A inulin gel carrying a naked flower of source of extracellular vesicle of purple olive and a preparation method and application thereof

Abstract

The application discloses inulin gel loaded with extracellular vesicles derived from S. nudiflora, a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The gel comprises extracellular vesicles derived from S. nudiflora (EVLP), an inulin gel matrix and zinc ions. The inulin gel is prepared by a heating-cooling method, and the EVLP is encapsulated and the zinc ions are loaded. Through oral administration, the inulin gel is used to precisely release the drug at an inflammation site, and the multiple effects of the EVLP, the inulin and the zinc ions are synergistically exerted, that is, the anti-inflammatory and antioxidant effects of the EVLP, the regulation of intestinal flora by the inulin and the enhancement of anti-inflammatory and barrier repair by the zinc ions. The gel improves the drug targeting, prolongs the local residence time, and can more efficiently and with less side effects treat ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to an inulin gel loaded with extracellular vesicles derived from *Callicarpa nudiflora*, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC), a chronic idiopathic inflammatory bowel disease, is one of the main forms of inflammatory bowel disease (IBD). Its pathogenesis is closely related to gut microbiota dysbiosis, genetic susceptibility, epithelial barrier defects, immune dysregulation, and environmental factors. Patients present with symptoms such as bloody diarrhea, abdominal pain, and urgency. Mucosal inflammation begins in the rectum and can spread to the proximal colon. Current clinical treatment is primarily drug-based, with 5-aminosalicylic acid used for mild to moderate cases and biologics or small molecule drugs for moderate to severe cases. However, this approach has significant limitations: non-targeted administration leads to hepatotoxicity and systemic immunosuppression; the mechanisms of action are singular, making it difficult to address complex pathological processes, resulting in a high relapse rate; the problem of gut microbiota imbalance is not addressed, and the deficiency of short-chain fatty acids, a metabolic product of gut microbiota, is associated with persistent inflammation; oral medications are affected by the gastrointestinal environment, resulting in low bioavailability, and injectable medications have poor adherence. Therefore, developing targeted and multi-pathway strategies to inhibit disease progression is crucial.

[0003] Plant-derived extracellular vesicle-like particles (PEVLPs) have shown great potential in the treatment of ulcerative colitis (UC) due to their biocompatibility, low immunogenicity, and anti-inflammatory and antioxidant activities. While extracts from the traditional medicinal plant *Callicarpa nudiflora* Hook. & Arn. have been shown to possess anti-inflammatory and antioxidant activities, the application of its extracellular vesicles (EVLPs) in UC treatment has not yet been reported. Furthermore, oral medications face challenges related to gastrointestinal disruption and intestinal motility, necessitating the development of colon-targeted delivery systems.

[0004] Inulin is a natural fructan. Because its β-configuration is hydrolyzed only by inulinase secreted by colonic flora, it can specifically release loaded drugs in the colon, making it an ideal colon-targeting carrier. Simultaneously, inulin can be fermented by bacteria to produce short-chain fatty acids, regulating intestinal flora balance and promoting intestinal epithelial repair. Zinc ions, as an essential trace element for the human body, can alleviate UC inflammation through pathways such as inhibiting nuclear factor κB (NF-κB) activation, scavenging reactive oxygen species (ROS), and enhancing tight junction protein expression, and can also improve the stability of the gel matrix. Summary of the Invention

[0005] This invention aims to provide an inulin gel loaded with extracellular vesicles derived from *Callicarpa nudiflora*, its preparation method, and its application. The gel is delivered to the colon via oral administration, and through the synergistic effects of EVLP, inulin gel, and zinc ions, it enhances the therapeutic efficacy of UC.

[0006] Firstly, inulin gels carrying extracellular vesicles derived from *Callicarpa nudiflora*.

[0007] The gel provided by this invention comprises extracellular vesicles derived from *Callicarpa nudiflora* (EVLP), an inulin gel matrix, and zinc ions. The mass ratio of EVLP to the inulin gel matrix is ​​1:(160-240), and the zinc ion concentration is 36.7-220 mM.

[0008] Preferably, the mass ratio of EVLP to inulin gel matrix is ​​1:220, and the zinc ion concentration is 73.4 mM.

[0009] The EVLPs were extracted using differential centrifugation and sucrose gradient centrifugation, with an average particle size of 50-150 nm, a Zeta potential of -1 to -30 mV, and exhibited regular spherical shape and uniform dispersion. They exert anti-inflammatory and antioxidant effects by inhibiting the release of pro-inflammatory factors (TNF-α, IL-6) and scavenging ROS. The inulin gel matrix is ​​prepared from 40-60% (w / v) inulin via a heating-cooling method. It possesses a loose, porous structure that enhances drug encapsulation and colonic retention. Because it is hydrolyzed only by colonic flora, it exhibits specific colonic targeting, while the fermentation produces short-chain fatty acids that regulate the intestinal flora. Zinc ions: Concentration 36.7-220 mM, added in the form of zinc chloride, can enhance gel stability and exert anti-inflammatory and barrier repair effects by promoting the expression of tight junction proteins (ZO-1, occludin).

[0010] Secondly, the preparation method of inulin gel loaded with extracellular vesicles derived from *Callicarpa nudiflora*. The preparation method includes three steps: EVLP extraction, inulin gel preparation, and composite gel formation. First, the leaves of *Callicarpa nudiflora* were homogenized, filtered through an 80-mesh filter, and then centrifuged at different speeds (1000×g for 15 min, 5000×g for 30 min, 10000×g for 1 h, and 150,000×g for 2 h). The precipitate was then subjected to 8-60% sucrose gradient centrifugation for 2 h, and the EVLP was collected and characterized.

[0011] Next, prepare an inulin solution of 40-60% (w / v), heat it at 60-80℃ for 10-30 minutes, cool it to 30-50℃, add zinc chloride and stir well, then add EVLP and let it solidify at room temperature to form a gel.

[0012] Preferably, the inulin concentration is 55% (w / v), the heating temperature is 80°C, the heating time is 20 min, the temperature is cooled to 40°C, the zinc ion concentration is 73.4 mM, the stirring speed is 300 rpm, and the stirring time is 15 min.

[0013] The preparation method provided by this invention utilizes a heating-cooling method to prepare inulin gel. Zinc ions are added during the cooling process to improve the stability of the gel matrix and load EVLP to enhance the targeting of the formulation. Furthermore, the preparation method provided by this invention is simple, convenient, and controllable.

[0014] Thirdly, the application of inulin gels carrying extracellular vesicles derived from *Callicarpa nudiflora*. Compared with the prior art, the present invention has the following beneficial effects: The gel provided by this invention can be used to prepare drugs for treating ulcerative colitis. Through oral administration, the colon-targeting properties of the inulin gel allow for precise drug release at the site of inflammation. EVLP exerts anti-inflammatory and antioxidant effects, inulin regulates the intestinal flora and promotes epithelial repair, and zinc ions enhance the expression of tight junction proteins. These three components work together to alleviate UC symptoms. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments are briefly introduced below.

[0016] Figure 1 A photograph of the inulin gel prepared in Example 1; Figure 2 This is a scanning electron microscope image of the gel used in performance testing. Figure 3 EV / Zn in performance testing 2+ Results of storage stability measurements for @INU; Figure 4 The results represent the steady-state rheological properties measured during performance testing. Figure 5 The results represent the dynamic rheological properties measured during performance testing. Figure 6 The results of in vitro release of the formulation in performance testing are shown in Figure a, where a is EV@INU and b is EV / Zn. 2+ @INU; Figure 7 In in vitro antioxidant experiments for performance testing, ZnCl2, EVLP, and Zn were used at concentrations of 30 μg / mL, 60 μg / mL, 120 μg / mL, 240 μg / mL, 300 μg / mL, and 480 μg / mL. 2+ @INU, EV@INU and EV / Zn 2+@INU's ability to clear DPPH; Figure 8 EVLP and Zn were used for performance evaluation at concentrations of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL. 2+ and EV / Zn 2+ @INU effect on RAW 264.7 cell viability, n=6; Figure 9 EVLP and Zn were used for performance evaluation at concentrations of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL. 2+ and EV / Zn 2+ The effect of INU on the survival rate of Caco-2 cells, n=6; Figure 10 EVLP and Zn were evaluated for performance testing at concentrations of 5 μg / mL, 15 μg / mL, and 25 μg / mL. 2+ Zn 2+ @INU, EV@INU and EV / Zn 2+ Effect of INU on NO concentration in RAW 264.7 cells (n=3; " "P<0.05; " "P<0.01; " "P<0.001; significance compared with the LPS group" Figure 11 15 μg / mL EVLP and Zn were used for cytological evaluation in performance testing. 2+ Zn 2+ @INU, EV@INU and EV / Zn 2+ Effect of INU on IL-6 concentration in RAW264.7 cells (n=3; " "P<0.05; " "P<0.01; " "P<0.001; significance compared with the LPS group" Figure 12 For performance testing, 5 μg / mL EVLP and Zn were used in cytological evaluation. 2+ Zn 2+ @INU, EV@INU and EV / Zn 2 +Effect of INU on TNF-α concentration in RAW264.7 cells (n=3; " "P<0.05; " "P<0.01; " "P<0.001; significance compared with the LPS group" Figure 13 15 μg / mL EVLP and Zn were used for cytological evaluation in performance testing. 2+ EV / Zn 2+ The effect of @IN on tight junction proteins (Occludin, ZO-1) in Caco-2 cells. a is the Western blot result, b is the quantitative statistical graph (n=3, " "P<0.05; " "P<0.01; " "P<0.001; significance compared with the LPS group" Figure 14 For each group in the pharmacodynamic study (Control group, DSS group, 5-ASA, Zn) 2+ EVLP group, Zn 2+ @INU Group, EV@INU Group and EV / Zn 2+ @INU group) Average body weight change trend of mice over 11 days (n=5; P < 0.5 P < 0.01, P < 0.001; significance compared with the model DSS group). Figure 15 This is a trend graph showing the change of DAI over time in each group of mice over 11 days in a pharmacodynamic study (n=5). P < 0.5 P < 0.01, P < 0.001; significance compared with the model DSS group). Figure 16 A quantitative statistical graph of colon length in mice for pharmacodynamic studies; Figure 17 This is a pathological section of mouse colon tissue from a pharmacodynamic study (scale bar = 50 μm). Figure 18This is a graph showing the results of pharmacodynamic studies on inflammatory factors in the mouse colon. a represents the expression level of TNF-α, b represents the expression level of IL-6, and c represents the expression level of IL-10 (n=5). P < 0.5 P < 0.01, P < 0.001; significance compared with the model DSS group). Figure 19 It is the mouse organ index (n=5) in pharmacodynamic studies. Figure 20 Figure showing the results of the α-diversity index of the mouse gut microbiota in a microbiome study (Note: A is the 5-ASA group; C is the Control group; D is the DSS group; EV is the EV group; EG is the EV / Zn ratio). 2+ @INU Group; EG- is EV@INU Group). Figure 21 This is a bar chart showing the abundance of gut microbiota in mice during a microbiome study (Note: A is the 5-ASA group; C is the Control group; D is the DSS group; EV is the EV group; EG is the EV / Zn ratio). 2+ @INU Group; EG- is EV@INU Group). Figure 22 This is a graph showing the results of Beta diversity analysis of the mouse gut microbiota in a microbiome study. Figure a is the non-metric multidimensional mapping analysis of the mouse gut microbiota; figure b is the principal component analysis of the mouse gut microbiota; and figure c is the principal coordinate analysis of the mouse gut microbiota. (Note: A is the 5-ASA group; C is the Control group; D is the DSS group; EV is the EV group; EG is the EV / Zn ratio.) 2+ @INU Group; EG- is EV@INU Group). Detailed Implementation

[0017] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention fall within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0018] The present invention provides an inulin gel carrying extracellular vesicles derived from *Callicarpa nudiflora*, the gel comprising extracellular vesicles derived from *Callicarpa nudiflora* (EVLP), an inulin gel matrix, and zinc ions.

[0019] In some embodiments, the mass ratio of the EVLP to the inulin gel matrix is ​​1:(160-240). In a preferred embodiment (such as Example 1), the mass ratio is 1:220.

[0020] In some embodiments, the concentration of zinc ions in the gel is 36.7-220 mM. In a preferred embodiment (such as Example 1), the concentration is 73.4 mM.

[0021] This invention also provides a method for preparing inulin gel loaded with extracellular vesicles derived from *Callicarpa nudiflora*, comprising the following steps: S1. Juice of Callicarpa nudiflora was extracted and filtered to obtain filtrate. The filtrate was then subjected to differential centrifugation and sucrose density gradient centrifugation to obtain extracellular vesicles derived from Callicarpa nudiflora. S2. Dissolve inulin in deionized water by heating, and then add zinc chloride and stir until completely dissolved; S3. After cooling, add extracellular vesicles from *Callicarpa nudiflora* to the above solution and let stand at room temperature.

[0022] In some embodiments, when performing step S1, differential centrifugation is performed through the following steps: 1000×g for 15 min, 5000×g for 30 min, 10000×g for 1 h, and 150,000×g for 2 h. The precipitate is then subjected to 8-60% sucrose gradient centrifugation for 2 h. 30-45% of the bands are taken, mixed with PBS solution, and centrifuged at 150,000×g for 1 h at 4°C.

[0023] In some embodiments, when performing step S2, heating and dissolving includes: adding a stir bar and heating and stirring at 60-80°C for 10-30 minutes, preferably heating and stirring at 80°C for 20 minutes.

[0024] The present invention also provides the application of the above-mentioned inulin gel containing extracellular vesicles derived from Callicarpa nudiflora in the treatment of ulcerative colitis.

[0025] Unless otherwise specified, all reagents and raw materials used in the embodiments of the present invention are commercially available products or products that can be prepared by known methods.

[0026] Example 1: Example 1 of this invention provides a method for preparing inulin gel loaded with EVLP, comprising the following steps: S1: Take 500 g of Callicarpa nudiflora leaves, cut them into small pieces, add 500 mL of PBS to homogenize, filter through an 80-mesh filter to obtain the filtrate, centrifuge the filtrate at 1000×g for 15 min at 4℃, take the supernatant and centrifuge at 5000×g for 30 min, then take the supernatant and centrifuge at 10000×g for 1 h, finally take the supernatant and centrifuge at 150,000×g for 2 h to obtain the crude extract of extracellular vesicles from Callicarpa nudiflora. Take the crude extract and resuspend it in 2-5 mL of PBS solution, centrifuge with an 8-60% sucrose gradient for 2 h, collect 30-45% of the bands, mix with PBS solution, centrifuge at 150,000×g for 1 h at 4℃, take 3 mL of PBS solution, blow off the precipitate and filter through a 450 nm filter membrane to obtain EVLP solution. The particle size was measured to be 126.72±1.65 nm and the Zeta potential was -1.98±0.49 by dynamic light scattering.

[0027] S2: Weigh 55 g of inulin and dissolve it in 45 mL of deionized water. Heat at 80°C for 20 min (stirring at 300 rpm) until completely dissolved. Cool to 40°C (cooling rate 5°C / min) to obtain a 55% (w / v) inulin solution. Add 1 g of zinc chloride (zinc ion concentration 73.4 mM) and stir at 300 rpm for 15 min.

[0028] S3: Add 0.25 g of EVLP suspension to the above inulin-zinc solution, continue stirring for 10 min, and let stand at room temperature for 2 h to form a pale yellow semi-fluid gel (e.g. Figure 1 (As shown).

[0029] Example 2 Example 2 of this invention provides a method for preparing an inulin gel loaded with EVLP. The difference from Example 1 is that in step S2, 40 g of inulin is weighed and dissolved in 60 mL of deionized water. The subsequent steps are the same as in Example 1, resulting in a 40% (w / v) pale yellow free-flowing inulin solution.

[0030] Example 3 Example 3 of the present invention provides a method for preparing an inulin gel loaded with EVLP. The difference from Example 1 is that in step S2, 50 g of inulin is weighed and dissolved in 50 mL of deionized water. The subsequent steps are the same as in Example 1, to obtain a 50% (w / v) pale yellow free-flowing inulin solution.

[0031] Example 4 Example 4 of this invention provides a method for preparing inulin gel loaded with EVLP. The difference from Example 1 is that in step S2, 60 g of inulin is weighed and dissolved in 40 mL of deionized water. The subsequent steps are the same as in Example 1, resulting in a 60% (w / v) pale yellow non-flowing inulin gel.

[0032] Example 5 Example 5 of the present invention provides a method for preparing an inulin gel loaded with EVLP. The difference from Example 1 is that in step S2, 0.5g of zinc chloride (zinc ion concentration 36.7mM) is added, and the subsequent steps are the same as in Example 1, to obtain a 55% (w / v) pale yellow flowable inulin gel.

[0033] Example 6 Example 6 of the present invention provides a method for preparing an inulin gel loaded with EVLP. The difference from Example 1 is that in step S2, 2g of zinc chloride (zinc ion concentration 146.8mM) is added, and the subsequent steps are the same as in Example 1, to obtain a 55% (w / v) pale yellow non-flowing inulin gel.

[0034] Example 7 Example 7 of the invention provides a method for preparing an inulin gel loaded with EVLP. The difference from Example 1 is that in step S2, 3g of zinc chloride (zinc ion concentration 220mM) is added, and the subsequent steps are the same as in Example 1, to obtain a 55% (w / v) pale yellow non-flowing inulin gel.

[0035] Performance testing EVLP and EV / Zn prepared in Example 1 2+ @INU performs performance testing, including: (1) The average particle size, polydispersity index and zeta potential of EVLP were determined by dynamic laser scattering particle size analyzer. The measurements were performed in parallel three times and the results are shown in Table 1.

[0036] The results showed that the polydispersity index of EVLP was less than 0.3, indicating that the particle size was uniform and the particles were evenly dispersed.

[0037] Table 1. Average particle size, polydispersity index, and potential value of EVLP

[0038] (2) Characterization analysis: INU, EV@INU, Zn 2+ @INU and EV / Zn 2+ @INU performed scanning electron microscopy observations. Add 0.1g of INU, EV@INU, and Zn 2+ @INU and EV / Zn 2+@INU freeze-dried powder was spread evenly on an aluminum tip coated with double-layer conductive tape, and then sputtered with gold using an ion sputtering system. The coated sample was then randomly imaged using scanning electron microscopy to observe its morphological characteristics and take photographs. The results are as follows: Figure 2 As shown, scanning electron microscopy reveals that the gel has a loose, porous internal structure. (3) Stability study The gel was stored at 4°C for 7 days, and its appearance, state, and texture were observed. Figure 3 For EV / Zn 2+ The storage stability results from @INU showed that after 7 days, the gel color was uniform and pale yellow; during tilting and rotating of the sample vial, the gel remained in a uniform semi-solid state, without layering, precipitation, or phase separation, and no liquid precipitation occurred; the gel felt smooth, had a uniform texture, no clumping, and its flowability was consistent with that before storage, indicating that the EV / Zn ratio was normal. 2+ @INU has good storage stability.

[0039] (4) Rheological properties The inulin blank gel (INU), inulin gel loaded with EVLP (EV@INU), and inulin gel loaded with ZnCl2 (ZnCl2) were investigated using a DHR-2 rheometer. 2+ @INU) and inulin gel loaded with EVLP and ZnCl2 (EV / Zn 2+ Steady-state and dynamic rheological properties of @INU.

[0040] Steady-state rheology: Continuous shear rate scans (shear rate γ from 0.1 s⁻¹) were performed using a rotational rheometer (cone-plate system, 40 mm diameter, 1° cone angle, 50 μm gap) at 25 °C. －1 ~100 －1 Record the shear stress-shear rate curve and the change law of apparent viscosity to analyze the non-Newtonian fluid properties of the gel.

[0041] Dynamic rheology: Under the same temperature conditions, the linear viscoelastic region (strain 0.1%) was determined by amplitude scanning, followed by frequency scanning (angular frequency ω from 0.1 rad / s to 100 rad / s, logarithmic scanning, flat plate system, gap 2 mm) to determine the frequency dependence of storage modulus (G') and loss modulus (G'').

[0042] Figure 4 and Figure 5 The results are from gel rheological tests.

[0043] The phenomenon that the viscosity of a gel decreases with increasing shear stress is called shear thinning, a typical non-Newtonian fluid behavior. At rest, the polymer chains in a gel form a three-dimensional network structure through physical or chemical cross-linking, encapsulating a large amount of water, thus resulting in high viscosity. When subjected to shear stress, the external force disrupts the weak interactions between the polymer chains (such as physical cross-linking points), causing partial dissociation of the network structure. Simultaneously, the polymer chains or particles orient themselves along the shear direction, thereby reducing internal resistance and leading to a decrease in viscosity. Figure 4 As shown, the gel with low cross-linking density exhibits significant shear thinning. Because weak interactions are easily destroyed by shear, the INU group shows a more obvious shear thinning phenomenon compared to other groups.

[0044] Figure 5 The results show that the storage modulus of INU and EV@INU is lower than the loss modulus at angular frequencies of 0.1–1 rad / s, possibly because the molecular motion adjustment time is sufficient at low frequencies, resulting in a slower network structure response and more pronounced viscous properties. At angular frequencies of 1–100 rad / s, the storage modulus is greater than the loss modulus, possibly because the crosslinking points and molecular chain interactions can recover rapidly at high frequencies, resulting in stronger elasticity. (Zn) 2+ @INU and EV / Zn 2+ @INU exhibits a storage modulus greater than a loss modulus at angular frequencies ranging from 0.1 to 100 rad / s. This is because zinc ions increase the crosslinking density, resulting in a tighter network structure, restricted molecular chain movement, and weaker viscous behavior.

[0045] (5) In vitro release experiment Incubate Dil and EVLP together in the dark, remove unbound dye by centrifugation (12000g, 10min), and resuspend after washing with PBS. Take equal amounts of labeled EVLP, as well as the prepared EV@INU and EV / Zn. 2+ Add 100 μL of INU to each well of a 96-well plate containing 100 μL of LGF, SIF, SCF, and SCFm (SCF containing inulinase). Set up three replicates for each group. Add an equal volume of EVLP (EVLP) to the same environment as the experimental group and measure the fluorescence intensity as the total fluorescence intensity. Place the 96-well plate in a temperature-controlled shaker to simulate physiological temperature. Take 70 μL samples at predetermined time points of 1, 2, 4, 6, 8, 12, 24, and 48 hours (avoiding gel aspiration) and measure the fluorescence intensity using a multi-mode microplate reader under light-protected conditions. After measurement, aspirate the samples back into the original 96-well plate. Figure 6 This is a graph showing the in vitro release results. The results indicate that EV@INU and EV / Zn... 2+@INU showed high release rates in SCFm, reaching 91.77% and 71.06% respectively at 48h, while the release rates in SIF were the lowest, reaching 52.29% and 37.47% respectively at 48h. Furthermore, the release was rapid in the first 12h, then slowed down. This indicates that inulinase can decompose inulin gel, promoting the release of EVLP from it. Overall, the EV / Zn ratio... 2 + The release rate of @INU was lower than that of EV@INU, indicating that the addition of ZnCl2 can improve the stability of inulin gel, make its three-dimensional structure more compact, and slow down the release of EVLP.

[0046] (6) In vitro antioxidant experiment To evaluate ZnCl2, EVLP, and Zn 2+ @INU, EV@INU and EV / Zn 2+ The antioxidant capacity of @INU was assessed by scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. ZnCl2, EVLP, and Zn were measured at concentrations of 30 μg / mL, 60 μg / mL, 120 μg / mL, 240 μg / mL, 300 μg / mL, and 480 μg / mL under simulated physiological free radical conditions. 2+ @INU, EV@INU and EV / Zn 2+ Antioxidant activity of @INU (calculated based on EVLP concentration).

[0047] DPPH exists stably in organic solvents as a nitrogen-containing free radical at room temperature. It turns purple in ethanol and is reduced to a colorless state in the presence of antioxidant molecules. Different concentrations of EVLP, EV@INU, and EV / Zn were compared. 2+ @INU was mixed with an equal volume of 0.05 mg / mL DPPH ethanol solution and incubated in the dark for 30 min. The absorbance of the sample was measured at 517 nm, and the DPPH scavenging rate was calculated using the following formula: DPPH clearance rate (%) = [1 - (Asample - A blank) / A control] 100%, where A sample is the absorbance of the drug-treated group, A control is the absorbance of the control group, and A blank is the absorbance of the blank group.

[0048] DPPH removal rate Figure 7 As shown, the results indicated that the antioxidant capacity of all five groups in the DPPH experiment exhibited a concentration-dependent effect within the range of 30–480 μg / mL. The antioxidant capacity, from highest to lowest, was EV / Zn. 2+ @INU、EV@INU、Zn2+ @INU、EVLP、ZnCl 2。 EVLP and ZnCl2 each exhibit certain antioxidant capabilities, while EV / Zn 2+ The @INU group exhibited the strongest free radical scavenging ability, indicating that the three components can work synergistically to alleviate UC symptoms.

[0049] (7) Cytological evaluation ① CCK-8 method for screening EVLP, ZnCl2 and EV / Zn 2+ The safe concentration of @INU for Caco-2 and RAW264.7 cells, and the specific steps include: Caco-2 cells were loaded at 5 × 10 3 Cells per well, RAW264.7 at 2 × 10⁻⁶ 4 After seeding cells at a density of 1 cell / well in 96-well plates, the plates were incubated in a cell culture incubator. Once cells adhered, the old culture medium was removed, and EVLP solution, ZnCl2 solution, and EV / Zn were added at concentrations of 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL, respectively. 2+ @INU extract (based on ZnCl2 concentration for calculation), six replicates were set up for different concentrations of different samples. After incubation for 24 h, the supernatant was discarded, and the cells were washed twice with PBS. 100 μL CCK-8 medium solution (basic medium: CCK-8 = 9:1) was added to each well, and the cells were incubated in the dark for 4 h. After incubation in the dark, the 96-well plate was removed, and the absorbance at 450 nm was measured. Cell viability was calculated using the following formula: [(OD sample-OD blank) / (OD control-OD blank)] 100% Wherein, OD sample is the OD value of the drug-treated group, OD control is the OD value of the control group, and OD blank is the OD value of the blank group.

[0050] The cell viability of RAW264.7 and Caco-2 cells were as follows: Figure 8 and Figure 9 As shown, the results indicate that Zn 2+ At concentrations of 5–10 μg / mL, the survival rate of Raw264.7 cells was above 85%, but subsequently fell below 85%; the EVLP group and EV / Zn 2+ In the @INU group, cell viability was above 85% in the concentration range of 5-25 μg / mL, while Caco-2 cells also showed high cell viability within this concentration range.

[0051] ② Take RAW264.7 cells that are in good growth condition and in the logarithmic growth phase, and use 2×10⁻⁶ cells. 4 Cells were seeded at a density of 1 cell per well in 24-well plates. 0.5 mL of cell suspension was added to each well and the plates were incubated in a cell culture incubator. After the cells adhered, they were washed three times with PBS and 1 μg / mL of LPS was added to induce M1 phenotype macrophages. After the macrophages were polarized, the concentrations of NO, TNF-α and IL-6 were measured.

[0052] 1 μg / mL of LPS was reacted with different concentrations (5 μg / mL, 15 μg / mL and 25 μg / mL) of different formulations (Zn) 2+ EVLP group, Zn 2+ @INU, EV@INU group and EV / Zn 2+ After incubation for 24 hours (INU group), 50 μL of supernatant was taken and added to a 96-well plate along with serially diluted standard solutions. Griess Reagent I and II reagents were added and the absorbance of each group was measured at 540 nm. The corresponding NO content was calculated based on the standard curve.

[0053] 1 μg / mL LPS with 15 μg / mL Zn 2+ EVLP group, Zn 2+ @INU, EV@INU group and EV / Zn 2+ After incubation for 24 hours in the INU group, the samples were processed according to the steps of the ELISA kit, and the absorbance of each group was measured at 540 nm. The corresponding TNF-α and IL-6 contents were calculated based on the standard curve.

[0054] The results of NO, IL-6, and TNF-α levels are as follows: Figure 10 , Figure 11 and Figure 12 As shown, the concentrations of NO, TNF-α, and IL-6 in the cell supernatant of the LPS-stimulated group were significantly higher than those in the normal group, proving the successful establishment of the inflammation model. The results showed that all treatment groups significantly inhibited the secretion of NO, TNF-α, and IL-6 (EV / Zn). 2+ @INU showed the most significant inhibitory effect. Meanwhile, EVLP and Zn were also observed to have similar inhibitory effects. 2+ ,EV@INU,Zn 2+ @INU、EV / Zn 2+ @INU inhibits NO production in a concentration-dependent manner.

[0055] ③ Caco-2 cells were evenly seeded in six-well plates and divided into four groups: LPS group, EV group, and Zn group. 2+ Group, EV / Zn 2+@INU Group. After Caco2 cells adhered, each group was incubated with 1 μg / mL LPS and the corresponding drug (5 μg / mL) for 24 h. After lysis with RIPA lysis buffer, the protein concentration in the supernatant was measured using a BCA kit, and the concentrations were adjusted to be consistent across groups. Loading buffer diluted five times was added to the supernatant, and the mixture was boiled at 100°C for 5 min to denature the proteins. 10% and 7.5% gels were prepared, and 20 μg of protein was loaded into each well, along with 5 μL of marker. After electrophoresis at 150 V for 40 min, the cells were transferred to PVDF membranes. The transfer conditions for Occludin-1 were 400 mA for 0.5 h, and for ZO-1 were 400 mA for 2 h. After blocking with rapid blocking buffer for 15 min, the sample was washed three times with Tween-containing tris(hydroxymethyl)aminomethane buffer (TBS-T), followed by incubation overnight at 4°C with primary antibodies against Anti-Occludin, Anti-ZO-1, and β-Actin (all diluted 1:1000). The sample was then washed four times and incubated for 2 h at room temperature with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000). Finally, the sample was washed again and developed using enhanced chemiluminescence immunoassay.

[0056] Western blot analysis Figure 13 The results showed that, compared with the LPS group, all treatment groups upregulated the expression of Occludin and ZO-1. Although Zn 2+ The group showed a significant repair effect, but EV / Zn 2+ The protein expression levels in the INU group (full-component gel) were numerically superior to those in the Zn-only group. 2+ This indicates that loading zinc ions into the inulin gel system not only preserves the bioactivity of zinc ions, but may also achieve better barrier repair efficacy through the adhesion and sustained-release properties of the gel in the intestine, synergistically enhancing the anti-inflammatory microenvironment of vesicles.

[0057] (8) In vivo pharmacodynamic studies Animal model establishment: Balb / c mice were acclimatized for 7 days. A Balb / c mouse ulcerative colitis model was induced using sodium dextran sulfate (DSS), and mice were randomly divided into a blank control group, a model group, a 5-aminosalicylic acid (5-ASA) positive group, and a Zn group. 2+ EVLP group, Zn 2+ @INU, EV@INU group and EV / Zn 2+ @INU group, 5 animals per group.

[0058] Drug administration regimen: Except for the control group, all mice were given 3.5% DSS solution for free drinking for 7 days, with the solution changed every two days. The control group was given distilled water to establish the UC mouse model. Subsequently, starting on the fourth day, each group was treated orally for 7 days. The control and model groups were simultaneously administered distilled water by gavage, while the other groups were administered the corresponding drugs by gavage. Simultaneously, the daily disease activity index (DAI) was calculated by comprehensively assessing changes in body weight, fecal morphology, and rectal bleeding. The DAI is a clinical activity index that reflects the combined characteristics of weight loss, rectal bleeding, diarrhea, etc., and is used as an indicator to assess the treatment or severity of colitis. The specific criteria for DAI scoring are shown in Table 2.

[0059] Table 2 DAI Scoring Criteria

[0060] The changes in body weight of mice in each group during the experiment are as follows: Figure 14 As shown in the figure. The results showed that the body weight of the control group mice remained relatively stable, and the body weight on the tenth day was 105.61% ± 2.09% of the initial body weight. The other groups induced by DSS all showed a trend of weight loss on the fourth day. The DSS group had a body weight of only 74.66% ± 1.87% of the initial body weight on the tenth day, and showed typical symptoms of diarrhea and bloody stool, indicating that the UC model was successfully established.

[0061] All treatment groups showed an improving trend, demonstrating varying degrees of therapeutic effect. Specifically, the 5-ASA group achieved a final body weight of 82.64% ± 4.18% of its initial body weight, and Zn... 2+ The percentage was 82.84% ± 4.32%, EVLP was 83.31% ± 3.3%, and Zn 2+ The percentage of EV@INU was 83.85% ± 3.93%, while that of EV@INU was 85.88% ± 3.44%, and that of EV / Zn... 2+ The final weight of the INU group was 91.01% ± 2.17% of the initial weight, which was better than the other three drug-treated groups.

[0062] DAI score as follows Figure 15 The results showed that the healthy control group maintained a score of essentially zero, while the DSS model group mice began to exhibit loose stools and rectal bleeding from day 3, ultimately achieving a DAI score of 7.6 ± 0.55. Symptoms improved in all groups after treatment. The EV / Zn ratio was [not specified in the original text]. 2+ The significance was most significant in the @INU group, at 3.2 ± 0.84.

[0063] Colon length was measured after drug administration, and the degree of histopathological damage to the colon was assessed using HE staining. The levels of inflammatory factors in the colon tissue were detected by ELISA. Simultaneously, the indices of major organs (heart, liver, spleen, lung, and kidney) were assessed to comprehensively evaluate the safety of the nano-formulation. The colon length of mice is statistically analyzed as follows: Figure 16 As shown, the average colon length in the healthy control group was 12.16±1.05 cm, while in the DSS group it was only 7.10±0.66 cm, a significant reduction in length. Inflammatory symptoms such as congestion, edema, or mild erosion were observed in the DSS group during the experiment. These symptoms improved after drug administration, with the EV / Zn ratio... 2+ The @INU group showed the most significant results.

[0064] Mouse colon tissue pathological sections as shown Figure 17 As shown, the overall structure of the colon in the Control group was basically normal, with regular arrangement of mucosal epithelial cells. The columnar epithelium in the mucosal layer was arranged in tubular formations, with numerous goblet cells interspersed among the epithelial cells. No obvious loose, edematous necrosis was observed in the submucosa, and no obvious inflammatory cell infiltration was observed. In the DSS group, the overall structure of the colon was abnormal. Numerous mucosal epithelial cells showed ulceration, degeneration, and necrosis. The crypt structure was severely damaged, and the normal intestinal structure was almost completely lost. The number of goblet cells in the necrotic areas was almost entirely absent, and a large number of inflammatory cells were observed infiltrating the tissue. After treatment, all groups showed an improvement trend, with the EV / Zn ratio showing the following improvement. 2+ The INU group showed the most significant effect. The overall structure of the colonic tissue was basically normal, the epithelial cells of the mucosal layer were arranged regularly, the columnar epithelium of the mucosal layer was arranged in a tubular shape, and a large number of goblet cells were interspersed between the epithelial cells. No obvious loose edema or necrosis was observed in the submucosa of the mucosal layer, and no obvious inflammatory cell infiltration was observed in the tissue.

[0065] Figure 18 This is a graph showing the results of inflammatory factors in the mouse colon. As can be seen from the graph, compared with the normal group, the DSS group mice showed increased levels of both TNF-α and IL-6 pro-inflammatory factors in their colon tissue, while the drug-treated groups showed varying degrees of decrease in pro-inflammatory factor levels, particularly in the EV / Zn ratio. 2+ The INU group showed the most significant reduction, demonstrating its effective inhibition of pro-inflammatory cytokine expression and reduction of inflammatory response. IL-10, an important anti-inflammatory factor, was lowest in the DSS group, but levels recovered somewhat after administration. Among other things, EV / Zn... 2+ The reduction was most significant in the @INU group, demonstrating that it can alleviate the inflammatory response by promoting the expression of anti-inflammatory factors.

[0066] The safety of the formulation was assessed using organ index analysis, and the results showed that ( Figure 19 The weight indices (organ coefficients) of major organs such as heart, liver, spleen, lungs, and kidneys in mice in each experimental group showed no statistically significant differences compared with the control group (p>0.05), indicating that the preparation has biological safety.

[0067] (9) Microbiome research Balb / c mice were used as experimental animals. An ulcerative colitis model was induced by DSS. After treatment by gavage on day 7, fecal samples of mice in each group were collected and preserved. The regulatory effect of the drug on the intestinal flora of each group of mice was investigated by 16S rDNA high-throughput sequencing. Figure 20 This is a graph showing the results of the α-diversity index of the mouse gut microbiota. The results indicate that DSS-induced colitis significantly reduced the richness and diversity of the gut microbiota. Drug intervention could partially restore the richness, diversity, and evenness of the microbiota, causing the microbiota structure to shift from "dominated by a single dominant bacterium" to "evenly distributed across multiple species." The goods_coverage values ​​for each group were close to 1, indicating that sequencing provided high coverage of the microbiota across all groups, and the results are reliable.

[0068] Figure 21 This is a bar chart of the abundance of gut microbiota in mice. The control group showed abundant and evenly distributed genus-level flora, with relatively high abundance of beneficial genera such as some Bacteroides. The DSS group showed a sharp increase in the relative abundance of orange taxa, which includes pathogenic bacteria such as Shigella and Escherichia coli; this increase is an important marker of intestinal inflammation. Drug administration reduced the proportion of harmful genera and improved the genus-level flora structure.

[0069] Figure 22 Figure 1 shows the results of the Beta diversity analysis of the gut microbiota. Figure 2a is the Non-Metric Multidimensional Diagnostic Method (NMDS) analysis, where the Stress value is used to measure the goodness of fit of the NMDS analysis. A Stress value less than 0.2 indicates that the NMDS plot can reflect the true relationship between samples well; the smaller the Stress value, the better the fit. Here, the Stress value is 0.1431, which is within a good range, indicating that the NMDS plot can reliably show the differences in the microbial community structure between samples. The control group samples show some clustering, indicating that the gut microbiota structure of healthy mice is relatively consistent and stable. The DSS-induced colitis mouse group shows obvious separation from the control group, indicating that the gut microbiota structure of mice has changed significantly after DSS-induced colitis, and is significantly different from the microbiota structure in the healthy state. The trend of the gut microbiota structure recovering to the healthy state after drug administration preliminarily suggests that the drug may have a regulatory effect on the gut microbiota. Figure 3b is the principal component analysis plot, a commonly used method to show the overall differences and similarities in microbial community structure between samples. Figure c is the principal coordinate analysis diagram, which is also used to show the differences and similarities in the microbial community structure among samples. Both figures show that after DSS-induced colitis, the structure of the intestinal microbial community in mice was significantly altered, and the difference from the microbial community structure in the healthy state was large. After drug administration, it may have affected the intestinal microbial community of mice with DSS-induced colitis, and there is a tendency to restore the intestinal microbial community structure to the healthy state, which initially suggests that the drug may have the effect of regulating the intestinal microbial community.

[0070] In summary, the inulin gel containing extracellular vesicles derived from *Callicarpa nudiflora* provided by this invention can improve the therapeutic effect of ulcerative colitis.

[0071] Although the present invention has been described through specific embodiments, those skilled in the art can make modifications and variations without departing from the core concept of the present invention, and such modifications and variations are all within the protection scope of the present invention.

Claims

1. An inulin gel loaded with extracellular vesicles derived from *Callicarpa nudiflora*, characterized in that, The gel comprises extracellular vesicles derived from *Callicarpa nudiflora*, an inulin gel matrix, and zinc ions. The mass ratio of the extracellular vesicles derived from *Callicarpa nudiflora* to the inulin gel matrix is ​​1:

220. The concentration of zinc ions in the gel is 73.4 mM. The extracellular vesicles derived from *Callicarpa nudiflora* have a bilayer phospholipid vesicle structure. The extracellular vesicles derived from *Callicarpa nudiflora* were prepared by the following method: 500 g of *Callicarpa nudiflora* leaves were taken, chopped, and homogenized with 500 mL of PBS. The mixture was filtered through an 80-mesh filter to obtain the filtrate. The filtrate was centrifuged at 1000×g for 15 min at 4 °C. The supernatant was centrifuged at 5000×g for 30 min, and then centrifuged at 10000×g for 1 h. Finally, the supernatant was centrifuged at 150,000×g for 2 h to obtain the crude extract of extracellular vesicles derived from *Callicarpa nudiflora*. The crude extract was resuspended in 2-5 mL of PBS solution and centrifuged with an 8-60% sucrose gradient for 2 h. 30-45% of the bands were collected, mixed with PBS solution, and centrifuged at 150,000×g for 1 h at 4 °C. The precipitate was precipitated by pipetting with 3 mL of PBS solution and filtered through a 450 nm filter membrane to obtain the extracellular vesicle solution derived from *Callicarpa nudiflora*. The extracellular vesicles derived from *Callicarpa nudiflora* had a particle size of 126.72 ± 1.65 nm and a Zeta potential of -1.98 ± 0.49 mV. The gel was prepared by the following method: 55 g of inulin was dissolved in 45 mL of deionized water, heated at 80°C for 20 min, stirred at 300 rpm until completely dissolved, cooled to 40°C at a cooling rate of 5°C / min to obtain a 55% w / v inulin solution; 1 g of zinc chloride was added, and stirred at 300 rpm for 15 min; 0.25 g of extracellular vesicle solution derived from Callicarpa nudiflora was added to the above inulin-zinc solution, and stirring was continued for 10 min. The solution was then left at room temperature for 2 h to form a gel. The gel exhibits a storage modulus greater than its loss modulus under the following conditions: 25°C, 0.1% strain, using a flat plate system with a gap of 2 mm, and an angular frequency of 0.1 rad / s to 100 rad / s. The release rate of the gel in simulated intestinal fluid was 37.47% after 48 hours, and the release rate in simulated colonic fluid containing inulinase was 71.06% after 48 hours.

2. A method for preparing the gel as described in claim 1, characterized in that, Includes the following steps: S1: Take 500 g of Callicarpa nudiflora leaves, cut them into small pieces, add 500 mL of PBS to homogenize, filter through an 80-mesh filter to obtain the filtrate, centrifuge the filtrate at 1000×g for 15 min at 4℃, take the supernatant and centrifuge at 5000×g for 30 min, then take the supernatant and centrifuge at 10000×g for 1 h, finally take the supernatant and centrifuge at 150,000×g for 2 h to obtain the crude extract of extracellular vesicles from Callicarpa nudiflora, resuspend the crude extract in 2-5 mL of PBS solution, centrifuge at an 8-60% sucrose gradient for 2 h, collect 30-45% of the bands, mix with PBS solution, centrifuge at 150,000×g for 1 h at 4℃, take 3 mL of PBS solution, blow the precipitate and filter through a 450 nm filter membrane to obtain the extracellular vesicle solution from Callicarpa nudiflora; S2: Weigh 55 g of inulin and dissolve it in 45 mL of deionized water. Heat at 80°C for 20 min with a stirring rate of 300 rpm until completely dissolved. Cool to 40°C at a cooling rate of 5°C / min to obtain a 55% w / v inulin solution. Add 1 g of zinc chloride with a zinc ion concentration of 73.4 mM and stir at 300 rpm for 15 min. S3: Add 0.25g of extracellular vesicle solution derived from Callicarpa nudiflora to the above inulin-zinc solution, continue stirring for 10min, and let stand at room temperature for 2h to form a gel.

3. The use of a gel as described in claim 1, or a gel prepared by the method described in claim 2, in the preparation of a medicament for treating ulcerative colitis.

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