Taraxacum extract capable of neutralizing bacterial toxin and use thereof

Abstract

The application discloses a dandelion extract capable of neutralizing bacterial toxins and application thereof. The dandelion extract is mainly prepared by the following method: fresh dandelion juice is squeezed, the juice is collected, the juice is centrifuged, the supernatant is collected, the supernatant is ultra-filtered, and components capable of passing through a filter membrane with a molecular weight cut-off of 100 kD and incapable of passing through a filter membrane with a molecular weight cut-off of 3 kD are collected, namely the dandelion extract. The dandelion extract obtained by the application is natural in composition, free of toxic side effects and high in biological activity. Experimental evidence shows that the dandelion extract can protect host cells from attack by directly and specifically combining with toxins, effectively neutralizes the toxicity of bacterial toxins, and has effective prevention and treatment effects on diseases such as systemic bacterial infection, wound infection and bacterial pneumonia. Therefore, the dandelion extract can be used as a new type of anti-microbial and anti-infection functional material and applied to the fields of functional food, health products, medicines or functional cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial infection drug technology, specifically relating to a dandelion extract that can neutralize bacterial toxins and its application. Background Technology

[0002] Bacterial infections are one of the leading causes of death worldwide. Statistics show that in 2019, bacterial infections caused 7.7 million deaths globally, accounting for 13.6% of all deaths. Current strategies for dealing with challenging clinical bacterial infections primarily involve symptomatic treatment with antibiotics, antimicrobial agents, and hormones. However, these strategies suffer from significant side effects and drug resistance, while the development of new antibiotics is becoming increasingly difficult. Although vaccines promise to reduce the risk of infection, their development cycle and cost remain limited in addressing emerging and sudden public health emergencies. Furthermore, due to the rapid mutation rate of pathogens, effective vaccines are still lacking for most bacterial infections, including the most common Staphylococcus aureus. Therefore, there is an urgent need to find new alternative drugs to address the global health crisis caused by bacterial infections.

[0003] Studies show that bacterial toxins are the main virulence factors in the bacterial infection process, which can cause hemolysis of erythrocytes, immune escape, or tissue necrosis (Cohen TS et al., Cell Rep., 2018, 22:2431-2441). In severe cases, they can trigger cytokine storms, leading to organ failure and even death. Therefore, treatments targeting the virulence factors that cause host damage and disease are an alternative to antibacterial infection (“antiviral therapy”). This approach inhibits bacterial growth and proliferation by intervening in, modulating, or disrupting bacterial virulence rather than “directly” killing bacteria, thus avoiding bacterial damage to the host and reducing the likelihood of drug resistance (M. Sakari et al., ACS Infect Dis., 2022, 8:433-456). Currently reported antiviral therapy strategies include small molecule inhibitors, engineered antibodies or receptors, and biomimetic cell membrane preparations, which can bind to toxin molecules or toxin receptor molecules, preventing toxins from contacting and damaging target cells (Dal Peraro M et al., Nat Rev Microbiol. 2016, 14:77-92). However, the above methods require customized design for the specific conformation of the toxin molecule, resulting in complex preparation methods and high costs. Furthermore, patents have reported the use of artificial liposomes for neutralizing methicillin-resistant Staphylococcus aureus toxins (CN113041346A, CN115054577A, CN109549925A). However, simple artificial lipid membranes cannot fully mimic the composition, structure, and function of cell membranes, exhibiting limitations such as a narrow antitoxic spectrum, nonspecificity, weak binding affinity, and poor in vivo stability. In summary, developing novel drugs with high detoxification efficiency, high stability, and ease of production is particularly important for the detoxification of bacterial toxins and the fight against bacterial infections. Summary of the Invention

[0004] Objective of the Invention: To address the aforementioned technical problems, the objective of this invention is to provide a dandelion extract capable of neutralizing bacterial toxins and its applications. This dandelion extract is natural in composition, has no toxic side effects, exhibits high biological activity, and its preparation and production methods are simple and easy, showing promise for clinical translation.

[0005] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] A dandelion extract capable of neutralizing bacterial toxins is prepared primarily by the following method: juicing fresh dandelion, collecting the juice, centrifuging the juice, collecting the supernatant, ultrafiltration of the supernatant, and collecting the components that can pass through a 100kD molecular weight cutoff membrane but cannot pass through a 3kD molecular weight cutoff membrane, which is the dandelion extract.

[0007] The dandelion extract is a naturally derived exosome-like nanovesicle with a lipid membrane structure. It is rich in nucleic acids, proteins and lipids and appears as a saucer-shaped, cup-shaped or spherical shape under an electron microscope, with a particle size of about 100-200 nm.

[0008] The dandelion extract mentioned above contains a concentration of molecules with a neutralizing effect that is mainly retained by the filter membrane with a molecular weight cutoff of 3 to 100 kDa, while the molecules with a molecular weight cutoff of >100 kDa and <3 kDa are essentially ineffective.

[0009] As a specific implementation plan, the juice is centrifuged under the following conditions: at 0-4℃, first centrifuge with a centrifugal force of (3000-5000)g for 40-80 minutes, and then centrifuge with a centrifugal force of (8000-12000)g for 40-80 minutes.

[0010] As a specific implementation scheme, the method for ultrafiltration of the supernatant to collect components that can pass through a filter membrane with a molecular weight cutoff of 100kD but cannot pass through a filter membrane with a molecular weight cutoff of 3kD includes the following steps:

[0011] First, ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 100 kD, and the filtrate is collected. Then, the filtrate is ultrafiltered again using an ultrafiltration membrane with a molecular weight cutoff of 3 kD, and the retentate that does not pass through the ultrafiltration membrane is collected.

[0012] This invention also provides a method for preparing the dandelion extract capable of neutralizing bacterial toxins, comprising the following steps:

[0013] Fresh dandelion is juiced, the juice is collected, the juice is centrifuged, the supernatant is collected, the supernatant is ultrafiltered, and the components that can pass through a filter membrane with a molecular weight cutoff of 100kD and cannot pass through a filter membrane with a molecular weight cutoff of 3kD are collected, which is the dandelion extract.

[0014] The present invention also provides a pharmaceutical composition comprising the dandelion extract that can neutralize bacterial toxins.

[0015] As a specific implementation, the dosage form of the pharmaceutical composition is an injection, an inhalation preparation, an oral preparation, or a topical preparation.

[0016] This invention also provides the dandelion extract and the application of the pharmaceutical composition in the preparation of drugs that neutralize bacterial toxins. The specific neutralization mechanism of the dandelion extract is to protect host cells from attack through direct and specific binding to bacterial toxins. The toxins include endotoxins, exotoxins, and other toxic substances that can induce pathological reactions such as cell damage, rupture, inflammation, and tissue damage; the substances constituting the toxins include proteins, polypeptides, lipopolysaccharides, exosomes, vesicles, and combinations thereof.

[0017] Furthermore, the bacterial toxin is a bacterial endotoxin or a bacterial exotoxin; the bacterial toxin originates from Gram-positive or Gram-negative bacteria. More specifically, the bacterial toxin originates from Staphylococcus aureus or methicillin-resistant Staphylococcus aureus, etc.

[0018] Finally, this invention provides the use of the dandelion extract and the pharmaceutical composition thereof in the preparation of remedies for the prevention and / or treatment of bacterial infections, bacterial pneumonia, or skin wound infections. When used as a medicine, the dandelion extract or pharmaceutical composition thereof of this invention can be administered to treat and / or prevent diseases or symptoms via any suitable dosage form and route of administration. Routes of administration include nasal, oral mucosal, oral, inhalation, intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, skin, or topical application to wounds.

[0019] Beneficial Effects: The dandelion extract of this invention is obtained by separating fresh dandelion juice through differential centrifugation and ultrafiltration, a simple preparation process. It is natural, non-toxic, and has high bioactivity, making it a novel antimicrobial and anti-infective agent applicable to functional foods, health products, pharmaceuticals, and cosmetics. Experimental evidence shows that this dandelion extract can protect host cells from attack by directly and specifically binding to toxins, effectively neutralizing the toxicity of bacterial toxins, and has effective preventative / therapeutic effects on systemic bacterial infections, wound infections, and bacterial pneumonia. This invention holds promise for development into a natural plant-derived antitoxin preparation, which is of great significance for the prevention and treatment of clinically relevant infectious diseases and for reducing antibiotic resistance. Attached Figure Description

[0020] Figure 1 The particle size, concentration, morphology, and chemical composition of TH-EVNs are shown.

[0021] Figure 2Evaluation of the antihemolytic activity of different isolated components from fresh dandelion.

[0022] Figure 3 The composition and antihemolytic activity of extracts from fresh dandelion juice, decoction, and decoction of dried dandelion were evaluated.

[0023] Figure 4 The effect of different media on the anti-hemolytic ability of TH-EVNs.

[0024] Figure 5 To evaluate the anti-hemolytic ability of different components in TH-EVNs.

[0025] Figure 6 To investigate the neutralization mechanism of TH-EVNs.

[0026] Figure 7 To evaluate the efficacy of subcutaneous injection of TH-EVNs in neutralizing S. aureus exotoxin.

[0027] Figure 8 The intervention effect of intraperitoneal injection of TH-EVNs on systemic infection of S. aureus.

[0028] Figure 9 Characterization of TH-EVNs@GelMA.

[0029] Figure 10 TH-EVNs@GelMA promote wound healing from S. aureus exotoxin infection.

[0030] Figure 11 To investigate the intervention effect of nebulized TH-EVNs on S. aureus-infected pneumonia mice. Detailed Implementation

[0031] The invention is further illustrated by the following embodiments. These embodiments are purely illustrative and are used only to specifically describe the invention, and should not be construed as limiting the invention. The invention is further described below with reference to the accompanying drawings and embodiments:

[0032] (1) Dandelion extract (TH-EVNs) was prepared by differential centrifugation-ultrafiltration and its physicochemical properties were characterized. The particle size and concentration were determined by nanoparticle tracking analyzer (NTA), and its morphology and size were observed by scanning electron microscopy (SEM). The lipid, protein and RNA components were analyzed and their componentomics were studied.

[0033] (2) The chemical composition and bacterial toxin neutralization activity of different extracts of fresh dandelion and dried dandelion were compared by HPLC and in vitro hemolysis experiments.

[0034] (3) The toxin neutralization capacity of TH-EVNs (3-100kD) was compared with that of other isolated components (>100kD and <3kD) and different components in TH-EVNs by in vitro hemolysis experiments, and the effects of different physiological media on the toxin neutralization capacity of TH-EVNs were investigated.

[0035] (4) The mechanism by which TH-EVNs neutralize toxins was investigated using techniques such as micro thermophoresis (MST) and nanoparticle tracking analysis (NTA).

[0036] (5) Evaluation of antitoxin treatment in a local bacterial infection model of the skin by subcutaneous injection of TH-EVNs.

[0037] (6) Evaluation of antitoxin treatment in bacterial peritonitis and systemic bacterial infection models by intraperitoneal injection of TH-EVNs.

[0038] (7) A hydrogel formulation loaded with TH-EVNs (TH-EVNs@GelMA) was prepared by photocuring.

[0039] (8) The antitoxin treatment effect of TH-EVNs@GelMA in a wound infection model was investigated.

[0040] (9) Evaluate the efficacy of TH-EVNs in preventing and treating bacterial pneumonia by nebulized inhalation.

[0041] In this embodiment, exotoxins secreted by Staphylococcus aureus (S. aureus) were selected as representatives to explore the neutralizing and scavenging effect of fresh dandelion extract (TH-EVNs) on S. aureus exotoxins (S. aureus EVs).

[0042] Example 1: Preparation and physicochemical characterization of fresh dandelion extract (TH-EVNs)

[0043] (1) Preparation of TH-EVNs

[0044] Juice 50g of fresh dandelion, centrifuge the juice at 4000g and 4℃ for 60min, collect the supernatant and centrifuge again at 10000g and 4℃ for 60min, and collect the supernatant; first ultrafilter it with a 100kD molecular weight ultrafiltration membrane, the retentate with >100kD is called Rententate, collect the filtrate, and then concentrate it with a 3kD ultrafiltration membrane, the filtrate with <3kD is called Filtrate, the obtained retentate is the fresh dandelion extract (TH-EVNs).

[0045] (2) Physicochemical characterization of TH-EVNs

[0046] The separated TH-EVNs were diluted to an appropriate concentration with 1×PBS, and the particle size and concentration of TH-EVNs were determined using NTA. TH-EVNs were fixed with 2.5% glutaraldehyde and 1% tannic acid for 2 h, and then diluted with ultrapure water at a ratio of 1:10 (v / v). 5 μL of the solution was pipetted onto the sample stage, allowed to dry naturally, sputtered to coat with gold, and the morphology of TH-EVNs was observed under SEM.

[0047] (3) Composition analysis of TH-EVNs

[0048] RNA was extracted from TH-EVNs using the Trizol Total RNA Extraction Kit and developed on 1% agarose gels. Proteins in TH-EVNs were denatured by adding 5×SDS-PAGE loading buffer and incubated at 100°C for 10 min. Samples were then developed on 12% SDS-PAGE gels and silver-stained. Lipids were extracted from TH-EVNs using the Folch method and stained with 10% CuSO4 in 8% phosphoric acid on silica gel thin-layer chromatography (TLC) plates using chloroform / methanol / acetic acid (190:9:1) as the developing solvent. Further lipidomics, proteomics, and small RNA (sRNA) sequencing analyses were performed on TH-EVNs.

[0049] like Figure 1 As shown in Figure A, the average particle size of TH-EVNs is 187 nm, and the particle number is 5.75 × 10⁻⁶. 11 The cells / mL were found to be spherical under electron microscopy. Component analysis revealed that TH-EVNs contained lipids, RNAs, and proteins. The RNAs were primarily small RNAs (…). Figure 1 B), can be degraded by RNase; the protein is mainly distributed in the 10-70 kDa range ( Figure 1 C); TLC analysis showed that TH-EVNs contained a variety of lipids ( Figure 1 D). Further omics analysis identified a total of 112 proteins and 353 lipids. Figure 1 RNAs with a length of 21-27 nucleotides (nt) were the most abundant (E, G). Figure 1 F).

[0050] Example 2: Evaluation of the in vitro neutralization capacity of different isolated components of fresh dandelion against bacterial toxins

[0051] The in vitro neutralization capacity of different isolated fractions of fresh dandelion, namely TH-EVNs (3-100kD), >100kD retentate, and <3kD filtrate, against *S. aureus* EVs was evaluated using an in vitro hemolysis assay. The specific procedure was as follows: 20 μL of *S. aureus* EVs were incubated with 100 μL of each isolated fraction at 37°C for 1 h, followed by incubation with 100 μL of 2% fresh mouse red blood cells for another 1 h. PBS and Triton X-100 were used as negative and positive controls, respectively. OD was measured using a microplate reader. 543 The relative hemolysis rate is calculated using the following formula:

[0052]

[0053] The results showed that the isolated fractions from fresh dandelion in the 3–100 kDa range, namely TH-EVNs, significantly inhibited S. aureus EV-induced hemolysis, reducing the hemolysis rate from 80% to 12%. Other isolated fractions (>100 kDa or <3 kDa) did not exhibit sufficient anti-hemolytic activity (hemolysis rate >60%), indicating that TH-EVNs are the main antitoxin. Figure 2 ).

[0054] Example 3: Effects of dried and fresh dandelion and the decoction process on their ability to combat bacterial toxins

[0055] Currently, dandelion is mostly used in medicine as dried medicinal slices, which are then decocted. To evaluate the effects of fresh and dried dandelion and the decoction process on the composition and efficacy of the extract, the following experiment was designed: ① The preparation steps of fresh dandelion extract (TH-EVNs, named fTH-EVNs in this example for easy differentiation from other groups) are as follows: 50g of fresh dandelion is juiced, and the juice is centrifuged at 4000g and 4℃ for 60min. The supernatant is collected and then centrifuged at 10000g and 4℃ for 60min. The supernatant is collected. First, it is ultrafiltered using a 100kD molecular weight ultrafiltration membrane, and the filtrate is collected and then concentrated to 50mL using a 3kD ultrafiltration membrane. ② The preparation steps of fresh dandelion decoction (hfTH-EVNs) are as follows: Take 50g of fresh dandelion, add 300mL of water and boil over high heat, then simmer over low heat for 1 hour. Filter the filtrate through gauze. Add another 200mL of water and boil over high heat, then simmer over low heat for 1 hour. Filter the decoction through gauze. Combine the two filtrates, concentrate them using a rotary evaporator, and centrifuge at 4000g and 4℃ for 60min. Collect the supernatant and centrifuge at 10000g and 4℃ for 60min. Take the supernatant and pass it through 100kD and 3kDa ultrafiltration membranes in sequence. Take 50mL of the 3-100kD fraction. ③ Preparation steps of dried dandelion decoction (hdTH-EVNs): Take 50g of fresh dandelion, dry it, add 300mL of water, bring to a boil over high heat, then simmer over low heat for 1 hour, filter the filtrate through gauze; add another 200mL of water, bring to a boil over high heat, then simmer over low heat for 1 hour, filter the decoction through gauze, combine the two filtrates, concentrate them using a rotary evaporator, centrifuge at 4000g and 4℃ for 60min, collect the supernatant, centrifuge at 10000g and 4℃ for 60min, take the supernatant; pass it through 100kD and 3kDa ultrafiltration membranes in sequence, and take 50mL of the 3-100kD range fraction.

[0056] High-performance liquid chromatography (HPLC) analysis revealed significant differences in the chemical components obtained from different extraction methods among various dandelion extracts. Figure 3 A); Hemolysis test results showed that the ability of fresh dandelion extract to neutralize toxins (anti-hemolytic effect) was significantly better than that of fresh dandelion decoction and dried dandelion decoction. Figure 3 BC).

[0057] Example 4: Effects of different media on the toxin neutralization capacity of fresh dandelion TH-EVNs bacteria

[0058] The effects of different physiological mediators on the neutralizing capacity of TH-EVNs were investigated. TH-EVNs (5.0 × 10⁻⁶) were used. 10 The NPs / mL were diluted to 5.0 × 10⁻⁶ with different concentrations of physiological media (140, 150 μM NaCl; 0.5%, 1%, 5%, 10% FBS; 5%, 10%, 20%, 50% mouse plasma). 9 NPs / mL, incubated at 37℃ for 30 min. Results showed that physiological saline ( Figure 4A) and fetal bovine serum (FBS) Figure 4 B) It had no effect on TH-EVNs activity; even high-concentration plasma (50%) did not reduce the hemolytic inhibitory ability of TH-EVNs. Figure 4 C). These results indicate that TH-EVNs exhibit strong anti-interference ability in their neutralizing activity.

[0059] Example 5: Study on the active ingredients in fresh dandelion TH-EVNs

[0060] To investigate the roles of different components in TH-EVNs in anti-hemolysis, total lipids were first extracted from TH-EVNs using the Folch method. The specific extraction steps were as follows: TH-EVNs were taken and added to a methanol-chloroform (2:1, v / v) mixture (3.75 times the volume of TH-EVNs), vortexed, and mixed thoroughly. Then, equal volumes of chloroform and deionized water were added separately and mixed thoroughly. The mixture was centrifuged at 2000 rpm for 10 min, and the lower organic layer was placed in a rotary evaporator to remove the solvent. The remaining solvent was then dried in a fume hood to obtain the total lipids from the TH-EVNs. Hemolysis experiments were then performed.

[0061] Next, to investigate the roles of RNAs and proteins in TH-EVNs in anti-hemolysis, RNase A and proteinase K were used to enzymatically digest the RNAs and proteins in TH-EVNs, respectively. 1 mL of TH-EVNs (3.69 × 10⁻⁶) was taken from each sample. 11 TH-EVNs were incubated in 1.5 mL epoxide tubes with 100 mM RNase K (50 μg / mL) for 2 hours at 37°C. Then, the following steps were performed: ① No treatment was performed, and the tubes were incubated at 37°C for 2 hours. ② 20 mg / mL of proteinase K was added to achieve a final concentration of 50 μg / mL, and the tubes were incubated at 37°C for 2 hours. Then, 100 mM PMSF was added to achieve a final concentration of 1 mM, and the reaction was terminated. ③ 10 mg / mL of RNase A was added to achieve a final concentration of 50 μg / mL, and the tubes were incubated at 37°C for 2 hours. ④ 50 μg / mL of RNase A was added, and the tubes were incubated at 37°C for 2 hours. Then, 50 μg / mL of proteinase K was added, and the tubes were incubated at 37°C for 2 hours. Then, 1 mM PMSF was added to terminate the reaction. Then, 1 mL of 1×PBS was used instead of TH-EVNs, and the same procedure was performed. All samples were diluted 20-fold with 1×PBS after treatment to achieve a TH-EVNs particle concentration of 1.84 × 10⁻⁶. 10 per mL. Perform a hemolysis test on the diluted sample.

[0062] The results showed that TH-EVN lipids alone had no anti-hemolytic effect, and the hemolysis rate was almost the same as that of the S. aureus EVs group. Figure 5A). RNase A or proteinase K themselves did not affect the hemolytic activity of S. aureus EVs, with no significant difference; and incubating them alone with erythrocytes did not induce hemolysis of erythrocytes. Figure 5 B). Based on this, when TH-EVNs were treated with RNase A, their anti-hemolytic ability remained almost unchanged, with hemolysis rates consistently around 3.6% (p>0.05); however, when TH-EVNs were treated with proteinase K or simultaneously with proteinase K and RNase A, the hemolysis rate significantly increased, rising from 3.76% to 13.84% (p=0.0103) and 21.01% (p=0.0275), respectively. This indicates that protein is its effective component. Figure 5 B).

[0063] Example 6: Study on the mechanism of TH-EVNs neutralizing bacterial toxins

[0064] The inhibition of hemolysis by TH-EVNs by *S. aureus* EVs may be due to three mechanisms: (1) simple physical barrier reducing the probability of *S. aureus* EVs attacking erythrocytes; (2) binding to erythrocytes to form a protective barrier against attack by *S. aureus* EVs; and (3) directly acting on *S. aureus* EVs to render them unable to attack erythrocytes. These mechanisms were verified separately.

[0065] (1) Since the physical barrier effect is related to particle size and concentration, extracts from three other different fresh plants were screened. The extraction method was as follows: fresh plants were juiced, the juice was collected, the juice was centrifuged, the supernatant was collected, the supernatant was ultrafiltered, and the fractions that could pass through a 100kD molecular weight cutoff filter membrane and could not pass through a 3kD molecular weight cutoff filter membrane were collected. The results showed that Artemisia annua extract (AB-EVNs, 177.9±5.5nm) and Portulaca oleracea extract (PL-EVNs, 177.4±5.2nm) had similar particle sizes to TH-EVNs (187.2±1.9nm), while grapefruit extract (CM-EVNs) had smaller particle sizes, with a particle size of 95.2±2.3nm. Figure 6 A).

[0066] The four extracts were diluted to the same concentration (5 × 10⁻⁶). 9 Hemolysis tests were conducted using samples (number of samples per mL). The results showed that fresh artemisia annua extract and TH-EVNs had better anti-hemolytic effects, with hemolysis rates below 5%; while fresh purslane extract and grapefruit extract had weaker anti-hemolytic abilities, with hemolysis rates exceeding 65%. Figure 6 B) indicates that particle size and concentration are not directly related to the hemolytic effect, thus ruling out the anti-hemolytic effect caused by physical barriers.

[0067] (2) We further verified whether TH-EVNs would bind to erythrocytes, thus reducing the chance of *S. aureus* EVs coming into contact with erythrocytes. TH-EVNs were labeled with FITC, and 100 μL of the labeled TH-EVNs was incubated with 100 μL of erythrocytes for 1 h. The erythrocytes were then centrifuged to precipitate, and the fluorescence intensity in the supernatant was measured. The results showed no significant change in fluorescence intensity in the supernatant. Figure 6 C) indicates that TH-EVNs do not bind to erythrocytes and thus prevent S. aureus EVs from contacting erythrocytes.

[0068] (3) We used MST to verify whether TH-EVNs directly interact with S. aureus EVs. FITC-labeled S. aureus EVs (4.9 × 10⁻⁶) were used. -3 Adding different concentrations (4.7 × 10 nmol / L) -5 In TH-EVNs containing -1.55 nmol / L, the laser start-up and shut-off times were set to 35 and 5 s, respectively, and the MST curves were recorded. The dissociation constants (Kaureus) of TH-EVNs and S. aureus EVs were calculated using NT analysis software. d The results show that TH-EVNs and S. aureus EVs have a strong binding force, and their equilibrium dissociation constant K d The calculated value is 0.78 ± 0.188 nmol / L. Figure 6 (DE). Therefore, analysis of the above results leads to the conclusion that TH-EVNs can mediate the anti-hemolytic effect of TH-EVNs by directly binding to S. aureus EVs, preventing S. aureus EVs from contacting erythrocytes.

[0069] Example 7: Subcutaneous injection of TH-EVNs for the prevention and treatment of local infection in S. aureus EVs

[0070] A mouse model of S. aureus EV infection was established by subcutaneous injection of S. aureus EVs. Before the experiment, male ICR mice (22-25g) were acclimatized for one week and then randomly divided into four groups of five mice each.

[0071] Model group: 50 μL S. aureus EVs (1 × 10⁻⁶) 9 The solution (number per mL) was diluted with 100 μL of PBS and injected subcutaneously into the hind limbs of mice.

[0072] Treatment group: 100 μL of TH-EVNs (1×10⁻⁶) was administered. 9 (cells / mL) and 50 μL S. aureus EVs (1×10⁻⁶) 9After pre-incubating at 37°C for 30 min, the mice were subcutaneously injected into their hind limbs.

[0073] Control group: 150 μL of PBS or TH-EVNs (1×10⁻⁶) were injected at the same site. 9 (units / mL).

[0074] The appearance of the subcutaneous injection area was observed by taking photographs, and the damage area was quantified using ImageJ. The results are as follows: Figure 7 As shown in AB, the S. aureus EVs model group showed severe skin damage, while the PBS control group and the TH-EVNs treatment group showed no significant skin damage. ELISA results showed ( Figure 7 CE), the TH-EVNs treatment group significantly inhibited the expression of inflammatory factors TNF-α, IL-6, and IL-1β in the subcutaneous injection area. H&E and TUNEL staining results of tissue sections showed ( Figure 7 In the *S. aureus* EV model group, significant inflammatory infiltration, necrosis, and extensive apoptosis were observed in the skin and muscle tissue. In contrast, the control group and the TH-EVNs treatment group showed normal epithelial structure and no significant apoptosis. These results indicate that TH-EVNs can effectively neutralize *S. aureus* EVs and reduce their virulence.

[0075] Example 8: Intraperitoneal injection of TH-EVNs for the prevention and treatment of systemic infection of S. aureus

[0076] A systemic infection model of S. aureus was established in mice by intraperitoneal injection. Before the experiment, male ICR mice (22-25g) were acclimatized for one week and then randomly divided into four groups of seven mice each.

[0077] Control group: Intraperitoneal injection of sterile PBS 0.1mL / 10g for 3 consecutive days, with sterile PBS injected 30 minutes after the third day.

[0078] Model group: Intraperitoneal injection of sterile PBS 0.1mL / 10g for 3 consecutive days, followed by injection of S. aureus bacterial solution 0.1mL / 10g 30min after the third day.

[0079] TH-EVNs group: intraperitoneal injection of 10 for 3 consecutive days 10 0.1 mL / 10 g of TH-EVNs were administered, followed by an intraperitoneal injection of 0.1 mL / 10 g of S. aureus bacterial solution 30 minutes after the injection on the third day.

[0080] Dexamethasone (DEX) group: DEX 5mg / kg was injected intraperitoneally for 3 consecutive days. On the third day, 0.1mL / 10g of S. aureus bacterial solution was injected intraperitoneally 30 minutes after the injection.

[0081] ELISA results showed that the levels of inflammatory factors rapidly increased in the S. aureus infection group. TH-EVNs had a strong inhibitory effect on the levels of IL-1β, IL-6, and TNF-α in peritoneal fluid and serum, and the effect was superior to that of the positive drug dexamethasone group. Figure 8 A). Q-PCR results showed that the expression levels of NLRP3 inflammasome-related mRNAs (IL-1β, TNF-α, IL-6, ASC, NLRP3, and IL-18) in the lung tissue of mice infected with S. aureus were significantly increased, while the levels of related mRNAs in mice treated with TH-EVNs were decreased to varying degrees. Figure 8 B). Western blot analysis of relevant protein expression levels showed that infection with *S. aureus* in mice increased NLRP3 expression and caspase-1 activation in the lungs. TH-EVNs treatment inhibited caspase-1 activation and suppressed NLRP3 inflammasome activation in the lungs. Figure 8 C). Tissue plate smear and Gram staining results showed that the bacterial load in the peritoneal fluid and lung tissue of mice in the TH-EVNs treatment group was significantly reduced compared with the model group, while the positive control drug DEX had no such effect. Figure 8 DF). H&E staining analysis of the lungs showed that after S. aureus infection, the lung tissue exhibited significant inflammatory cell infiltration and alveolar wall thickening; the TH-EVNs treatment group showed reduced inflammatory cell infiltration and decreased alveolar wall thickness compared to the model group, demonstrating superior efficacy compared to DEX ( ). Figure 8 G). The above results indicate that intraperitoneal injection of TH-EVNs can prevent systemic infection of S. aureus.

[0082] Example 9: Preparation and characterization of TH-EVNs hydrogels

[0083] The preparation method of TH-EVNs hydrogel is as follows: Weigh an appropriate amount of gelatin into a beaker, add 1×PBS, heat in a water bath at 50℃ and stir until the gelatin is completely dissolved to prepare a 10% (w / v) solution. Then, slowly add 0.1 mL of MA to 10 mL of Gelatin solution (10%, w / v), stir continuously for 2 h, add 40 mL of 1×PBS dilution solution to terminate the reaction. Dialyze the diluted solution in deionized water for 36 h using an 8-14 kDa dialysis bag to remove unreacted MA. Freeze-dry the dialyzed solution to obtain white solid GelMA, and store the product at -20℃ for later use. Weigh 1 g of freeze-dried GelMA with a degree of substitution of 20%, dissolve it in 10 mL of PBS, add TH-EVNs and mix to make a final concentration of 10%. 9TH-EVNs were added at a concentration of 10 g / mL. Photoinitiator 2959 (0.5%, w / v) was added, and the mixture was irradiated at 365 nm for 40 s using a ZF-20D darkroom UV analyzer to obtain GelMA hydrogel loaded with TH-EVNs (TH-EVNs@GelMA). 1 g of Gelatin was weighed, dissolved in 10 mL of PBS, and TH-EVNs were added and mixed to achieve a final concentration of 10 g / mL. 9 The gelatin hydrogel loaded with TH-EVNs (TH-EVNs@Gelatin) was prepared by cooling at room temperature for 30 min with a density of 1 / mL.

[0084] The mechanical properties of TH-EVNs@GelMA were analyzed and characterized using a rotational rheometer on a parallel rough plate with a diameter of 50 mm. Mechanical spectra were recorded in a constant strain mode, maintaining a low deformation of 0.01 rad / s within an angular frequency range of 0.1–100 rad / s at 25 °C. The temperature dependence of gel storage energy and loss modulus was determined by oscillatory shear deformation (dynamic rheological observation) at constant angular frequency (10 rad / s) and constant shear strain (γ = 0.1), with a temperature scan range of 25–45 °C (heating rate 3 °C / min). Irradiation was performed at a constant angular frequency (5 rad / s) and constant shear strain (γ = 0.05) using a UV 365 nm source at 70 MW / cm². 2 The storage modulus and loss modulus of the gel were tested as a function of light irradiation. Rheological results showed that after 30 seconds of UV irradiation, the storage modulus (G') of GelMA was significantly higher than the loss modulus (G”), confirming the rapid photocuring properties of GelMA. Figure 9 A).

[0085] Lyophilized TH-EVNs@GelMA were longitudinally sectioned, sputter-coated with gold, and then directly observed using SEM. The pore size of ten pores was randomly measured using ImageJ software, and the mean pore size and variance were calculated. SEM images showed that the gel formed after adding TH-EVNs to GelMA had a denser and slightly coarser porous structure. Figure 9 B), the pore size of TH-EVNs@GelMA is 124.2±13.1μm.

[0086] Millicell insert cell culture dishes were used to assess the in vitro release of TH-EVNs. Simply put, FITC-labeled TH-EVNs@GelMA and TH-EVNs@Gelatin were added to the upper chamber (0.4 μm) of a Transwell plate, and 1.5 mL of PBS (pH 7.4) was added to the lower chamber of each plate. The plate was placed in a temperature-controlled shaker at 32°C and 100 rpm. At predetermined time points, the release medium from the lower chamber was aspirated, and quantitative analysis was performed using a microplate reader at 488 nm. The release of TH-EVNs from TH-EVNs@GelMA was as follows: Figure 9 As shown in Figure C, compared with TH-EVNs@Gelatin, the release curve of GelMA (DS = 20%) is more sustained and stable, indicating that GelMA can achieve sustained release of TH-EVNs.

[0087] Example 10: TH-EVNs hydrogel for treating wounds infected with S. aureus EVs

[0088] A mouse model of S. aureus EV infection was established by instilling S. aureus EVs into the skin. Male ICR mice (22-25g) were acclimatized for one week prior to the experiment, and their backs were shaved one day in advance. After anesthetizing with 4% chloral hydrate, two full-thickness skin defects (6mm in diameter) were created on the backs of the mice using a punch. 30μL of S. aureus EVs were instilled into the wounds, and the mice were then randomly divided into four groups of eight each:

[0089] Control group: 30 μL of sterile PBS was infused into the wound.

[0090] GelMA group: 30 μL of GelMA hydrogel was cross-linked with ultraviolet light in vitro for 40 seconds and then placed on the wound of mice.

[0091] TH-EVNs@GelMA group: 30 μL of TH-EVNs@GelMA was cross-linked with ultraviolet light in vitro for 40 seconds and then placed on the wound of mice.

[0092] TH-EVNs group: 30 μL TH-EVNs (1×10⁻⁶) were infused into the wound. 9 (units / mL).

[0093] After administration, the wound was covered with a sterile dressing. Mice were housed separately and the dressing was changed every 3 days.

[0094] The wound was photographed on days 0, 3, 7, 9, 12, and 15, and the wound area was measured using ImageJ image analysis software to calculate the wound healing rate.

[0095] Wound healing rate = (area) 第0天 -area特定天数 ) / area 第0天 ×100

[0096] The results are as follows Figure 10 As shown in A and B, the TH-EVNs@GelMA treatment group healed the fastest. Figure 10 CE showed that GelMA, TH-EVNs@GelMA, and TH-EVNs all reduced the expression of TNF-α, IL-6, and IL-1β mRNA in skin wounds, with TH-EVNs@GelMA showing the best effect. Furthermore, TH-EVNs@GelMA also promoted the expression of IL-10 mRNA, a marker of M2 macrophages. Figure 10 F) and VEGF mRNA expression in the wound ( Figure 10 G). H&E staining and Masson staining results showed that, compared with the control group, the TH-EVNs@GelMA group had no obvious inflammatory response, more complete epithelial regeneration, mature collagen matrix and good tissue in the wound, and the effect was better than that of the GelMA and TH-EVNs groups. Figure 8 These results indicate that TH-EVNs@GelMA can effectively promote wound healing.

[0097] Example 11: Nebulized administration of TH-EVNs for the treatment of pneumonia caused by S. aureus infection

[0098] A mouse model of bacterial pneumonia was established using intratracheal instillation of *S. aureus*. Before the experiment, male ICR mice (22-25g) were acclimatized for one week and randomly divided into four groups of six mice each:

[0099] Control group: 30 μL of normal saline was administered via nebulization into the trachea for 3 consecutive days. 2 hours after nebulization on the third day, 30 μL of normal saline was instilled into the trachea.

[0100] Model group: 30 μL of physiological saline was administered via intratracheal nebulization for three consecutive days. Two hours after nebulization on the third day, 30 μL of 2.2 × 10⁻⁶ mg / L of saline solution was infused intratracheally. 8 Staphylococcus aureus suspension at CFUs / mL.

[0101] TH-EVNs group: 30 μL of TH-EVNs (5 × 10⁻⁶) was administered via intranebulization for 3 consecutive days. 9 (number / mL), 2 hours after nebulization on the third day, 30 μL of 2.2 × 10⁻⁶ nitroglycerin was infused into the trachea. 8 Staphylococcus aureus suspension at CFUs / mL.

[0102] Dexamethasone (DEX) group: Dexamethasone (5 mg / kg) was administered intraperitoneally for 3 consecutive days. Two hours after the intraperitoneal injection on the third day, 30 μL of 2.2 × 10⁻⁶ mg / kg was infused intratracheally. 8Staphylococcus aureus suspension at CFUs / mL.

[0103] All mice in each group were sacrificed 6 hours after modeling and their samples were collected.

[0104] The results of lung imaging in mice showed that after infection with S. aureus, the lungs of mice were enlarged and congested, appearing dark red and showing a solid texture. In contrast, the lungs of the TH-EVNs nebulized inhalation group and the DEX group showed normal color, appearing light pink, with less lung tissue damage. The TH-EVNs nebulized inhalation group showed better results. Figure 11 A). Tissue plate smear, colony count, and Gram staining results showed that TH-EVN nebulized inhalation therapy could reduce the number of colonies in lung tissue and alleviate S. aureus infection in lung tissue, while DEX treatment had no significant inhibitory effect on colony growth in lung tissue. Figure 11 BD). Q-PCR ( Figure 11 EG) and ELISA Figure 11 The results (HJ) showed that TH-EVN nebulization significantly reduced the expression of pulmonary inflammatory factors TNF-α, IL-6, and IL-1β, with better effects than the DEX group. The total protein concentration in bronchoalveolar lavage fluid showed that the model group exhibited significant protein exudation; TH-EVN nebulization and DEX treatment alleviated S. aureus infection-induced protein exudation and significantly reduced the protein concentration in bronchoalveolar lavage fluid. Figure 11 Histological results showed that after TH-EVNs nebulization inhalation treatment, the lung lesions in S. aureus-infected mice were alleviated, the inflammatory cell infiltration in the alveolar cavity was significantly reduced, and the alveolar structure was relatively intact. The therapeutic effect of TH-EVNs nebulization inhalation was superior to that of DEX (K). Figure 11 L).

Claims

1. A dandelion extract capable of neutralizing bacterial toxins, characterized in that, The dandelion extract is mainly prepared by the following method: fresh dandelion is squeezed to collect juice, the juice is centrifuged to collect supernatant, the supernatant is ultrafiltrated to collect components capable of passing through a filter membrane with a molecular weight cut-off of 100 kD and incapable of passing through a filter membrane with a molecular weight cut-off of 3 kD, and the components are the dandelion extract; the juice is centrifuged under the following conditions: first, 3000-5000g centrifugal force is used for centrifugation at 0-4 ℃ for 40-80 minutes, and then 8000-12000g centrifugal force is used for centrifugation for 40-80 minutes; and the dandelion extract is an exosome-like nanovesicle of dandelion.

2. The dandelion extract capable of neutralizing bacterial toxin according to claim 1, characterized by, The method for ultrafiltrating the supernatant to collect components capable of passing through a filter membrane with a molecular weight cut-off of 100 kD and incapable of passing through a filter membrane with a molecular weight cut-off of 3 kD comprises the following steps: The method comprises the following steps:

3. The method of producing a dandelion extract capable of neutralizing bacterial toxins according to any one of claims 1 to 2, characterized in that, The method comprises the following steps: The dandelion extract is mainly prepared by the following method: fresh dandelion is squeezed to collect juice, the juice is centrifuged to collect supernatant, the supernatant is ultrafiltrated to collect components capable of passing through a filter membrane with a molecular weight cut-off of 100 kD and incapable of passing through a filter membrane with a molecular weight cut-off of 3 kD, and the components are the dandelion extract; the juice is centrifuged under the following conditions: first, 3000-5000g centrifugal force is used for centrifugation at 0-4 ℃ for 40-80 minutes, and then 8000-12000g centrifugal force is used for centrifugation for 40-80 minutes; and the dandelion extract is an exosome-like nanovesicle of dandelion.

4. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the dandelion extract capable of neutralizing bacterial toxins according to any one of claims 1-2.

5. The pharmaceutical composition of claim 4, wherein, The dosage form of the pharmaceutical composition is injection, inhalation preparation, oral preparation or external preparation.

6. Use of the dandelion extract according to any one of claims 1-2 or the pharmaceutical composition according to claim 4 or 5 in the preparation of a drug for neutralizing bacterial toxins.

7. Use according to claim 6, characterized in that, The bacterial toxin is bacterial endotoxin or bacterial exotoxin; and the bacterial toxin is derived from gram-positive bacteria or gram-negative bacteria.

8. Use according to claim 6, characterized in that, The bacterial toxin is derived from Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

9. Use of the dandelion extract according to any one of claims 1-2 or the pharmaceutical composition according to claim 5 or 6 in the preparation of a drug for preventing and / or treating bacterial infection, bacterial pneumonia or skin wound infection.

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