Application of honeysuckle-derived nano-vesicles in preparation of products for preventing, improving or treating clostridium difficile infection-related diseases

Abstract

The invention relates to application of honeysuckle-derived nano-vesicles in preparation of products for preventing, improving or treating clostridium difficile infection related diseases. Specifically, the nano-vesicles (HNVs) from honeysuckle have a remarkable direct bacteriostatic effect on clostridium difficile, can effectively relieve clostridium difficile infection, can effectively relieve colitis caused by clostridium difficile infection, and can effectively relieve body inflammation caused by clostridium difficile infection. Experiments show that the HNVs have excellent biocompatibility and safety, and important guarantee is provided for clinical application of the HNVs. The invention provides an innovative solution for developing a novel clostridium difficile infection resistant preparation, and has a wide application prospect in the field of anti-infection treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of honeysuckle-derived nanovesicles in the preparation of products for preventing, improving or treating diseases related to Clostridioides difficile infection. Background Art

[0002] Clostridioides difficile (C. difficile) is a strictly anaerobic, spore-forming Gram-positive bacterium that is widely distributed in the natural environment and also exists in the digestive tracts of humans and animals. C. difficile is an opportunistic pathogen that can overgrow and release toxins in the intestines of patients with long-term antibiotic use, low immunity, or intestinal flora disorders, thereby causing intestinal infectious diseases, namely Clostridioides difficile infection. Clostridioides difficile infection is one of the global public health problems, which is usually clinically related to the extensive use of broad-spectrum antibiotics and the outbreak of C. difficile. Its symptoms include mild to severe diarrhea, and in severe cases, it can cause life-threatening colon perforation, pseudomembranous colitis, and toxic megacolon, and it is one of the common causes of nosocomial infectious diarrhea and enteritis.

[0003] At present, the clinical treatment methods for Clostridioides difficile infection mainly include antibiotic treatment (vancomycin or fidaxomicin) and fecal microbiota transplantation therapy. However, about 20-25% of patients with Clostridioides difficile infection treated with antibiotics experience recurrent infection after cure. At the same time, although fecal microbiota transplantation therapy has good efficacy for recurrent and refractory Clostridioides difficile infection, there are still problems such as donor source selection, fecal microbiota production standards, transplantation methods, and the psychological burden of patients. Therefore, it is of great significance to find new and safe prevention and treatment means that can effectively target Clostridioides difficile infection.

[0004] Honeysuckle (Lonicera japonica Thunb.), also known as Japanese honeysuckle, is a medicinal and edible herbaceous plant belonging to the genus Lonicera of the family Caprifoliaceae. Approximately 1,500 years ago, the ancient Chinese already used honeysuckle as a traditional Chinese medicine to treat exogenous wind-heat, warm diseases, sores, carbuncles, boils and other diseases. At the same time, plant-derived nanovesicles (PNVs) are a general term for vesicle-like structural nanoparticles isolated from plants. Plants release PELNVs, which serve as extracellular messengers and respond to various pathological diseases. PELNVs entering the animal gastrointestinal tract can cross the intestinal barrier and be absorbed by immune cells such as intestinal macrophages, inducing the production of various cytokines to mediate cell communication between species. Existing studies have shown that PELNVs have functions such as regulating gene / protein expression in recipient cells, anti-inflammatory, antioxidant, regulating the intestinal flora, and promoting intestinal tissue regeneration. However, to date, the nanovesicles derived from honeysuckle and their application in the treatment of Clostridioides difficile infection have not been fully studied. Revealing their potential pharmacological mechanisms and clinical benefits will provide new treatment hopes for patients with Clostridioides difficile infection. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of honeysuckle-derived nanovesicles (HNVs) in the preparation of products for preventing, improving or treating diseases related to Clostridioides difficile infection. This exosome can be used to effectively prevent, improve or treat diseases related to Clostridioides difficile infection.

[0006] Specifically, the present invention provides the application of honeysuckle-derived nanovesicles in the preparation of products for preventing, improving or treating diseases related to Clostridioides difficile infection or in products for inhibiting the growth of Clostridioides difficile.

[0007] In some specific embodiments of the present invention, the products include but are not limited to one or more of drugs, health foods, foods for special medical purposes, foods, functional foods, feeds, feed additives or pet foods.

[0008] In some specific embodiments of the present invention, the product is a drug.

[0009] In some specific embodiments of the present invention, the diseases include but are not limited to antibiotic-associated diarrhea, colitis, recurrent Clostridioides difficile infection, pyelonephritis, meningitis, abdominal and vaginal infections, bacteremia and gas gangrene.

[0010] In some specific embodiments of the present invention, the disease is pseudomembranous colitis or fulminant enteritis.

[0011] In some specific embodiments of the present invention, the related symptoms of Clostridium difficile infectious diseases include watery diarrhea (typical manifestation), abdominal pain, abdominal distension, fever, colon inflammation, impaired intestinal mucus barrier, damage to the intestinal mechanical barrier, nausea and vomiting, weakness, and loss of appetite; the possible complications include severe dehydration, hypoproteinemia with peripheral edema, paralytic ileus, toxic megacolon, colon perforation, circulatory shock, renal failure, systemic inflammatory response syndrome, sepsis, and even death.

[0012] In some specific embodiments of the present invention, the honeysuckle-derived nanovesicles are isolated and extracted from honeysuckle. The honeysuckle-derived nanovesicles exhibit a uniform membrane-enclosed vesicle-like structure, and the average particle size of the nanovesicles is 83 nm.

[0013] In some specific embodiments of the present invention, the preparation of the honeysuckle-derived nanovesicles comprises the following steps:

[0014] Step 1: Honeysuckle is soaked in sterile PBS and homogenized into a slurry. After filtering to remove impurities, the juice is collected;

[0015] Step 2: The juice is purified and extracted by differential centrifugation and tangential flow filtration to obtain honeysuckle-derived nanovesicles.

[0016] In some specific embodiments of the present invention, the specific operation method of Step 2 is as follows: The juice is centrifuged at 10,000×g for 1 hour, and the supernatant is collected. The above centrifugation operation is repeated until there is no precipitate; subsequently, the obtained supernatant is injected into the storage tank of the tangential flow filtration system, passed through a 200 nm membrane column, and the sample with a particle size ≤200 nm is collected; the crude sample is injected into the storage tank of the tangential flow filtration system again, passed through a 750 kD membrane column to remove small molecule impurities, and the sample with a molecular weight >750 kD is collected to obtain purified honeysuckle-derived nanovesicles.

[0017] The present invention also provides a composition, drug, health food, food for special medical purposes, food, functional food, feed, feed additive, or pet food for preventing, improving, or treating Clostridium difficile infection-related diseases or inhibiting the growth of Clostridium difficile, which is characterized by comprising honeysuckle-derived nanovesicles.

[0018] In some specific embodiments of the present invention, the diseases include but are not limited to antibiotic-associated diarrhea, colitis, recurrent Clostridium difficile infection, pyelonephritis, meningitis, abdominal and vaginal infections, bacteremia, and gas gangrene.

[0019] In some specific embodiments of the present invention, the honeysuckle-derived nanovesicles are isolated and extracted from honeysuckle. The honeysuckle-derived nanovesicles exhibit a uniform membrane-enclosed vesicle-like structure, and the average particle size of the nanovesicles is 83 nm.

[0020] In some specific embodiments of the present invention, the preparation of the honeysuckle-derived nanovesicles comprises the following steps:

[0021] Step 1: Soak honeysuckle in sterile PBS to make a slurry, filter to remove impurities, and collect the juice;

[0022] Step 2: The juice is extracted using differential centrifugation and tangential flow purification to obtain honeysuckle-derived nanovesicles.

[0023] In some specific embodiments of the present invention, the specific operation method of step 2 is: centrifuging the juice at 10,000×g for 1 hour, collecting the supernatant, and repeating the above centrifugation operation until there is no precipitation; then injecting the obtained supernatant into the liquid storage tank of the tangential flow filtration system, passing through a 200nm membrane column, and collecting samples with a particle size of ≤200nm; injecting the crude sample into the liquid storage tank of the tangential flow filtration system again, passing through a 750kD membrane column to remove small molecule impurities, collecting samples with a molecular weight of >750kD, and obtaining purified honeysuckle-derived nanovesicles.

[0024] Compared with the prior art, the present invention has the following significant advantages and effects:

[0025] (1) Significant therapeutic effect: HNVs have a significant direct antibacterial effect on Clostridium difficile and can effectively inhibit the proliferation of Clostridium difficile in brain heart infusion broth; they can significantly reduce the fecal Clostridium difficile load and effectively alleviate Clostridium difficile infection; they can significantly alleviate the reduced survival rate, weight loss and shortened colon length of long-clawed gerbils caused by Clostridium difficile infection, indicating that HNVs have a good alleviating effect on Clostridium difficile infection; they can effectively alleviate the histopathological changes such as colon crypt loss, submucosal edema and inflammatory cell infiltration caused by Clostridium difficile infection, indicating that HNVs can effectively alleviate colitis caused by Clostridium difficile infection; oral administration of HNVs can effectively alleviate the upregulation of proinflammatory cytokines IL-1β, TNF-α, IL-6 and IFN-γ in serum caused by Clostridium difficile infection, and can also significantly increase the expression level of anti-inflammatory cytokine IL-10 in serum, indicating that HNVs can effectively alleviate the inflammation caused by Clostridium difficile infection. In short, these findings provide a basis for HNVs as potential functional foods or drugs to alleviate Clostridium difficile infection.

[0026] (2) Safety and applicability: Continuous intragastric administration of HNVs (200 mg / kg) for 8 days did not cause significant changes in the body weight, colon length, liver index, and spleen index of Mongolian gerbils. At the same time, it did not cause significant changes in the liver and kidney function levels in the serum. Continuous intragastric administration of HNVs (200 mg / kg) for 8 days did not cause changes in the tissue morphology of the heart, liver, spleen, lungs, kidneys, and colon of Mongolian gerbils, indicating that HNVs have good biosafety, which is a solid foundation for the application of HNVs in relieving Clostridioides difficile infection.

[0027] (3) Easy access and cost-effectiveness: Honeysuckle, as a raw material, is rich in resources and low in cost. After the preparation method of honeysuckle-derived nanovesicles is optimized, the yield and efficiency are greatly improved, and the production cost is reduced. This not only makes honeysuckle-derived exosomes an affordable treatment option but also creates conditions for their large-scale production and popular application.

[0028] In summary, the present invention positions honeysuckle-derived nanovesicles as an efficient, safe, and economical treatment tool for relieving Clostridioides difficile infection. Its comprehensive advantages bring new hope to patients with Clostridioides difficile infection and also inject innovative vitality into the field of health management. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0030] Figure 1 is the flow chart for the preparation of honeysuckle-derived nanovesicles HNVs;

[0031] Figure 2 is the morphological structure of honeysuckle-derived nanovesicles HNVs;

[0032] Figure 3 is the particle size of honeysuckle-derived nanovesicles HNVs;

[0033] Figure 4 is the Zeta potential of honeysuckle-derived nanovesicles HNVs;

[0034] Figure 5 is the determination of the in vivo distribution of honeysuckle-derived nanovesicles HNVs;

[0035] Figure 6 is the construction of a Clostridioides difficile infection model;

[0036] Figure 7Determination of the direct antibacterial effect of honeysuckle-derived nanovesicles HNVs;

[0037] Figure 8 Determination of the fecal Clostridium difficile load in each group for the treatment of Clostridium difficile infection with honeysuckle-derived nanovesicles HNVs;

[0038] Figure 9 Mortality rate of Mongolian gerbils in each group for the treatment of Clostridium difficile infection with honeysuckle-derived nanovesicles HNVs;

[0039] Figure 10 Change in body weight of Mongolian gerbils in each group for the treatment of Clostridium difficile infection with honeysuckle-derived nanovesicles HNVs;

[0040] Figure 11 Colon length of Mongolian gerbils in each group for the treatment of Clostridium difficile infection with honeysuckle-derived nanovesicles HNVs;

[0041] Figure 12 Histopathological examination of colon tissues in each group for the treatment of Clostridium difficile infection with honeysuckle-derived nanovesicles HNVs;

[0042] Figure 13 Detection of serum immune factors of honeysuckle-derived nanovesicles HNVs;

[0043] Figure 14 Biological safety indicators of honeysuckle-derived nanovesicles HNVs;

[0044] Figure 15 Histological examination of the biological safety of honeysuckle-derived nanovesicles HNVs. Detailed implementation manners

[0045] Next, the technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention and should not be used to limit the protection scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the protection scope of the present invention.

[0046] Example 1 Preparation of honeysuckle-derived nanovesicles (HNVs)

[0047] The preparation steps are as follows (the preparation process schematic diagram is as Figure 1As shown in the figure: Weigh 100 g of honeysuckle samples and soak them in 500 mL of sterile PBS for 1 h. Homogenize thoroughly with a homogenizer, filter out impurities with gauze, collect the filtrate and soak it overnight at 4°C. Centrifuge the filtrate at 10,000 g and 4°C for 1 h, collect the supernatant, and repeat the above centrifugation operation until there is no precipitate. Subsequently, inject the obtained supernatant into the storage tank of the tangential flow filtration system, pass through a 200 nm membrane column, and collect the samples with a particle size ≤ 200 nm. Inject the crude extract sample again into the storage tank of the tangential flow filtration system, pass through a 750 kD membrane column to remove small molecule impurities, and collect the samples with a molecular weight > 750 kD to obtain purified honeysuckle-derived nanovesicles, namely HNVs, and store them in an ultra-low temperature refrigerator at -80°C.

[0048] Example 2 Characterization of Honeysuckle-Derived Nanovesicles

[0049] Aspirate the honeysuckle-derived nanovesicles prepared in Example 1 and load them onto a grid covered with a porous carbon film. Then place the grid into liquid ethane cooled with liquid nitrogen. Load the frozen sample onto a cryo-electron microscope sample stage cooled with liquid nitrogen, observe its microstructure and take pictures; at the same time, dilute it 100 times with a diluent and inject it into a nanoparticle Coulter counter to measure its particle size; aspirate HNVs and inject them into a dynamic light scattering particle size analyzer to measure its Zeta potential.

[0050] It should be noted that as shown in the appendix Figure 2 As shown, it was found by cryo-electron microscopy that the structure of HNVs is a lipid membrane-encapsulated vesicle.

[0051] Furthermore, as shown in the appendix Figure 3 、 4 As shown, the average particle size of HNVs measured by a nanoparticle Coulter counter is 83 nm, and the particle concentration is 3.89×10 11 particles / mL; the Zeta potential of HNVs measured by a dynamic light scattering particle size analyzer is -6.94 ± 0.62 mV.

[0052] Example 3: Determination of the In Vivo Distribution of Honeysuckle-Derived Nanovesicles HNVs

[0053] Aspirate 1 mL of HNVs and mix well with 10 μL of DiR dye by pipetting. Incubate in the dark at room temperature for 30 min. Inject the incubated liquid into an ultrafiltration tube and centrifuge at 5000 g and 4°C for 20 min in a high-speed centrifuge to remove free DiR dye; intragastrically administer the long-clawed gerbils at a dose of 200 mg / kg (HNVs protein concentration / body weight), and collect the digestive tract (from the stomach to the anus) of the long-clawed gerbils at 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h after intragastric administration, and observe the in vivo distribution of HNVs with a small animal in vivo imaging system.

[0054] It should be noted that as shown in the appendixFigure 5 As shown, fluorescence signals carried by HNVs were detected in the stomach, duodenum, jejunum, and ileum 0.5 h after intragastric administration. Fluorescence signals could be detected in the cecum and colon 1 h after intragastric administration, indicating that HNVs could reach the cecum and colon 1 h after intragastric administration, and the fluorescence signal intensity at this site gradually weakened over time. 24 h after intragastric administration, only weak fluorescence signals were detected in the colon, and no fluorescence signals were detected in the other parts, indicating that most of the HNVs had been excreted from the body 24 h after intragastric administration.

[0055] Example 4: Functional detection of honeysuckle-derived nanovesicles HNVs in alleviating Clostridium difficile infection

[0056] Experimental protocol 1: Construction of an animal model of Clostridium difficile infection

[0057] As shown in the appendix Figure 6 Six-week-old male Mongolian gerbils were purchased from the Experimental Animal Center of Hangzhou Medical College and housed in an SPF-class animal house. There were 4-5 gerbils in each cage. The room temperature was 23±2°C. They had free access to standard feed and drinking water, and the light was on for 12 h per day. Fifty-six gerbils were randomly divided into 4 groups, namely the control group (Control), the model group (Model), the HNVs treatment group (HNVs), and the vancomycin positive treatment group (Vancomycin), with 14 gerbils in each group.

[0058] After the gerbils had adapted for 1 week, kanamycin (0.4 mg / mL), gentamicin (0.035 mg / mL), polymyxin (850 U / mL), metronidazole (0.215 mg / mL), and vancomycin (0.045 mg / mL) were added to the drinking water. After 3 days of drinking, the water was changed to normal drinking water. Two days after changing the water, clindamycin (10 mg / kg) was injected intraperitoneally. Twenty-four hours after the intraperitoneal injection, the gerbils were given an intragastric administration of 5×104 CFU of Clostridium difficile spores. The treatments for each group in the experiment were as follows:

[0059] Control group: The gerbils in this group were not treated with Clostridium difficile infection (antibiotics were not added to the drinking water, and aseptic PBS was used instead of experimental operations such as intraperitoneal injection / intragastric administration).

[0060] Model group: The gerbils in this group were treated with Clostridium difficile infection and were intragastrically administered sterile PBS at regular intervals within 4 days after modeling.

[0061] HNVs treatment group: The gerbils in this group were treated with Clostridium difficile infection and were intragastrically administered HNVs (200 mg / kg) at regular intervals within 4 days after modeling.

[0062] Vancomycin positive treatment group: The gerbils in this group were treated with Clostridium difficile infection and were intragastrically administered vancomycin (50 mg / kg) at regular intervals within 4 days after modeling.

[0063] The administration interval was 24 h. Before gavage, the mortality and body weight of Mongolian gerbils were recorded. 12 h after the 4th weighing / gavage, the Mongolian gerbils were decapitated and samples were collected.

[0064] Experimental protocol 2: Determination of the direct antibacterial effect of HNVs against Clostridium difficile

[0065] 1 mL of sterile PBS or HNVs was respectively mixed with 9 mL of brain heart infusion broth. Subsequently, 100 μL of Clostridium difficile (105 CFU) was added into a 15 mL centrifuge tube, and the mixture was incubated statically at 37 °C in a sealed manner for 48 h (n = 6). Centrifuge at 5000 rpm / min for 10 min at 4 °C, and aspirate the supernatant. Add 10 mL of sterile PBS to wash the bacterial cells, centrifuge again and discard the supernatant, and repeat the washing twice. Add 10 mL of sterile PBS to resuspend the bacterial cells, aspirate 200 μL and inject it into a 96-well plate, and measure the absorbance value at OD600.

[0066] It should be noted that as shown in the appendix Figure 7 HNVs had a significant direct antibacterial effect against Clostridium difficile and could effectively inhibit the proliferation of Clostridium difficile in brain heart infusion broth.

[0067] Experimental protocol 3: Determination of fecal bacterial load

[0068] On the day of sampling, fresh feces of Mongolian gerbils were collected, about 0.1 g for each, and put into a 1.5 mL sterile centrifuge tube. Add 0.9 mL of sterile PBS to mix well with the feces under sterile conditions, and then perform 10-fold serial dilutions. The diluted liquid was evenly spread on CCFA agar (Clostridium difficile selective plate), and anaerobically cultured at 37 °C in a biochemical incubator for 48 h, and observed and counted.

[0069] It should be noted that the feces of Mongolian gerbils were collected and spread on the Clostridium difficile selective plate to determine the number of Clostridium difficile in the feces; the results were as shown in Figure 8 Gavage of Mongolian gerbils with 200 mg / kg of HNVs could significantly reduce the fecal Clostridium difficile load, indicating that HNVs could effectively relieve Clostridium difficile infection.

[0070] Experimental protocol 4: Determination of mortality, body weight and colon length of Mongolian gerbils

[0071] Within 4 days after modeling, the mortality and body weight of Mongolian gerbils were regularly gavaged / recorded.

[0072] Survival rate (%) = (number of surviving individuals on the day / number of surviving individuals before modeling) * 100%

[0073] Body weight change (%) = (body weight on the day / initial body weight before modeling) * 100%

[0074] During sampling, the intestine from the ileocecal junction to the anus of the Mongolian gerbil was separated with scissors, and the length of the colon was accurately measured.

[0075] It should be noted that, as shown in Figure 9 、 10 、11, intragastric administration of HNVs can significantly alleviate the reduction of survival rate, body weight and shortening of colon length caused by Clostridium difficile infection, indicating that HNVs have a good alleviating effect on Clostridium difficile infection.

[0076] Experimental protocol 5: Histopathological examination of colon

[0077] After measuring the colon length, a segment of the colon (the intestinal segment not previously clamped) was cut with scissors and immersed in 4% paraformaldehyde fixative for at least 72 h. The fixed colon tissue was taken out, dehydrated with ethanol solutions of gradient concentrations, then cleared with xylene, and subsequently embedded in paraffin. The embedded tissue was sectioned with a paraffin microtome, and the section thickness was 5 μm. The sections were fixed on glass slides and baked at 45 °C overnight. Subsequently, the sections were dewaxed to water, stained with hematoxylin staining solution and eosin staining solution, dehydrated and sealed, and observed and photographed with an optical microscope.

[0078] It should be noted that, as shown in Figure 12 , intragastric administration of HNVs can effectively alleviate histopathological changes such as colon crypt loss, submucosal edema and inflammatory cell infiltration caused by Clostridium difficile infection, indicating that HNVs can effectively alleviate colitis caused by Clostridium difficile infection.

[0079] Experimental protocol 6: Detection of serum immune factors

[0080] During sampling, blood was first collected from the eyeballs, and the whole blood was collected in a 1.5 mL centrifuge tube and left to stand in a 37 °C water bath for 30 min. Centrifuge at 3500 rpm for 10 min, and aspirate the upper layer of serum and aliquot it into 200 μL centrifuge tubes. An ELISA kit was used to measure the expression levels of various cytokines (IL-1β, TNF-α, IL-6, IFN-γ and IL-10) in the serum.

[0081] It should be noted that, as shown in Figure 13 , intragastric administration of HNVs can effectively alleviate the up-regulation of the expression levels of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and IFN-γ in the serum caused by Clostridium difficile infection, and at the same time can significantly increase the expression level of the anti-inflammatory cytokine IL-10 in the serum, indicating that HNVs can effectively alleviate the body inflammation caused by Clostridium difficile infection.

[0082] Example 5 Biosafety detection of HNVs

[0083] The long-clawed gerbils were given sterile PBS or HNVs (200 mg / kg) by gavage at regular intervals for 8 consecutive days, and their weights were measured every 2 days. After 8 days of gavage, the hearts, livers, spleens, lungs, kidneys, colons, and sera of the long-clawed gerbils were collected. The changes in the body weights of the mice were recorded respectively; the colon length was measured; the liver index (liver index = liver weight mg / body weight g) and spleen index (spleen index = spleen weight mg / mouse body weight g) were calculated; the levels of liver and kidney function indicators in the sera were measured (AST: aspartate aminotransferase; ALT: alanine aminotransferase; CREA: creatinine; UREA: urea); and the tissue morphology of the heart, liver, spleen, lungs, kidneys, and colon was observed.

[0084] It should be noted that as shown in Figure 14 , gavage with HNVs (200 mg / kg) for 8 consecutive days did not cause significant changes in the body weight, colon length, liver index, and spleen index of the long-clawed gerbils, and at the same time did not cause significant changes in the levels of liver and kidney functions in the sera.

[0085] Furthermore, as shown in Figure 15 , gavage with HNVs (200 mg / kg) for 8 consecutive days did not cause changes in the tissue morphology of the heart, liver, spleen, lungs, kidneys, and colon of the long-clawed gerbils. The above content indicates that HNVs have good biosafety, which is a solid foundation for the application of HNVs in relieving Clostridioides difficile infection.

[0086] Although the present invention has been described in the above embodiments, it should be understood that the present invention can be further modified and changed without departing from the spirit of the present invention, and these modifications and changes are all within the protection scope of the present invention.

Claims

1. Application of nanovesicles derived from honeysuckle in the preparation of products for preventing, improving or treating diseases related to Clostridium difficile infection or in products for inhibiting the growth of Clostridium difficile.

2. The use of the honeysuckle-derived nanovesicles according to claim 1 in the preparation of products for preventing, improving or treating diseases related to Clostridium difficile infection or in products for inhibiting the growth of Clostridium difficile, characterized in that: The products include, but are not limited to, one or more of medicines, health foods, foods for special medical purposes, foods, functional foods, feeds, feed additives or pet foods.

3. The use of the honeysuckle-derived nanovesicles according to claim 1 in the preparation of products for preventing, improving or treating diseases related to Clostridium difficile infection or in products for inhibiting the growth of Clostridium difficile, characterized in that: Such diseases include, but are not limited to, antibiotic-associated diarrhea, colitis, recurrent Clostridium difficile infection, pyelonephritis, meningitis, abdominal and vaginal infections, bacteremia, and gas gangrene.

4. The use of the honeysuckle-derived nanovesicles according to claim 3 in the preparation of products for preventing, improving or treating diseases related to Clostridium difficile infection or in products for inhibiting the growth of Clostridium difficile, characterized in that: The disease is pseudomembranous colitis or fulminant enteritis.

5. Use of the honeysuckle-derived nanovesicles according to any one of claims 1 to 4 in the preparation of products for preventing, improving or treating diseases related to Clostridium difficile infection or in products for inhibiting the growth of Clostridium difficile, characterized in that: The honeysuckle-derived nanovesicles are separated and extracted from honeysuckle. The honeysuckle-derived nanovesicles have a uniform membrane-enclosed vesicle-like structure, and the average particle size of the nanovesicles is 83 nm.

6. Use of the honeysuckle-derived nanovesicles according to claim 5 in preparing products for preventing, improving or treating diseases related to Clostridium difficile infection or in products for inhibiting the growth of Clostridium difficile, characterized in that: The preparation of the honeysuckle-derived nanovesicles comprises the following steps: Step 1: Soak honeysuckle in sterile PBS to make a slurry, filter to remove impurities, and collect the juice; Step 2: The juice is extracted using differential centrifugation and tangential flow purification to obtain honeysuckle-derived nanovesicles.

7. A composition, medicine, health food, food for special medical purposes, food, functional food, feed, feed additive or pet food for preventing, improving or treating diseases related to Clostridium difficile infection or inhibiting the growth of Clostridium difficile, characterized in that: Including nanovesicles derived from honeysuckle.

8. The composition, medicine, health food, food for special medical purposes, food, functional food, feed, feed additive or pet food for preventing, improving or treating Clostridium difficile infection-related diseases or inhibiting the growth of Clostridium difficile according to claim 7, characterized in that: Such diseases include, but are not limited to, antibiotic-associated diarrhea, colitis, recurrent Clostridium difficile infection, pyelonephritis, meningitis, abdominal and vaginal infections, bacteremia, and gas gangrene.

9. The composition, medicine, health food, food for special medical purposes, food, functional food, feed, feed additive or pet food for preventing, improving or treating Clostridium difficile infection-related diseases or inhibiting the growth of Clostridium difficile according to claim 7, characterized in that: The honeysuckle-derived nanovesicles are separated and extracted from honeysuckle. The honeysuckle-derived nanovesicles have a uniform membrane-enclosed vesicle-like structure, and the average particle size of the nanovesicles is 83 nm.

10. The composition, medicine, health food, food for special medical purposes, food, functional food, feed, feed additive or pet food for preventing, improving or treating Clostridium difficile infection-related diseases or inhibiting the growth of Clostridium difficile according to claim 9, characterized in that: The preparation of the honeysuckle-derived nanovesicles comprises the following steps: Step 1: Soak honeysuckle in sterile PBS to make a slurry, filter to remove impurities, and collect the juice; Step 2: The juice is extracted using differential centrifugation and tangential flow purification to obtain honeysuckle-derived nanovesicles.

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