Preparation method and application of warm turmeric external capsule

Abstract

The application discloses a preparation method of Wen-jujin-derived extracellular vesicles based on an affinity capture technology, in particular to a high-efficiency and mild preparation method using lectin affinity chromatography combined with tangential flow ultrafiltration and size exclusion chromatography, and belongs to the technical field of medicines. Embodiments of the application show that the Wen-jujin-derived extracellular vesicles have certain anti-ulcerative colitis activity in vivo and in vitro, and can be used for preparing medicines for treating ulcerative colitis.

Description

Technical Field

[0001] This invention relates to a method for preparing vesicles derived from Curcuma longa based on affinity capture technology, and in particular to a highly efficient and mild preparation method using lectin affinity chromatography combined with tangential flow ultrafiltration and size exclusion chromatography, belonging to the field of pharmaceutical technology. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory bowel disease with an incompletely understood etiology. It primarily affects the colonic and rectal mucosa, and its clinical manifestations include abdominal pain, diarrhea, bloody and mucous stools, and weight loss. The disease course is protracted and recurrent, severely impacting patients' quality of life. Epidemiological data shows a significant upward trend in the incidence of ulcerative colitis in my country in recent years. Long-term presence of this disease can increase the risk of colorectal cancer; therefore, its prevention and treatment have always been a key research focus in the field of digestive system diseases. Currently, it is believed that the pathogenesis of UC is closely related to multiple factors, including immune inflammatory imbalance, impaired intestinal mucosal barrier function, increased oxidative stress, and intestinal flora dysbiosis. Clinically, drugs used to treat UC mainly include aminosalicylic acid preparations, glucocorticoids, immunosuppressants, and biologics. While these drugs can alleviate symptoms and control inflammation progression to some extent, they still have many limitations: aminosalicylic acid preparations are only effective for mild to moderate cases. While ulcerative colitis (UC) is effective, its efficacy is limited for severe cases. Long-term use of glucocorticoids can lead to serious adverse reactions such as increased risk of infection and metabolic disorders. Immunosuppressants and biologics are expensive, and some patients experience insufficient response or secondary loss of response, making them prone to drug resistance or relapse. In recent years, plant-derived exovesicles have attracted widespread attention in the biomedical field as a novel natural nanoscale delivery system. Plant exovesicles are lipid bilayer vesicles secreted by plant cells, containing bioactive substances such as proteins, lipids, nucleic acids, and secondary metabolites from the source plant. Compared with chemically synthesized nanocarriers, plant exovesicles have advantages such as high biocompatibility, low immunogenicity, and ease of large-scale production. Currently, the extraction methods of plant exovesicles still face many technical bottlenecks, such as cumbersome extraction steps, low yield, insufficient purity, and uneven particle size, which limit their further application in the pharmaceutical field.

[0003] Wen Yujin ( Curcuma wenyujin YHChen et C.Ling has the effects of promoting blood circulation and relieving pain, regulating qi and relieving depression, clearing the heart and cooling the blood, and promoting bile secretion and reducing jaundice. It is one of the important authentic medicinal materials in the famous "Eight Treasures of Zhejiang". Modern pharmacological studies have shown that turmeric contains a variety of active ingredients such as sesquiterpenes, and has multiple biological activities such as anti-inflammatory, antioxidant and anti-tumor effects.

[0004] However, current common solutions have many drawbacks, including: existing drugs have problems such as large individual differences in efficacy, significant adverse reactions with long-term use, high cost, and easy drug resistance and relapse; the preparation of existing plant exovesicles mostly relies on differential ultracentrifugation, which has drawbacks such as cumbersome steps, high shear force, damage to vesicle integrity, low yield, and difficulty in large-scale production. This invention is the first to use solid-phase lectin for specific capture, followed by gentle elution to obtain high-purity, high-activity exovesicles, overcoming many technical bottlenecks of traditional methods.

[0005] Although Curcuma longa, a traditional Chinese medicine, has various biological activities such as anti-inflammatory and antioxidant effects, its effective components suffer from low solubility and poor bioavailability, limiting its direct application efficacy. Existing research has few reports on the preparation methods of Curcuma longa-derived exovesicles and their use in the preparation of anti-ulcerative colitis drugs. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the preparation method and application of the above-mentioned turmeric-derived exovesicles, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to provide a method for preparing Curcuma longa vesicles and their application, which is applicable to solving the problems of large individual differences in efficacy, significant adverse reactions with long-term use, high price, and easy drug resistance and relapse of existing drugs; existing extraction methods for plant-derived vesicles generally suffer from technical bottlenecks such as cumbersome operation, low yield, insufficient purity, and uneven particle size; although Curcuma longa, as a traditional Chinese medicine, has multiple biological activities such as anti-inflammatory and antioxidant effects, its effective components have problems such as low solubility and poor bioavailability, which limits the direct application effect; currently, there are few reports on the preparation method of Curcuma longa vesicles and their use in the treatment of ulcerative colitis.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing exovesicles derived from Curcuma longa, comprising the following steps: S1. Raw material pretreatment and crude extraction Take Curcuma longa raw material, wash and chop it, mix it with extraction buffer and homogenize it, and then filter it to obtain crude extract.

[0010] The raw material of Curcuma zedoaria is fresh Curcuma zedoaria tuberous roots or dried Curcuma zedoaria slices. The extraction buffer is a buffer with a pH of 7.2 to 7.6, preferably a buffer containing phosphate and NaCl (such as phosphate buffer, PBS). The mass-to-volume ratio of Curcuma zedoaria raw material to extraction buffer is 1 g : (4 to 8) mL.

[0011] S2, Remove large particles and cell debris The crude extract obtained in step S1 was centrifuged at low speed and medium speed to remove large particulate impurities and cell debris, and the supernatant was collected.

[0012] The centrifugal force of the low-speed centrifugation is 2,000–5,000 × g, and the centrifugation time is 10–30 min; the centrifugal force of the medium-speed centrifugation is 8,000–15,000 × g, and the centrifugation time is 20–60 min.

[0013] S3, Tangential flow ultrafiltration concentration The supernatant collected in step S2 is concentrated by tangential flow ultrafiltration to obtain a concentrated solution.

[0014] The tangential flow ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 100 kDa to 500 kDa, preferably 300 kDa.

[0015] S4, lectin affinity chromatography captures outer vesicles The concentrate obtained in step S3 is brought into contact with a solid-phase lectin affinity medium, so that the extracellular vesicles derived from Curcuma longa bind to the lectin affinity medium.

[0016] The lectin in the lectin affinity medium is selected from one or both of concanavalin A (Con A) and castor bean lectin (RCA120), preferably a lectin derived from concanavalin A (Con A).

[0017] S5, Size Exclusion Chromatography Purification The lectin affinity medium was eluted with phosphate buffer (pH 7.2–7.6) containing competitive sugar ligands, and the eluent containing extracellular vesicles derived from Curcuma longa was collected. The eluent was purified by size exclusion chromatography, and the extracellular vesicle components were collected.

[0018] The size exclusion chromatography uses Sepharose CL-4B, Sepharose CL-6B or Capto Core 700 packing material, eluted with phosphate buffer, preferably Sepharose CL-4B packing material.

[0019] The competitive glycoligand is selected from one or more of methyl-α-D-mannoside, D-galactose, or lactose, preferably methyl-α-D-mannoside; the concentration of the competitive glycoligand in phosphate buffer is 0.1 M to 1.0 M, preferably 0.5 M.

[0020] S6. Aseptic filtration and preservation The extracellular vesicle components collected in step S5 were sterilized by 0.22 μm filtration to obtain extracellular vesicles derived from Curcuma longa, which were then stored at -80℃ or freeze-dried.

[0021] The filtration and sterilization process uses a polyethersulfone membrane, a polyvinylidene fluoride membrane, or a cellulose acetate membrane with a pore size of 0.22 μm, preferably a polyethersulfone membrane.

[0022] Secondly, the present invention provides that the exovesicles derived from Curcuma longa prepared by the above method have complete structures and are enriched with anti-inflammatory active ingredients from Curcuma longa.

[0023] Thirdly, the present invention provides the use of the aforementioned turmeric-derived vesicles in the preparation of drugs for treating or alleviating ulcerative colitis.

[0024] The beneficial effects of this invention are as follows: 1. It exhibits high specificity and purity, utilizing glycosylation modifications commonly found on the surface of plant exovesicles, such as mannose and galactose, as affinity targets, thus avoiding the co-precipitation of impurities or vesicle loss caused by improper centrifugal force settings in traditional ultracentrifugation methods. BCA value evaluation shows that the obtained exovesicle concentration can be increased by 3–5 times.

[0025] 2. Gentle and efficient, maintaining vesicle integrity: No high-speed centrifugation (>100,000 g) is required throughout the process. Combined with tangential flow ultrafiltration and affinity chromatography, the lipid bilayer structure of the outer vesicles and the contained active molecules are preserved to the greatest extent, exhibiting in vitro anti-inflammatory activity.

[0026] 3. Scalability and reproducibility: Affinity chromatography media can be reused, and tangential flow ultrafiltration is easy to scale up linearly, making it suitable for industrial production.

[0027] 4. Currently, there are no reports on the preparation of Curcuma longa exovesicles using lectin affinity chromatography. Natural nanovesicles with intact morphology, uniform particle size (main peak 100-150 nm), high purity, and high stability can be obtained under mild conditions. The Curcuma longa exovesicles prepared by this method, after oral administration, can target and accumulate in the inflamed colonic tissue of mice with ulcerative colitis, significantly inhibiting the pro-inflammatory factor TNF-α. α IL-1 βIL-6 releases and upregulates the level of the anti-inflammatory factor IL-10, dose-dependently improving the disease activity index, restoring colon length, repairing mucosal damage, and reducing the expression of NLRP3 and IL-1β proteins in the colon tissue of DSS-induced colitis mice, suggesting that it can regulate inflammation-related proteins, thus providing further support for its application in the preparation of drugs for the treatment of ulcerative colitis, and providing a novel natural nanotherapy strategy for the prevention and treatment of ulcerative colitis. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is a flowchart of the process for preparing vesicles derived from Curcuma longa in Example 1.

[0029] Figure 2 This is a characterization diagram of the external vesicles (W-EVs) derived from Curcuma longa in Example 3.

[0030] Figure 3 This is a diagram showing the in vitro anti-inflammatory activity of Curcuma longa extravesicles (W-EVs) in RAW264.7 cells, as described in Example 4.

[0031] Figure 4 In Example 5, non-targeted metabolomics analysis was used to analyze the different components of extracellular vesicles and tissues derived from Curcuma longa.

[0032] Figure 5 Distribution of Curcuma longa extravesicles (W-EVs) in mice after gavage in Example 5.

[0033] Figure 6 The in vivo pharmacodynamics of the outer vesicles of Curcuma longa (W-EVs) in Example 6 in mice with DSS-induced colitis is shown in the figure.

[0034] Figure 7 The image shows the WB band diagram and grayscale analysis of the regulation of NLRP3 and IL-1β proteins in the colon tissue of mice with DSS-induced ulcerative colitis by external vesicles derived from Curcuma longa in Example 8. Detailed Implementation

[0035] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0036] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0037] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0038] Example 1 This example provides a method for preparing extracellular vesicles derived from Curcuma longa (process flow is shown in...). Figure 1 Specifically, it includes the following steps: Take 500 g of fresh Curcuma longa root, wash and chop it, and add 2.5 L of pre-cooled phosphate-buffered saline (PBS, pH 7.4) at a ratio of 1 g: 5 mL. Homogenize the mixture three times at 12,000 rpm for 30 s each time using a high-speed disperser. After coarse filtration through three layers of medical gauze, the homogenate is then filtered through a 0.45 μm filter membrane to obtain the crude extract.

[0039] The crude extract was centrifuged at 3,000×g for 20 min at 4℃, and the supernatant was collected; then it was centrifuged at 10,000×g for 30 min, and the supernatant was collected to obtain a clear supernatant.

[0040] The supernatant was concentrated to 250 mL (1 / 10 of the original volume) using a Millipore Pellicon 3 tangential flow ultrafiltration system (membrane pack molecular weight cutoff 300 kDa) to obtain the concentrate.

[0041] The concentrate was loaded onto a 20 mL Con A-Sepharose 4B affinity chromatography column (GE Healthcare) at a flow rate of 1.2 mL / min and incubated at room temperature for 45 min to allow extracellular vesicles derived from Curcuma longa to bind to the lectin medium. The column was washed with 10 column volumes of phosphate-buffered saline (PBS, pH 7.4) to remove non-specific adsorbed impurities. Elution was then performed with elution buffer (PBS containing 0.5 M methyl-α-D-mannoside, pH 7.4), and the elution peak (approximately 5 mL) was collected.

[0042] Concentrate the eluent to 1 mL and load it onto a Sepharose CL-4B size exclusion column (1.6 × 60 cm). Elute isocratically with phosphate buffer (PBS, pH 7.4) and collect fractions from 8 to 14 mL (elution peaks corresponding to outer vesicles).

[0043] The collected extracellular vesicle solution was filtered through a 0.22 μm polyethersulfone filter membrane for sterilization to obtain a suspension of extracellular vesicles derived from Curcuma longa. The suspension was then aliquoted and stored at -80℃ or freeze-dried.

[0044] Example 2, an embodiment of the present invention, provides an application of Curcuma longa vesicles, including: the application of Curcuma longa-derived vesicles in the preparation of a drug for treating ulcerative colitis.

[0045] Specifically, the drug is an oral preparation.

[0046] Furthermore, the dosage form of the drug is one or more of the following: oral liquid, lyophilized reconstituted preparation, or suspension.

[0047] Example 3: Characterization of external vesicles derived from Curcuma longa The W-EVs suspension prepared in Example 1 was characterized as follows: (1) Transmission electron microscopy (TEM) observation: W-EVs suspension was dropped onto a copper grid, negatively stained with phosphotungstic acid, and observed under TEM. The results showed that ( Figure 2 In the middle A section, W-EVs exhibit a typical cup-shaped or biconcave disk-shaped structure with a diameter of approximately 100-200 nm, and are morphologically complete with clear boundaries.

[0048] (2) Nanoparticle tracking analysis (NTA): W-EVs were diluted with PBS, and the particle size distribution was detected using an NTA instrument. The results showed that ( Figure 2 (Part B) The particle size exhibits a unimodal distribution, with the main peak concentrated in the 100-150 nm range and a peak concentration of approximately 4 × 10⁻⁶. 10 Particles / mL, uniform particle size, high purity.

[0049] (3) Zeta potential detection: The results were obtained using a Zeta potential analyzer and showed that ( Figure 2 (Part C), with an average potential of approximately -26 mV, exhibits a concentrated potential distribution and good colloidal stability.

[0050] (4) Protein quantification (BCA method): Protein concentration was determined using a BCA kit. The results showed that ( Figure 2 (Part D), with a protein concentration of approximately 1.05 mg / mL, has a high sample loading capacity, which can meet the needs of subsequent experiments.

[0051] Example 4: In vitro anti-inflammatory activity of Curcuma longa-derived vesicles RAW264.7 macrophages were harvested and processed at a concentration of 1×10⁻⁶ cells. 5100 cells / well were seeded into 96-well plates. The experimental groups were: blank control group (without LPS and W-EVs), model group (with 1 μg / mL LPS), and W-EVs administration group (with 1 μg / mL LPS and 10, 20, and 50 μg / mL W-EVs). After 24 h of culture, the NO and inflammatory factor content in the supernatant was detected.

[0052] The results show that ( Figure 3 Part A): W-EVs concentration-dependently inhibit LPS-induced NO release ( P <0.0001), ELISA detection indicates ( Figure 3 In the middle BC portion), 20 μg / mL W-EVs significantly reduced the levels of TNF-α, IL-1β, and IL-6. P < 0.001 or P < 0.0001).

[0053] Example 5: Non-target metabolomics analysis of different components of extracellular vesicles and tissues derived from Curcuma longa.

[0054] To compare the differences in composition between extracellular vesicles derived from Curcuma longa and Curcuma longa tissue, non-targeted metabolomics analysis was performed on both samples. Based on sample grouping information, the Curcuma longa tissue sample was designated TCM1, and the extracellular vesicle sample derived from Curcuma longa was designated EXO1.

[0055] The extracellular vesicle samples derived from Curcuma longa were prepared using the methods described in the preceding embodiments; the Curcuma longa tissue samples were obtained from the tuberous root tissue of Curcuma longa. Metabolites were extracted from the above samples, and then detected using liquid chromatography-mass spectrometry.

[0056] For sample pretreatment, for tissue samples, the samples were lyophilized and ground into powder. The sample powder was weighed and extracted with pre-cooled 70% methanol-water internal standard extraction solution. After vortexing, centrifugation, and filtration through a 0.22 μm microporous membrane, the samples were used for UPLC-MS / MS analysis. For exovesicle samples, the samples were lyophilized, and after extraction with 70% methanol internal standard extraction solution, the samples were vortexed, sonicated in an ice-water bath, centrifuged, and filtered through a 0.22 μm microporous membrane before being used for LC-MS / MS detection.

[0057] The chromatographic and mass spectrometric (MS / MS) detection conditions were as follows: a Waters ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm) was used; mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid; the column temperature was 40 ℃, the flow rate was 0.40 mL / min, and the injection volume was 4 μL. MS / MS detection was performed using an AB Triple TOF6600+ mass spectrometer in both positive and negative ion modes. Peak extraction, alignment, and retention time correction were performed on the raw MS / MS data, and missing values ​​and peak area correction were also performed. After metabolite identification, the positive and negative ion mode data were merged to obtain the data file used for subsequent analysis.

[0058] Further intergroup comparison analysis was performed between extracellular vesicle sample EXO1 and tissue sample TCM1 derived from Curcuma longa. Principal component analysis results showed that ( Figure 4 In Part A), EXO1 and TCM1 were clearly separated in the principal component space, suggesting that there are significant differences in the overall metabolite composition between extracellular vesicles derived from Curcuma longa and Curcuma longa tissue.

[0059] In the differential metabolite screening, a fold change ≥2 or fold change ≤0.5 was used as the screening criteria to screen metabolites between EXO1 and TCM1. A total of 6073 differential metabolites were obtained by comparing EXO1 and TCM1, including 3934 upregulated metabolites and 2139 downregulated metabolites, indicating that there are extensive differences in metabolite composition and relative abundance between extracellular vesicles derived from Curcuma longa and Curcuma longa tissue.

[0060] This invention systematically analyzed the differences in metabolomic characteristics between Curcuma longa tissue and its derived external vesicles (W-Evs) using non-targeted metabolomics technology, clarifying the significant metabolite sorting patterns between the two types of samples. Figure 4 B). Figure 4The metabolic components in B, from top to bottom, are the following from the Curcuma aromatica plant tissue (TCM1, red): 4-Dimethylaminocinnamaldehyde, Neocarrabiose, 8-Hydroxyeicosa-9,11,14-trienoic acid, N-Acetylputrescine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine, 9S-HOTrE, Atalaphylline, Ala-Val-Val-Pro-Leu, and L-Allysine ethylene acetal. Metabolic components of exovesicles derived from Curcuma longa (EXO1, green): Rosmarinic acid, 7-hydroxy-2-(4-hydroxyphenyl)-5-methoxy-6,8-dimethyl-3,4-dihydro-2H-1-benzopyran-4-one, Heudelottin, Carnitine C2:0, N-linoleoylglycine, Trileucine, Scymnol, Ile-Cys-Arg, Val-Tyr, Huperzine-A. L-Leucyl-L-alanine (L-Leucyl-L-alanine). Figure 4B shows a significant difference in metabolite composition between Curcuma longa tissue and its derived external vesicles (W-Evs), suggesting that external vesicles may selectively load or enrich specific metabolites. Unlike the relatively enriched lipids, amino acids / peptides, nitrogenous metabolites, and some tissue-related secondary metabolites in plant tissues, W-Evs are enriched with function-related metabolites such as rosmarinic acid, acetylcarnitine, N-linoleylglycine, huperzine A, and various small peptides. Some of these components have been reported to possess anti-inflammatory, antioxidant, neuroprotective, or metabolic regulatory potential, suggesting their possible involvement in the formation of the bioactivity of W-Evs. These results provide metabolomics evidence for revealing the potential material basis and metabolite sorting characteristics of Curcuma longa external vesicles, and offer a reference for subsequent quality control, activity evaluation, and development as a natural active ingredient delivery system.

[0061] This study systematically analyzed the differences in metabolic pathways between Curcuma longa tissue and its derived external vesicles (W-Evs) using KEGG pathway enrichment analysis. The results showed ( Figure 4 C). Figure 4The metabolic pathways in C, from top to bottom, are: Biosynthesis of various plant secondary metabolites, Glycerophospholipid metabolism, Purine metabolism, Biosynthesis of cofactors, Nucleotide metabolism, Pyrimidine metabolism, Lysine degradation, Linoleic acid metabolism, Phenylalanine, tyrosine, and tryptophan biosynthesis, Biosynthesis of various alkaloids, Zeatin biosynthesis, Flavonoid biosynthesis, Starch and sucrose metabolism, Tropane, Piperidine, and Pyridine alkaloids. Biosynthesis of alkaloids (hyoscyamine, piperidine, and pyridine), Caffeine metabolism, Neomycin, kanamycin, and gentamicin biosynthesis, Biosynthesis of secondary metabolites, Glycerolipid metabolism, Lysine biosynthesis, and Arginine and proline metabolism. Figure 4Cc analysis revealed that the differentially metabolites between *Curcuma wenyujin* tissue and W-Evs mainly involved pathways such as plant secondary metabolite biosynthesis, glycerol-phospholipid metabolism, nucleotide metabolism, amino acid metabolism, linoleic acid metabolism, flavonoid biosynthesis, and alkaloid biosynthesis, suggesting significant differences between the two in secondary metabolism, lipid metabolism, and basic substance metabolism. Enrichment of related pathways indicates that W-Evs may carry or be enriched in some functional metabolites related to anti-inflammatory, antioxidant, and metabolic regulation, providing a material basis for its potential biological activities. These results provide metabolomics evidence at the pathway level for revealing the metabolic characteristics of W-Evs, screening for quality control biomarkers, and subsequent pharmacodynamic mechanism studies.

[0062] The above results indicate that there are significant differences between extracellular vesicles derived from Curcuma longa and Curcuma longa tissue in terms of metabolite composition, relative content, and distribution of related metabolic pathways. This suggests that extracellular vesicles derived from Curcuma longa are not simply replicates of Curcuma longa tissue components, but rather possess metabolite characteristics distinct from Curcuma longa tissue. These results provide metabolomics evidence for the study of the component characteristics and biological functions of extracellular vesicles derived from Curcuma longa.

[0063] Example 6: Study on the in vivo distribution of extravesicles derived from Curcuma longa W-EVs were labeled with Cy7 fluorescence and administered by gavage to healthy mice and mice with DSS-induced ulcerative colitis. In vivo imaging showed that ( Figure 5 Part A): Cy7-W-EVs were significantly enriched at sites of colonic inflammation in mice, with fluorescence intensity higher than in the healthy group, and the signal could still be detected after 24 h. Ex vivo imaging ( Figure 5 The results (from the middle BC section) confirmed that the fluorescence was mainly concentrated in the colon, with almost no signal in other organs, indicating that oral W-EVs can target the inflamed colon and have little distribution throughout the body.

[0064] Example 7: Therapeutic effect of Curcuma longa-derived exovesicles on DSS-induced ulcerative colitis mice An acute colitis model in mice was established using 3% DSS in the drinking water for 7 days. The mice were divided into three groups: normal control group, model control group (PBS), positive control group (5-ASA 100 mg / kg), and low (5 mg / kg), medium (15 mg / kg), and high (45 mg / kg) dose groups of W-EVs. The mice were administered the drugs by gavage. Body weight and DAI scores were monitored daily. Colon length and spleen index were measured at the experimental endpoint. Colon tissue was collected for HE staining and detection of inflammatory factors.

[0065] result( Figure 6 W-EVs dose-dependently improve weight loss ( Figure 6 Part A), reducing spleen index ( Figure 5 Part B) and DAI score ( Figure 6Part C of the middle section significantly restored colon length. Figure 6 Part D in middle 5 and part E in middle 5), HE staining shows ( Figure 5 In the middle and high-dose groups of W-EVs, the colonic mucosal structure was basically intact, crypts were restored, and inflammatory cell infiltration was significantly reduced. ELISA showed ( Figure 6 In the GJ group, W-EVs significantly reduced pro-inflammatory factors TNF-α, IL-1β, and IL-6, and increased anti-inflammatory factor IL-10. The high-dose group was more effective than the 5-ASA group.

[0066] Example 8: Regulation of NLRP3 and IL-1β proteins in colon tissue of mice with DSS-induced ulcerative colitis by external vesicles derived from Curcuma longa. Extracellular vesicles derived from Curcuma longa prepared in Example 1 were used for animal experiments. An acute colitis model in mice was established using 3% DSS in the drinking water. The experiment was divided into a normal control group (CON), a model group (DSS), a positive drug control group (DSS+5-ASA), and low-dose (DSS+W-EVs-L), medium-dose (DSS+W-EVs-M), and high-dose (DSS+W-EVs-H) groups of extracellular vesicles derived from Curcuma longa. After treatment of each group, colon tissue was collected, and total protein was extracted using lysis buffer. Protein concentration was determined using the BCA method. Equal amounts of protein samples from each group were separated by SDS-PAGE electrophoresis and transferred to membranes, which were then incubated with NLRP3 and IL-1, respectively. β and β -actin antibody was used, and chemiluminescence immunoassay was employed to develop and record band results. β-actin was used as an internal control protein. Results ( Figure 7 The results of Western blot analysis of NLRP3 and IL-1β proteins in colon tissues of each group are shown. Compared with the normal control group, the expression of NLRP3 and IL-1β proteins in the colon tissues of the model group was significantly increased; compared with the model group, the expression of NLRP3 and IL-1β proteins in the low, medium, and high dose groups of extracellular vesicles derived from Curcuma longa was significantly decreased, suggesting that extracellular vesicles derived from Curcuma longa can regulate the expression of inflammatory proteins related to DSS-induced colitis. Figure 7 middle,***, P <0.001, **, P <0.01.

[0067] Example 9: Pharmaceutical preparations derived from the outer vesicles of Curcuma longa The W-EVs suspension prepared in Example 1 was mixed with pharmaceutically acceptable excipients (such as mannitol, trehalose, etc.) and freeze-dried to produce a lyophilized powder or a lyophilized reconstituted preparation; or it was mixed with an appropriate amount of excipients to produce an oral liquid or suspension.

[0068] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing extracellular vesicles derived from Curcuma longa, characterized in that, Includes the following steps: S1. After washing and chopping the raw material of Curcuma longa, mix it with phosphate buffer and homogenize it. After coarse filtration, the crude extract is obtained. S2. Centrifuge the extract at low speed and medium speed to remove large particulate impurities and cell debris, and collect the supernatant. S3. The supernatant is concentrated by tangential flow ultrafiltration to obtain a concentrated solution; S4. The concentrated solution is brought into contact with a solid-phase lectin affinity medium to allow extracellular vesicles derived from Curcuma longa to bind to the lectin affinity medium. S5. The lectin affinity medium is eluted with phosphate buffer containing competitive sugar ligands, and the eluent containing extracellular vesicles derived from Curcuma longa is collected; the eluent is purified by size exclusion chromatography, and the extracellular vesicle components are collected. The competitive sugar ligand is selected from methyl- α One or more of D-mannoside, D-galactose, or lactose, preferably methyl- α -D-Mannoside; the competitive glycoligand is present in phosphate buffer at a concentration of 0.1 M to 1.0 M, preferably 0.5 M; S6. The extracellular vesicle components are sterilized by 0.22 μm filtration to obtain extracellular vesicles derived from Curcuma longa.

2. The preparation method according to claim 1, characterized in that, The raw material for the herb *Curcuma longa* is either fresh *Curcuma longa* tuber or dried *Curcuma longa* slices.

3. The preparation method according to claim 1 or 2, characterized in that, In step S1, the extraction buffer is a buffer with pH 7.2 to 7.6, preferably a buffer containing phosphate and NaCl, and / or the mass-to-volume ratio of the Curcuma longa raw material to the extraction buffer is 1 g:(4 to 8) mL.

4. The preparation method according to claim 1, characterized in that, In step S2, the centrifugal force of the low-speed centrifugation is 2,000 to 5,000 × g, and the centrifugation time is 10 to 30 min; the centrifugal force of the medium-speed centrifugation is 8,000 to 15,000 × g, and the centrifugation time is 20 to 60 min.

5. The preparation method according to claim 1, characterized in that, In step S3, the ultrafiltration membrane used in the tangential flow ultrafiltration has a molecular weight cutoff of 100 kDa to 500 kDa, preferably 300 kDa.

6. The preparation method according to claim 1, characterized in that, In step S4, the lectin in the lectin affinity medium is selected from one or both of concanavalin A and castor bean lectin, preferably a lectin derived from concanavalin A.

7. The preparation method according to claim 1, characterized in that, In step S5, the size exclusion chromatography uses Sepharose CL-4B, Sepharose CL-6B or Capto Core 700 packing material and elutes with phosphate buffer, preferably Sepharose CL-4B.

8. The preparation method according to claim 1, characterized in that, In step S6, the filtration and sterilization uses a polyethersulfone membrane, a polyvinylidene fluoride membrane, or a cellulose acetate membrane with a pore size of 0.22 μm, preferably a polyethersulfone membrane.

9. An extracellular vesicle preparation derived from Curcuma longa, characterized in that, The extracellular vesicle preparation derived from Curcuma longa is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the extracellular vesicle preparation derived from Curcuma longa according to claim 9 in the preparation of a medicament for treating ulcerative colitis.

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