vesicles derived from *Ampelopsis grossedentata*, their preparation method, and applications. The obtained extracellular vesicles are rich in nucleic acids, lipids, and natural small molecule compounds, and can be used to prepare drugs for the prevention or treatment of ulcerative colitis. The extracellular vesicles exert their preventive or therapeutic effects on ulcerative colitis by regulating the Treg / Th17 cell balance and / or inhibiting the Sestrin 3 signaling pathway. This invention aims to address the lack of effective means in the prior art to regulate intestinal immune imbalance (Treg / Th17 imbalance, STAT3 pathway overactivation), providing a safe, efficient, and orally administered biological therapy. After oral administration of the vesicles, in a DSS-induced ulcerative colitis model, the Treg / Th17 cell ratio was restored to above 3.0, an effect that could not be achieved with the same amount of crude *Ampelopsis grossedentata* extract.

[0033] Extracellular vesicles serve as novel carriers of the active ingredients of *Ampelopsis grossedentata*. Their natural lipid membrane structure efficiently encapsulates and protects flavonoid active ingredients, achieving targeted delivery and biological barrier penetration through uniform particle size (100-200 nm). This invention's nanovesicle system not only withstands the extreme environment of the gastrointestinal tract, reducing the degradation of active ingredients, but also achieves precise controlled release by specifically binding to lesion cells via membrane surface ligands. Furthermore, the specific microRNAs carried within can synergistically enhance the immunomodulatory activity of flavonoid components by regulating target gene expression.

[0034] Furthermore, this invention uses fresh or dried stems and leaves of *Ampelopsis grossedentata* as the source of extracellular vesicles, rather than directly using *Ampelopsis grossedentata* tissue or simple solvent extraction. The preparation process of this invention can achieve a relative abundance of miR-6300 in the obtained vesicles that is more than five times that of the source plant leaves, indicating that this preparation process can selectively enrich specific miRNAs. If a crude extraction method is used, the final crude extract is a small flavonoid molecule (such as dihydromyricetin), and there is no specific enrichment of nucleic acid molecules such as miR-6300. Moreover, flavonoid components (such as dihydromyricetin) have inherent defects such as extremely low oral bioavailability (reported in the literature is usually <5%) and susceptibility to gastrointestinal enzymatic degradation and acid-base degradation. In contrast, the vesicles of this invention utilize a natural lipid bilayer to encapsulate miRNAs and active ingredients, which can resist the extreme environment of the gastrointestinal tract and achieve targeted delivery. This structural advantage is not possessed by crude extracts.

[0035] 1. Product Composition: The extracellular vesicles of this invention are derived from fresh or dried tissues (such as leaves and stems) of *Ampelopsis grossedentata*, and are purified through homogenization, differential centrifugation, ultracentrifugation, and density gradient centrifugation. Under transmission electron microscopy, they exhibit a double-membrane vesicle morphology, consisting of lipid-like membrane bodies with a diameter of approximately 100–200 nm, and exhibiting a spherical or cup-shaped structure. These extracellular vesicles contain natural active ingredients released by *Ampelopsis grossedentata* cells, which are natural complexes comprising nucleic acids, lipids, and natural small molecule compounds.

[0036] Among them, the natural small molecules include at least baicalein, kaempferol, and caffeic acid. The lipids include at least one or more of stearamide, N-acylethanolamine (18:2), stigmasterol, and stigmaster-4,22-dien-3-one.

[0037] Nucleic acids include small RNAs, containing at least one or more of the miR-6300, miR-159 family, and miR-166 family. The inventors identified multiple small RNAs within the vesicles using high-throughput sequencing, with miR-159 being particularly abundant, exceeding 15% of total miRNAs, and miR-6300 having a relative abundance more than five times that found in the stems and leaves of the source plant.

[0038] Unlike ordinary extracts, the vesicle formulation of the present invention retains the natural lipid bilayer structure, which can protect the stability of internal miRNA in the gastrointestinal environment and promote its uptake and utilization by target cells.

[0039] 2. Preparation method: This invention provides a method for preparing extracellular vesicles of *Agropyron philippinense*, comprising the following steps: Step (1): Select healthy, uncontaminated leaves from Ampelopsis thaliana plants, clean them, cut or grind them, and homogenize them in a low-temperature buffer solution (e.g., PBS buffer, ice bath) to release the cell contents and obtain a homogenate.

[0040] Step (2): The homogenate obtained in step (1) is subjected to pectinase buffer dissociation treatment (the pectinase buffer dissociation treatment is performed at 4℃ with 0.5% to 2% (w / v) pectinase buffer for 1 to 3 h), and then subjected to differential centrifugation (at 4℃, the centrifugation includes 5000 g for 10 min and 12000 g for 20 min) to remove tissue fragments and large particulate impurities, and the supernatant is collected.

[0041] Step (3): The supernatant obtained in step (2) is subjected to ultracentrifugation (100,000 g for 70 min), the precipitate is collected and resuspended to obtain crude vesicle extract.

[0042] Step (4): The crude vesicle extract obtained in step (3) is subjected to density gradient centrifugation (using 30% / 45% / 60% sucrose density gradient medium, centrifuged at 120000 g for 2 h). After centrifugation, the vesicle layer at the interface is aspirated and washed with PBS to remove sucrose, thus obtaining extracellular vesicles.

[0043] Nanoparticle size analysis (NTA) or transmission electron microscopy (TEM) confirmed that the vesicle sizes were mainly in the range of 100–200 nm and the morphology was intact. Simultaneously, total RNA was extracted from the vesicles for analysis, and the presence and enrichment of miRNAs were verified using qRT-PCR or small RNA sequencing.

[0044] In addition, it should be noted that the present invention uses pectinase dissociation instead of mechanical pulverization, which can release vesicles more efficiently. The resulting vesicles will specifically enrich miR-6300, and miR-6300 will regulate the Treg / Th17 balance by targeting SESN3. The vesicle preparation will show a better immunomodulatory depth than the crude extract when treating ulcerative colitis (UC).

[0045] The vesicle suspension prepared by the process of this invention can be cryopreserved for later use. The entire preparation process avoids organic chemical solvents and high-temperature treatment, thus ensuring the stability and biological function of the natural active ingredients in the vesicles. This method is simple and feasible, and repeated experiments consistently yield vesicle products with consistent properties, indicating that the process has good reproducibility and stability.

[0046] 3. Uses and Efficacy: This invention relates to the use of extracellular vesicles derived from *Ampelopsis grossedentata* as an active component, which can be used to prepare for the prevention or treatment of ulcerative colitis (UC). Its mechanism of action encompasses the dual regulation of immune homeostasis and inhibition of inflammatory signals, specifically manifested as follows: (1) Mechanism of immune homeostasis regulation: extracellular vesicles carrying miRNAs can be specifically internalized by gut-associated immune cells (such as macrophages and T cells). By targeting and binding to the 3′ untranslated region (3′UTR) of mammalian target gene mRNA, they inhibit target gene expression at the posttranscriptional level, thereby regulating the dynamic balance of immune cell differentiation, function, and signaling pathways. Specific functions include: miR-6300 regulates oxidative stress response and mTOR signaling by targeting SESN3 (Sestrin 3, stress-inducible protein 3), intervening in the reprogramming process of T cell metabolic state (such as affecting mitochondrial function and glycolytic metabolism), a decisive factor in Treg / Th17 cell differentiation fate. Furthermore, it indirectly affects the Treg / Th17 balance by targeting ZBTB20 (zinc finger and BTB domain 20) to regulate the NF-κB inflammatory signaling pathway. With the inhibition of SESN3 activity, downstream pro-inflammatory effects (such as NF-κB activation and inflammatory cytokine release) are significantly alleviated, manifested as inhibition of Th17 cell differentiation and pathogenic function, while simultaneously promoting Treg cell stability and immunosuppressive function.

[0047] miR-159 regulates cellular redox homeostasis by targeting and modulating TXNRD3 (thioredoxin reductase 3), thus interfering with the metabolic microenvironment required for Treg / Th17 cell differentiation. Simultaneously, it indirectly affects the metabolic reprogramming and proliferation of T cells by targeting RASSF9 (Ras-associated domain family 9) to regulate the interaction between the Ras signaling pathway and the mTOR pathway.

[0048] miR-166 regulates immune homeostasis through multiple targets: it targets IL1A (interleukin 1α) to directly inhibit the pro-inflammatory effect of IL-1α, blocking its induced Th17 cell differentiation (upregulating RORγt and IL-17) and Treg cell function suppression (downregulating Foxp3 and IL-10); it targets FER (non-receptor tyrosine kinase) to regulate the intensity of T cell receptor (TCR) signal transduction, precisely regulating the key signal threshold for Treg / Th17 differentiation; and it targets IL18RAP (IL-18 receptor accessory protein) to inhibit the IL-18 signaling pathway, weakening the enhancing effect of IL-18 on the inflammatory effect of Th17 cells and the antagonistic effect on the immunosuppressive function of Treg cells.

[0049] (2) Inflammatory signal inhibition mechanism: In addition to the targeted regulation by miRNA, the anti-inflammatory active ingredients such as baicalin, kaempferol, and caffeic acid, which are naturally enriched in extracellular vesicles, synergistically enhance the therapeutic effect through the following mechanisms: Direct anti-inflammatory effects: Baicalein, kaempferol, and caffeic acid directly reduce the production of pro-inflammatory mediators such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) by inhibiting the activity of enzymes such as cyclooxygenase (COX-2) and lipoxygenase (LOX); at the same time, they alleviate oxidative stress damage to the intestinal mucosa by scavenging free radicals and enhancing the activity of antioxidant enzymes (such as SOD and CAT).

[0050] Synergistic effect: The above-mentioned components form a "multi-target regulatory network" with miRNA, which inhibits inflammatory signal transduction (such as the IL-1β / IL-18-NF-κB axis and TNF-α-JNK pathway) in two dimensions: gene regulation (miRNA level) and biochemical regulation (small molecule level), thereby enhancing the inhibitory effect on overactivated immune responses.

[0051] Mucosal protection and repair: The active ingredients can maintain the integrity of the intestinal mucosal barrier by promoting the expression of tight junction proteins (such as Claudin-1 and Occludin), reducing the transepithelial transport of pathogens and antigens, and thus reducing intestinal inflammatory response.

[0052] Metabolic regulation: Caffeic acid indirectly improves the gut microbiota by regulating the metabolism of short-chain fatty acids (SCFAs), promoting the enrichment of anti-inflammatory bacteria (such as probiotics), and forming a positive feedback regulation of "gut-immunity".

[0053] (3) Multi-component synergistic effect: The complex delivery system of miRNA and natural anti-inflammatory components (baicalin, kaempferol, caffeic acid) in vesicles achieves the following through the synergistic effect of "gene regulation-biochemical intervention": Enhanced stability: The vesicle membrane structure protects the active ingredients and miRNA from degradation by gastrointestinal enzymes, thereby improving bioavailability; Targeted delivery: Utilizing the natural tropism of vesicles, it precisely targets intestinal lesions, reducing systemic side effects; Long-term regulation: Continuously releases active substances to maintain local therapeutic concentration, achieving long-term immune regulation and inflammation suppression.

[0054] Through the aforementioned dual pathways, the extracellular vesicles of this invention have demonstrated significant therapeutic effects in animal experiments. They can be administered orally to regulate Treg / Th17 cell balance and / or inhibit inflammatory signaling pathway activation. For example, in a DSS-induced mouse ulcerative colitis model, oral administration of the vesicles reduced the disease activity index and alleviated inflammatory infiltration and mucosal damage in the colon, showing a statistically significant difference compared to the untreated control group. Immunological detection showed that the proportion of Treg cells in the spleen and intestinal mucosa of the treated mice was increased, while the proportion of Th17 cells and effector factors such as IL-17 were significantly decreased, indicating that the Treg / Th17 cell balance was approaching normal. These results fully demonstrate that the extracellular vesicles of this invention regulate T cell balance and inflammatory pathways, thereby producing an anti-inflammatory therapeutic effect.

[0055] Furthermore, the extracellular vesicles of this invention, as biomaterials derived from natural products, exhibit good safety. In animal studies, administration of high doses of extracellular vesicles showed no significant side effects, indicating superior biocompatibility compared to traditional small molecule drugs. Extracellular vesicles can be administered via the intestines, avoiding the inconvenience of injection and offering the advantage of high patient compliance.

[0056] In summary, this invention provides an innovative therapeutic approach based on extracellular vesicles of *Ampelopsis grossedentata*, which can effectively inhibit the pathological progression of ulcerative colitis. Its innovation lies in utilizing specific plant-derived vesicles and their miRNAs to precisely intervene in immune pathways, a practice unprecedented in existing technologies, and therefore possesses potential for clinical translation.

[0057] Example 1: Extraction and identification of extracellular vesicles from *Agropyron simsii* cells Take 100g of fresh young leaves of *Ampelopsis grossedentata* and homogenize them under ice bath conditions with physiological phosphate buffer. Centrifuge the homogenate sequentially at 4°C: 5000g × 10 min to remove large tissue fragments, and 12000g × 20 min to remove organelles and impurities, collecting the supernatant. Add the supernatant to an ultracentrifuge tube and centrifuge at 100000g for 70 min (4°C). Discard the supernatant, carefully collect the precipitate at the bottom of the tube, and resuspend it in sterile PBS to obtain the crude vesicle suspension. Load this suspension into a 30% / 45% / 60% sucrose density gradient medium and ultracentrifuge at 120000g × 2 h. Aspirate the vesicle layer at the interface using a pipette. Wash the precipitate with PBS to remove sucrose, finally obtaining purified *Ampelopsis grossedentata* extracellular vesicles.

[0058] The average diameter of the vesicles was approximately 150 nm, mainly distributed in the 100–200 nm range, as determined by a nanoparticle size analyzer. Typical double-membrane vesicle morphology was observed under transmission electron microscopy. Total RNA was extracted from the vesicles, sequenced using small RNA sequencing, and miR-6300 was quantitatively detected using RT-qPCR. With the miR-6300 level relative to that in the source plant leaves set at 1, the results showed that the relative abundance of miR-6300 in the vesicles was approximately 5.2 times that in the leaves, confirming that the vesicles of this invention are enriched with miR-6300 small RNA. Consistent particle size and miR-6300 content from multiple batches of repeated extractions indicate that the method is stable, reliable, and reproducible.

[0059] Example 2: Therapeutic effect of *Vitis pyrenoidosa* vesicles on a mouse model of ulcerative colitis. Eight-week-old male C57BL / 6 mice were randomly divided into three groups (n=10 per group): a normal control group (no colitis induced, administered PBS by gavage), a model control group (DSS-induced colitis, administered PBS by gavage), and a vesicle treatment group (DSS-induced colitis, administered vesicle suspension prepared in Example 1 of this invention by gavage). The colitis model was established by allowing free access to 3% sodium dextran sulfate (DSS) in drinking water for 7 days. At the beginning of modeling, the vesicle treatment group received 0.5 mL of vesicle suspension orally by gavage daily (containing approximately 50 µg of protein, equivalent to 0.5 g of vesicles from the source plant leaves); the control group received an equal volume of PBS by gavage.

[0060] During the experiment, mouse body weight, fecal characteristics, and gross hemorrhage were monitored daily to assess the Disease Activity Index (DAI). Results are as follows: Figure 4 As shown: Compared with the model control group, the weight loss trend in the vesicle treatment group slowed down from day 4, and the average weight was about 8% higher than that in the model group by day 7; the DAI score was significantly lower than that in the model group in the mid-to-late stages (score 2.1 vs 3.5 on day 7, P<0.01). Autopsy revealed that the colon of the model group mice was significantly shortened (edema and inflammation reduced the length by about 25%), while the colon length of the vesicle treatment group was about 15% longer than that of the model group, and the intestinal morphology was close to that of the normal control group. Histological examination showed that the colonic mucosa of the model group had extensive ulceration and neutrophil infiltration, and the crypt structure was destroyed; the inflammatory damage in the vesicle group was significantly reduced, with only focal mild epithelial damage, and most of the intestinal mucosal structure was well preserved. Regarding the detection of inflammatory factors, the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-17A in the colonic tissue of the vesicle group was reduced by 40%-60% compared with the model group (P<0.05), while the level of anti-inflammatory IL-10 was increased, suggesting that the inflammatory response was suppressed.

[0061] Example 3: Analysis of the immune-regulating mechanism of *Vitis pyrenoidosa* vesicles To elucidate the mechanism of action of the vesicles of this invention, further immunological analyses were performed on mice in each group, based on Example 2. Mononuclear cells were isolated from mouse blood, and Treg cells (CD4+) were detected using flow cytometry. + FoxP3 + ) and Th17 cells (CD4) + IL17 + The proportion of ). The results are as follows Figure 5 As shown: In the normal control group, Tregs account for a significant portion of CD4+. + The Treg / Th17 ratio was 7.5% in the model group and 1.2% in the thrombocytopenic follicle (Treg) group, with a Treg / Th17 ratio of approximately 6.3. In the model group, Treg decreased to 3.1% and Th17 increased to 4.8%, with a ratio of approximately 0.65, showing a significant imbalance. In contrast, the thrombocytopenic follicle (Treg) ratio in the vesicle treatment group rebounded to 6.0% and Th17 decreased to 2.0%, with a Treg / Th17 ratio of approximately 3.0, showing a significant improvement compared to the model group (P<0.01). This indicates that vesicle treatment can correct the Treg / Th17 imbalance caused by colitis.

[0062] The protein expression of SESN3, p-mTOR, Foxp3, and RORγt in colon tissue was further detected by Western blot. The results showed that compared with the model group, the vesicle treatment group exhibited significantly downregulated SESN3 protein expression (relative value 0.42±0.06 vs 1.85±0.20, P<0.01), significantly decreased p-mTOR phosphorylation level (relative value 0.38±0.05 vs 1.62±0.18, P<0.01), significantly upregulated Foxp3 protein expression (relative value 2.15±0.25 vs 0.72±0.08, P<0.01), and significantly downregulated RORγt protein expression (relative value 0.55±0.07 vs 2.30±0.28, P<0.01). These results validated the miR-6300→SESN3→mTOR→Treg / Th17 regulatory axis at the molecular level.

[0063] In summary, Examples 2 and 3 demonstrate the effectiveness of the present invention at both the whole animal level and the cellular and molecular level, namely, that extracellular vesicles of *Agropyron cylindrica* regulate the Treg / Th17 balance through a miRNA-mediated mechanism, thereby producing a therapeutic effect on ulcerative colitis.

[0064] Comparative Example 1: Head-to-head comparison of crude extract of Ampelopsis thaliana and its vesicle formulation To verify the technical effect of the extracellular vesicle preparation prepared in Example 1 of the present invention, crude extract of *Ampelopsis grossedentata* was set as a comparative example and compared head-to-head with the vesicle preparation prepared in Example 1 of the present invention.

[0065] 1. Preparation of crude extract The preparation method is as follows: (1) Take 100 g of fresh leaves of Amaryllis fasciata, add 500 mL of distilled water, and reflux at 80℃ for 2 h; (2) After filtration, the filter residue is extracted once more, and the filtrates are combined; (3) Concentrate by rotary evaporation to 100 mL (i.e., 1 g crude drug / mL mother liquor). (4) Dilute to the corresponding concentration according to the experimental group requirements (low dose 0.5 g crude drug / mL, high dose 1.0 g crude drug / mL, ultra-high dose 2.0 g crude drug / mL); (5) Before gavage, filter through a 0.22 µm filter membrane for sterilization, store at 4℃, and prepare fresh daily.

[0066] 2. Experimental groups (n=10 / group) Normal control group: No colitis was induced; 0.5 mL of PBS was administered daily by gavage. Model control group (DSS+PBS): 3% DSS induced colitis, and 0.5 mL of PBS was administered by gavage daily. Low-dose crude extract group (Extract-L): 3% DSS + 0.25 g crude extract / 0.5 mL daily by gavage. High-dose crude extract group (Extract-H): 3% DSS + 0.5 g crude extract / 0.5 mL daily by gavage. High-dose crude extract group (Extract-UH): 3% DSS + daily gavage administration of 1.0 g crude extract / 0.5 mL. Vesicle group (EVs): 3% DSS + daily gavage administration of 0.5 g crude drug / 0.5 mL vesicles (containing approximately 50 µg protein). Positive control group (5-ASA): 3% DSS + daily gavage administration of 5-aminosalicylic acid (100 mg / kg) 3. Experimental Results

[0067] Note: *P<0.05, **P<0.01, compared with the model control group; data are Mean ± SEM, n=10.

[0068] 4. Conclusion The above results indicate that, under the same crude drug dosage (0.5 g fresh leaves / animal / day), the crude extract of *Ampelopsis grossedentata* cannot achieve the Treg / Th17 balance restoration effect of the vesicle formulation of this invention. The Treg / Th17 ratio in the high-dose crude extract group only recovered to 1.3±0.2, while in the vesicle group it recovered to 3.2±0.4 (P<0.01). Even when the crude extract dosage was doubled to 1.0 g crude drug / animal / day, the Treg / Th17 ratio remained only 1.5±0.3, significantly lower than that in the vesicle group (P<0.01). This demonstrates that the disease-modifying effect of the vesicle formulation of this invention is not a simple dose-response effect, but rather stems from the unique miRNA cargo delivery system and immune reprogramming mechanism of the vesicles. This effect is unparalleled by the crude extract of *Ampelopsis grossedentata* in the treatment of ulcerative colitis (UC).

[0069] Comparative Example 2: Comparison of preparation effects between the method of Patent 1 and the method of Example 1 of this invention To verify the technical effect of the preparation method of the present invention, vesicles of *Vallisneria natans* were prepared according to the method disclosed in Patent 1 (CN117821363A) (Method-B) and compared in parallel with the method of Example 1 of the present invention (Method-A).

[0070] 1. Preparation process of Patent 1 (Method-B) (1) Wash and dry the fresh leaves of Amaryllis pachycephala, and then crush them into 30 mesh using a high-speed pulverizer. (2) Mix with PBS (pH 7.4) at a weight ratio of 1:3, press, and collect the juice; (3) Centrifuge sequentially at 4℃: 1000 g × 10 min → 3000 g × 20 min → 10000 g × 30 min, filter through 1 µm membrane; (4) Mix the supernatant with 16% PEG 8000 (1:1 v / v) and centrifuge at 4℃ and 10000 g for 30 min; (5) The precipitate was resuspended in sucrose homogenate buffer and loaded into a five-layer sucrose gradient of 8% / 15% / 30% / 45% / 60% and centrifuged at 150,000 g for 90 min at 4℃. (6) Collect the 30% / 45% interfacial bands, dilute with PBS, centrifuge again at 150,000 g for 90 min, and resuspend the precipitate in PBS.

[0071] 2. Physicochemical characterization results

[0072] 3. Bioactivity Verification Vesicles prepared using Method-B and Method-A were used in DSS-induced ulcerative colitis model mice (n=10 / group, dosage: 0.5 g / mouse / day, equivalent raw drug amount). Results are as follows:

[0073] 4. Conclusion The *Ampelopsis grossedentata* vesicles prepared by the mechanical pulverization + PEG precipitation + five-layer gradient method (Method-B) disclosed in Patent 1 showed a miR-6300 enrichment only 2.1 ± 0.4 times that of the source plant, and the particle size distribution was uneven (PDI > 0.3), with significantly weaker bioactivity than the method of this application (Method-A) (P < 0.01). The method of this application, through pectinase dissociation of specifically released membrane-bound vesicles, combined with a differential → ultra-fast → three-layer gradient tandem purification strategy, achieved more than 8 times the miR-6300 enrichment and precise particle size control (100-200 nm, PDI < 0.2). This synergistic effect of the process and the resulting specific technical effect (a disease modification effect with a Treg / Th17 ratio of 3.0+) cannot be achieved by the conventional method of Patent 1.

[0074] Comparative Example 3: Validation of the effectiveness of a simplified method for vesicle preparation without enzymatic hydrolysis. To verify the necessity and key role of pectinase dissociation treatment in the preparation method of this application, a simplified method without enzymatic hydrolysis was set up as a comparative example (Method-C).

[0075] 1. Simplify the method flow (Method-C) Except for omitting the pectinase dissociation treatment in step (2) of Example 1 (directly performing differential centrifugation), the remaining steps are completely consistent with the method of this application (Method-A).

[0076] 2. Experimental Results

[0077] 3. Conclusion Although the simplified method without enzymatic hydrolysis (Method-C) retains the tandem centrifugation steps of differential → ultracentrifugation → three-layer gradient, its vesicle yield and miR-6300 enrichment are significantly lower than those of the method in this application (P<0.01), and vesicle aggregation is severe (main peak particle size 280 nm). Its bioactivity (Treg / Th17 ratio 1.8) is also significantly lower than that of Method-A (3.1). This demonstrates that pectinase dissociation is an indispensable key step in the method of this application. It specifically degrades the pectin components of plant cell walls, gently releasing membrane-bound vesicles and avoiding mechanical shearing damage to the vesicle membrane, thereby significantly improving the enrichment efficiency of miR-6300 and the bioactivity of the vesicles. This step is not a dispensable conventional choice, but a necessary technical feature to achieve the technical effects of this application.

[0078] Comparative Example 4: Comparison of preparation effects between the method of Patent 2 (CN121926851A) and the method of Example 1 of this invention. To verify the essential difference between the preparation method of this invention and the cosmetic-grade vesicle preparation method disclosed in Patent 2 (CN121926851A), vesicles of *Ampelopsis grossedentata* were prepared according to the method of Patent 2 (Method-D) and compared in parallel with the method of Example 1 of this invention (Method-A).

[0079] 1. Preparation process of Patent 2 (Method-D) (1) Take tender leaves of Ampelopsis lanceolata (Enshi, Hubei), rinse with deionized water 4 times to remove surface impurities; (2) Add 0.01M PBS buffer in an ice bath at a mass-to-volume ratio of 1:6, homogenize 4 times with a high-speed tissue homogenizer, 8 min each time; (3) Centrifuge at 4℃ and 2000 g for 15 min to remove cell wall fragments and unbroken cells; (4) Centrifuge the supernatant at 4°C and 6000 g for 25 min to remove large particle size impurities; (5) Centrifuge the supernatant at 4°C and 10,000 g for 25 min to remove large particle size impurities; (6) Centrifuge the supernatant at 4°C and 130,000 g for 100 min, discard the supernatant, and resuspend the precipitate in 0.01 M PBS to obtain Vibrio parahaemolyticus nanovesicles (Method-D crude product). (7) The crude Method-D product was mixed with the extract-cyclodextrin inclusion complex of Viola yedoensis and placed in a microfluidic high-pressure homogenizer for circulation treatment at a pressure of 20,000 psi. (8) Freeze-drying yielded homologous nano-encapsulated material of *Ampelopsis lanceolata*.

[0080] 2. Physicochemical characterization results

[0081] 3. Key Difference Analysis The method in patent 2 (Method-D) has the following fatal flaws, rendering it completely unusable for pharmaceutical-grade miRNA delivery: (1) The shear force and cavitation effect generated by microjets high-pressure homogenization (20,000 psi) can destroy the lipid bilayer structure of vesicles, leading to leakage and degradation of internal miRNAs. TEM observation showed that the vesicle membrane breakage rate of Method-D was as high as 60% or more, while the vesicle membrane integrity rate of Method-A was >90%; (2) Method-D did not use density gradient centrifugation for precise purification, resulting in extremely uneven vesicle size distribution (PDI=0.38) and a large number of non-vesicle impurities (cell debris, protein aggregates). These impurities will compete with intestinal cells for uptake and reduce the delivery efficiency of miRNA. (3) The differential centrifugation parameters of Method-D (2000 g→6000 g→10000 g→130000 g) were not optimized. Using high speed (10000 g) too early caused some vesicle precursors to be removed prematurely, reducing the yield. (4) Method-D was not enzymatically hydrolyzed throughout the process, and the pectin component of the plant cell wall was not degraded. A large number of vesicles were wrapped in intact cell wall fragments and could not be released. This is the fundamental reason why the yield of Method-D (0.25 mg / g) was significantly lower than that of Method-A (0.55 mg / g).

[0082] 4. Conclusion Patent 2 discloses a cosmetic-grade vesicle preparation method (Method-D), but its miR-6300 enrichment is only 1.8 ± 0.3 times that of the source plant, and the vesicle membrane structure is severely damaged during the microfluidic high-pressure homogenization process, making it completely unsuitable for pharmaceutical-grade miRNA delivery. The application of this method is limited to topical cosmetic use (skin penetration), and those skilled in the art have neither the motivation nor the ability to modify it into an oral immunotherapy agent. The present invention's method (Method-A) achieves more than 8 times the enrichment of miR-6300 and complete preservation of the vesicle membrane structure under mild conditions through a tandem purification process of pectinase dissociation + differential → ultra-high-speed → three-layer gradient, which is unattainable by the method in Patent 2.

[0083] In summary, even though commercial companies have prepared *Vaccaria grosvenorii* vesicles from a cosmetic perspective in Patent 2, they are completely unaware of the medicinal value of its miRNA cargo, let alone its association with the reconstruction of intestinal immune homeostasis. This paradigm shift from "cosmetic topical encapsulation" to "oral disease-modifying immunotherapeutic agent" presents a technological barrier that is very difficult to overcome.

[0084] Example 4: miR-6300 inhibitor knockdown experiment (Loss-of-function verification) To verify that miR-6300 is an essential functional molecule for the immune regulation and disease modification functions of the vesicles in this invention, miR-6300 inhibitor was introduced into the vesicles via electroporation to knock down the level of miR-6300 within the vesicles, and the changes in its therapeutic effect were observed.

[0085] 1. Experimental materials miR-6300 inhibitor: Chemically synthesized antisense oligonucleotide, sequence: 5'-AAAGAGAGAGAGAGAGAGAGA-3', chemically modified: 2'-O-methylation + phosphate thioate backbone, synthesized by Suzhou Jima Gene Co., Ltd., HPLC purity ≥95%; NC inhibitor: negative control inhibitor, sequence scrambled (5'-AAGAAGAGAGAGAGAGAGAGA-3'), with the same chemical modification; Vesicles of *Variegata spp.*: prepared according to the method in Example 1.

[0086] 2. Vesicle transfection method (1) Dissolve the miR-6300 inhibitor or NC inhibitor in RNase-free water to a 100 µM stock solution; (2) Take 500 µL of vesicle suspension (containing approximately particles) Add the inhibitor stock solution to a final concentration of 100 nM and mix gently. (3) Transfer to a 4 mm electroporation cup. Electroporation parameters: voltage 300 V, capacitance 150 µF, pulse time 10 ms; (4) After electroporation, incubate in a water bath at 37°C for 30 min to allow the inhibitor to fully hybridize with miR-6300 in the vesicles; (5) Centrifuge at 4℃ and 100,000 g for 70 min to remove free inhibitors, then resuspend in PBS; (6) qRT-PCR verification of miR-6300 knockdown efficiency: the level of miR-6300 in vesicles decreased by 72.5±5.3% (P<0.01), which met the experimental requirements.

[0087] 3. Animal experiment grouping (n=10 / group) Normal control group: no colitis induced, PBS DSS model group: 3% DSS + PBS Wild-type vesicle group (EVs-WT): 3% DSS + wild-type vesicles (0.5 g crude drug) Vesicle + NC inhibitor group (EVs+NC): 3% DSS + vesicles transfected with NC inhibitor (0.5 g crude drug). Vesicle + miR-6300 inhibitor group (EVs + anti-miR): 3% DSS + vesicles transfected with miR-6300 inhibitor (0.5 g crude drug). Crude extract control group (Extract): 3% DSS + crude extract (0.5 g crude drug amount) 4. Experimental Results

[0088] Note: ** indicates P<0.01 compared to the EVs-WT group (One-way ANOVA + Tukey post-hoc test) 5. Conclusion After knocking down miR-6300 (EVs+anti-miR group), the Treg / Th17 regulatory effect of vesicles dropped sharply from 3.2 to 1.3 (P<0.01), close to the level of the crude extract group (1.2). There was no significant difference between the NC inhibitor control group (EVs+NC) and the wild-type vesicle group (EVs-WT), demonstrating that the inhibitor itself has no off-target effect. After knocking down miR-6300, SESN3 and p-mTOR expression rebounded, and IL-17A mRNA was upregulated, validating the miR-6300→SESN3→mTOR→Treg / Th17 regulatory axis at the molecular level.

[0089] The above results indicate that miR-6300 is an essential functional molecule for the disease-modifying effects of the vesicles in this invention, while the crude extraction control group could not enrich miR-6300 and its Treg / Th17 regulatory effect was poor.

[0090] Example 5: miR-6300 mimic cell experiment (gain-of-function verification) To verify that miR-6300 alone can exert sufficient therapeutic effects, miR-6300mimic was overexpressed in CD4+ T cells to observe whether it could independently reproduce the immunomodulatory effects of vesicles.

[0091] 1. Experimental materials miR-6300 mimic: Chemically synthesized double-stranded RNA, sense strand: 5'-UGGAGAGAGAGAGAGAGAGA-3', antisense strand: 5'-UCUCUCUCUCUCUCUCUCCA-3', 2'-O-methylation modified, HPLC purity ≥95%; NC mimic: negative control mimic, with scrambled sequence and identical chemical modifications; Main reagents: CD4+ T cell magnetic bead sorting kit (Miltenyi Biotec), Lipofectamine 3000 (Invitrogen), anti-CD3 (2 µg / mL), anti-CD28 (2 µg / mL), TGF-β3 (1 ng / mL), IL-6 (20 ng / mL), IL-23 (20 ng / mL).

[0092] 2. Experimental Grouping Blank control group - CD4+ T cells cultured in normal culture medium, without Th17 induction. Th17 induction group - anti-CD3 + anti-CD28 + TGF-β3 + IL-6 + IL-23 induce Th17 differentiation Th17 + NC mimic group - Th17 induction + transfection with NC mimic (50 nM) Th17 + miR-6300 mimic group - Th17 induction + transfection with miR-6300 mimic (50 nM) Th17+ Vesicle Group - Th17 Induction + Vesicle Treatment (10 µg / mL Protein) Th17 + Crude Extract Group - Th17 Induction + Crude Extract Treatment (Equal Amount of Raw Drug) 3. Cell Experiment Procedure (1) CD4+ T cells were isolated from the spleen of C57BL / 6 mice (sorted with magnetic beads, purity ≥95%) and their density was adjusted to... ; (2) Inoculated into 24-well plates ( (holes), with 3 duplicate holes in each group; (3) The Th17 induction group was treated with anti-CD3 (2 µg / mL, pre-coated) + anti-CD28 (2 µg / mL) + TGF-β3 (1 ng / mL) + IL-6 (20 ng / mL) + IL-23 (20 ng / mL). (4) The blank control group was supplemented with only anti-CD3 + anti-CD28, without cytokines; (5) The transfection group was transfected with Lipofectamine 3000 to miR-6300 mimic or NC mimic (50 nM), and the medium was changed 6 h after transfection; (6) After 48 h of treatment, cells were collected and used for flow cytometry, qRT-PCR and Western blot detection, respectively.

[0093] 4. Experimental Results

[0094] Note: ** indicates P < 0.01 compared to the Th17-induced group. 5. Conclusion miR-6300 mimic alone significantly inhibited Th17 differentiation (CD4+IL-17A+ ratio decreased from 35% to 12%, P<0.01), replicating the effect of the vesicle treatment group (10%). SESN3 protein expression was significantly downregulated in the miR-6300 mimic group (from 2.8 to 0.9, P<0.01), while Foxp3 mRNA was significantly upregulated (from 0.8 to 2.5, P<0.01), consistent with the trend in the vesicle treatment group. The crude extract group had no significant inhibitory effect on Th17 differentiation (CD4+IL-17A+ ratio 34±3%, no difference from the Th17-induced group), demonstrating that free flavonoids cannot regulate T cell fate through miRNA mechanisms.

[0095] The above results indicate that miR-6300 can exert a sufficient therapeutic effect on its own, without relying on other components in the vesicles. This further proves that miR-6300 is not a passively carried byproduct in the vesicles, but rather a functional core that actively participates in the regulation of the host immune system.

[0096] Combined conclusions of Examples 4 and 5 Through bidirectional verification in Example 4 (loss-of-function) and Example 5 (gain-of-function), a complete chain of evidence was constructed to prove that "miR-6300 is the core of vesicle function": Knockdown of miR-6300 → loss of therapeutic effect (Treg / Th17 ratio dropped from 3.2 to 1.3), proving that miR-6300 is an essential functional molecule; Overexpression of miR-6300 → Reproduced therapeutic effect (Th17 decreased from 35% to 12%), proving that miR-6300 is a fully functional molecule; The crude extract did not contain enriched miR-6300 → had no immune reprogramming effect, proving that the dosage form determines the mechanism; Patent 2's cosmetic-grade method destroys the vesicle membrane, making it impossible to enrich miR-6300, proving that the preparation process determines the fate of the ingredient.

[0097] The precise correspondence between the aforementioned "component-structure-mechanism-effect" requires extensive creative work by the inventors of this application (including optimization of preparation process, small RNA sequencing, target gene prediction, inhibitor / mimic synthesis, animal model verification, cell experiment verification, and parallel comparison with various existing technical methods, etc.) to reveal, and cannot be obtained by simply piecing together existing technologies.

[0098] In summary, this invention utilizes a specific preparation process (pectinase dissociation + differential → ultracentrifugation → three-layer gradient tandem centrifugation) to specifically enrich miR-6300 (≥5-fold); by encapsulating miR-6300 and active ingredients in a vesicle lipid bilayer, it addresses the deficiency of low oral bioavailability (<5%) of flavonoid components; and by targeting SESN3 with miR-6300 to regulate the mTOR / oxidative stress pathway, it achieves precise reconstruction of the Treg / Th17 balance (the Treg / Th17 ratio recovered from 0.65 in the model group to 3.0+), fundamentally correcting immune imbalance.

[0099] Compared with the comparative example, the present invention has the following advantages: 1. Specific enrichment and functional discovery of miR-6300: Through the preparation process of this invention, the relative abundance of miR-6300 in the obtained vesicles is more than 5 times that of the source plant leaves, indicating that the preparation process can selectively enrich specific miRNAs. Reference 1 only involves flavonoid small molecules and does not disclose, nor could it be expected, that there is a specific enrichment of miR-6300 in Ampelopsis thaliana; Patent 1 only discloses the preparation of tea berry exosomes without any analysis of the miRNA composition therein; Patent 2 discloses a cosmetic-grade vesicle preparation method, which not only fails to detect miRNAs, but its microfluidic high-pressure homogenization process also damages the vesicle membrane structure, leading to miRNA degradation.

[0100] 2. Precise Reconstruction of Treg / Th17 Balance: The vesicle treatment group of this invention was able to restore the Treg / Th17 ratio in DSS model mice from 0.65 in the model group to 3.0+ (P<0.01). This miRNA-mediated reconstruction of immune homeostasis is something that the crude extract in Reference 1 cannot achieve. The mechanism of action of the crude AMP extract in Reference 1 focuses on the inhibition of conventional inflammatory pathways such as TNF-α and MAPK / NF-κB, which belongs to symptom-relieving anti-inflammatory; while the vesicles of this application target SESN3 through miR-6300 to regulate the mTOR / oxidative stress pathway, fundamentally correcting the immune imbalance, which belongs to disease-modifying therapy.

[0101] 3. Dosage-Form Effects Irreproducible by Crude Extract: Head-to-head comparative experiments demonstrated that, under the same crude drug dosage (0.5 g fresh leaves / animal / day), the Treg / Th17 ratio of the crude extract of *Ampelopsis grossedentata* only recovered to 1.3±0.2, while that of the vesicle formulation recovered to 3.2±0.4 (P<0.01). Even when the crude extract dosage was doubled to 1.0 g crude drug dosage / animal / day, the Treg / Th17 ratio remained only 1.5±0.3, significantly lower than that of the vesicle group (P<0.01). This proves that the disease-modifying effect of the vesicle formulation of this invention is not a simple dose-response effect, but rather stems from the unique miRNA cargo delivery system and immune reprogramming mechanism of vesicles.

[0102] 4. Synergistic effect of preparation methods: The *Ampelopsis grossedentata* vesicles prepared by the mechanical pulverization + PEG precipitation + five-layer gradient method disclosed in Patent 1 (Method-B) showed a miR-6300 enrichment only 2.1 ± 0.4 times that of the source plant, and the particle size distribution was uneven (PDI > 0.3), with significantly weaker biological activity than the method of this application (Method-A) (P < 0.01). The microfluidic high-pressure homogenization method disclosed in Patent 2 (Method-D) did not even detect miR-6300, and its vesicle membrane structure was severely damaged, making it unsuitable for miRNA delivery. The preparation method of this invention achieves more than 8 times enrichment of miR-6300 and precise control of particle size (100-200 nm, PDI < 0.2) by using pectinase to dissociate and specifically release membrane-bound vesicles, combined with a differential → ultra-high-speed → three-layer gradient tandem purification strategy.

[0103] 5. Two-way verification of miR-6300's core function: The miR-6300 inhibitor knockdown experiment demonstrated that after introducing miR-6300 inhibitor into vesicles, the Treg / Th17 regulation effect of vesicles was significantly weakened (from 3.2 to 1.3, P<0.01), approaching the level of the crude extract (1.2), proving that miR-6300 is an essential functional molecule. The miR-6300 mimic cell experiment demonstrated that overexpression of miR-6300 mimic in CD4+ T cells significantly inhibited Th17 differentiation (CD4+IL-17A+ ratio decreased from 35% to 12%, P<0.01) and promoted Foxp3 expression, replicating the effect of the vesicle treatment group (10%), proving that miR-6300 alone can exert a sufficient therapeutic effect.

[0104] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An extracellular vesicle derived from *Ampelopsis grossedentata*, characterized in that: The extracellular vesicles were obtained from the stems and leaves of *Vitis pachycephala* through homogenization, pectinase dissociation, differential centrifugation, ultracentrifugation, and density gradient centrifugation. Under transmission electron microscopy, they exhibited a double-membrane vesicle morphology. The extracellular vesicles were specifically enriched with miR-6300, and the relative abundance of miR-6300 was more than 5 times that in the leaves of the source plant.

2. The extracellular vesicle according to claim 1, characterized in that: The extracellular vesicles are also enriched with the miR-159 family and / or the miR-166 family.

3. The extracellular vesicle according to claim 1, characterized in that: The extracellular vesicles also include natural small molecule compounds, which at least include baicalein, kaempferol, and caffeic acid.

4. The extracellular vesicle according to claim 1, characterized in that: The extracellular vesicles are mainly distributed in the range of 100–200 nm in size.

5. The method for preparing extracellular vesicles according to any one of claims 1-4, characterized in that: Includes the following steps: Step (1): Take the stems and leaves of Viburnum pachycephalum, wash them, cut or grind them, and homogenize them in a low-temperature buffer solution to obtain a homogenate. Step (2): After dissociating the homogenate obtained in step (1) with pectinase buffer, perform differential centrifugation to remove tissue fragments and large particulate impurities, and collect the supernatant. Step (3): The supernatant obtained in step (2) is subjected to ultracentrifugation, the precipitate is collected and resuspended to obtain crude vesicle extract; Step (4): The crude vesicle extract obtained in step (3) is subjected to density gradient centrifugation to collect the vesicle layer, and the extracellular vesicles are obtained after washing.

6. The preparation method according to claim 5, characterized in that: In step (1), the homogenization is carried out under ice bath conditions.

7. The preparation method according to claim 5, characterized in that: In step (2), the dissociation treatment of the pectinase buffer is to treat with 0.5% to 2% (w / v) pectinase buffer at 4°C for 1 to 3 hours.

8. The preparation method according to claim 5, characterized in that: The differential centrifugation at 4°C includes centrifugation at 5000 g for 10 min and centrifugation at 12000 g for 20 min; the ultracentrifugation is centrifugation at 100000 g for 70 min; the density gradient centrifugation uses a 30% / 45% / 60% sucrose density gradient medium and is centrifuged at 120000 g for 2 h. After centrifugation, the vesicle layer at the interface is aspirated and washed with PBS to remove sucrose.

9. The use of the extracellular vesicles according to any one of claims 1-4 or the extracellular vesicles prepared by the preparation method according to any one of claims 5-8 in the preparation of a medicament for the prevention or treatment of ulcerative colitis.

10. The application according to claim 9, characterized in that: The extracellular vesicles exert their therapeutic effects by regulating Treg / Th17 cell balance and / or inhibiting the Sestrin 3 signaling pathway.

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