Preparation method of eugenol-loaded quercetin-zinc nano-enzyme for treating livestock enteritis

Abstract

The invention discloses a preparation method of eugenol-loaded quercetin-zinc nano-enzyme for treating enteritis of livestock. The nano-enzyme is constructed by forming nano-enzyme particles by virtue of coordination of quercetin and zinc ions and loading eugenol by virtue of metal-hydroxyl interaction, the nano-enzyme has the high-efficiency antioxidant activity of quercetin-zinc nano-enzyme and the anti-inflammatory and antibacterial functions of eugenol, and can synergistically inhibit pathogenic bacteria in intestinal tracts, remove excessive reactive oxygen species (ROS) at intestinal inflammation parts and promote the recovery of the intestinal tracts. The nano-enzyme is good in biocompatibility and high in stability, can slowly release active ingredients in an intestinal environment, is lasting in effect, can effectively relieve symptoms of bacterial enteritis and promote intestinal tissue repair, provides a novel efficient treatment strategy for treatment of bacterial enteritis, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of veterinary biomedicine technology, specifically to a method for preparing quercetin-zinc nanoenzymes loaded with eugenol, and particularly to a method for preparing eugenol-loaded quercetin-zinc nanoenzymes for treating bacterial enteritis in livestock. Background Technology

[0002] Diarrhea-associated enteritis (DAD) is a common digestive tract disease in livestock and poultry farming. It is often caused by bacterial infection, feed mutations, stress, intestinal microecological imbalance, and oxidative stress. It has a rapid onset, spreads quickly, and has a high mortality rate, leading to stunted growth and reduced feed utilization, causing serious economic losses to the livestock industry. The core pathogenesis of this disease is oxidative stress and intestinal flora imbalance. Pathogens or stress stimulation cause the intestine to produce large amounts of reactive oxygen species (ROS), damaging the intestinal mucosal barrier and inducing inflammation. Simultaneously, pathogenic bacteria proliferate and form biofilms, exacerbating the flora imbalance, ultimately creating a vicious cycle of oxidative stress—inflammation—floral flora imbalance, further aggravating diarrhea and intestinal damage.

[0003] Currently, in the livestock industry, the treatment of diarrhea-related enteritis in livestock still primarily relies on antibiotics and chemical antidiarrheal drugs, but this approach is accompanied by several prominent problems. Long-term, high-dose use of antibiotics not only leads to drug resistance in pathogens, gradually reducing treatment effectiveness, but also allows drug-resistant genes to spread through the food chain, posing a threat to public health and safety. Furthermore, while antibiotics suppress pathogens, they also kill beneficial bacteria in the gut, exacerbating intestinal flora imbalance and causing recurrent illness in livestock. In addition, antibiotics and chemical drugs easily leave residues in livestock products, which not only harm human health but also fail to meet current food safety standards. Traditional treatments mostly target only a single antibacterial or antidiarrheal point, failing to simultaneously address multiple pathological issues such as oxidative stress, intestinal mucosal damage, and biofilm formation, making it difficult to achieve effective treatment for this type of enteritis at its root.

[0004] Although nanozymes and plant-based active ingredients have shown application potential in the field of veterinary intestinal diseases, existing technologies still have significant drawbacks: single nanozyme materials only have antioxidant functions and lack synergistic effects such as antibacterial, anti-inflammatory, and gut microbiota regulation, failing to break the vicious cycle of "oxidative stress-inflammation-microbiota imbalance"; quercetin has extremely poor water solubility, is easily metabolized in vivo, and has low bioavailability, making it difficult to achieve effective concentrations at intestinal lesions when administered alone; eugenol is highly volatile, has poor stability, short gastrointestinal residence time, and lacks targeted delivery capabilities, easily causing local irritation and waste of effective ingredients; existing nano-drug delivery systems mostly adopt two-step / multi-step preparation methods, which are cumbersome, have poor batch stability, and are not environmentally friendly, making them difficult to adapt to large-scale veterinary drug production.

[0005] Chinese patent CN116235903A (A method for preparing and applying nano-zinc feed for the prevention and treatment of colitis) focuses only on the antibacterial properties of metal oxides, lacking antioxidant and anti-inflammatory functions, and has a large particle size and low absorption and utilization rate; Chinese patent CN1875960A (An eugenol nanoemulsion drug and its preparation method) only solves the problem of eugenol solubilization, lacks the antioxidant and intestinal mucosal repair functions of nanoenzymes, and fails to achieve targeted release and long-term retention; Chinese patent CN111053776A (A pharmaceutical composition of afatinib and its application) uses a quercetin metal complex that only improves the stability of quercetin, without loading antibacterial components, and cannot deal with pathogenic bacteria and biofilms; existing veterinary nanoenzyme preparations have not achieved the synergistic effect of "nanozyme + quercetin + eugenol", and have obvious shortcomings in clearing ROS, repairing mucosa, inhibiting pathogenic bacteria / biofilms, and regulating the flora. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a quercetin-zinc nanozyme loaded with eugenol that is safe, residue-free, multi-target synergistic, targeted, and suitable for large-scale production for treating bacterial enteritis in livestock. The method includes: Quercetin-zinc nanoenzyme particles formed by coordination between quercetin and zinc ions; And eugenol loaded onto the quercetin-zinc nanoenzyme particles via metal-hydroxyl interaction; The quercetin-zinc nanozyme loaded with eugenol is used to treat bacterial enteritis in livestock.

[0007] Furthermore, the livestock are dairy sheep and meat sheep, and the pathogens causing bacterial enteritis are Escherichia coli and Clostridium perfringens.

[0008] Furthermore, the specific steps of the preparation method are as follows: Step S1: Prepare 90 ml of a deionized water-ethanol mixed solvent with a volume ratio of 2:1; Step S2: Add 10 mg of quercetin, 8.02-10.69 mg of zinc sulfate, and 1.5 mg of eugenol to the mixed solvent in sequence, and sonicate for 15 min to completely dissolve and disperse the raw materials; adjust the pH of the reaction system to 7.0 with 0.1 mol / L phosphate buffer. Step S3: Place the system in a 37°C constant temperature water bath and stir magnetically for 4 hours to simultaneously achieve coordination assembly of quercetin and zinc ions and co-loading of eugenol. After the reaction, centrifuge at 8000 rpm for 10 minutes to collect the precipitate, wash it three times alternately with deionized water and ethanol, and then vacuum dry it at 60°C for 4 hours to obtain quercetin-zinc nanozyme loaded with eugenol.

[0009] Furthermore, in step S2, the amount of zinc sulfate is 10 mg.

[0010] Furthermore, the molar ratio of quercetin to zinc ions is 1:1.5-1:2. This ratio range ensures that quercetin and zinc ions are fully coordinated to form nanoenzyme particles with stable structure and excellent enzyme activity. If the molar ratio is too high, quercetin is prone to excessive aggregation, leading to nanoparticle agglomeration. If the molar ratio is too low, insufficient zinc ions will affect the sufficiency of the coordination reaction and reduce the catalytic activity of the nanoenzyme. The mass ratio of quercetin-zinc nanoenzyme to eugenol is 12:1-14:1.

[0011] Furthermore, the loading of eugenol is 8.4%-13.2% of the total mass of the nanozyme. This loading range can ensure the inhibitory effect of eugenol on common pathogenic bacteria in the intestines of livestock and its anti-biofilm activity, while avoiding instability of the nanozyme structure or intestinal irritation caused by sudden release of eugenol due to excessive loading. At the same time, it ensures the slow release of active ingredients and prolongs the action time.

[0012] Furthermore, the quercetin-zinc nanoenzyme particles have a particle size of 60-210 nm. This particle size range is conducive to the retention and distribution of nanoenzymes in the intestines of livestock and poultry, preventing them from being rapidly excreted. At the same time, it facilitates the penetration of the intestinal mucus layer and full contact with the infected site, thereby improving bioavailability.

[0013] Furthermore, the zinc ion source is selected from zinc sulfate, which has good water solubility and strong coordination ability. Zinc is an essential trace element for livestock and will not cause residues in the body.

[0014] Furthermore, the mixed solvent is a mixture of water and ethanol in a volume ratio of 1:1 to 2:1. This solvent system can simultaneously dissolve quercetin, zinc ion source, and eugenol, and the solvent is easily volatilized and removed without any residue risk. The concentration of quercetin is 0.11 mg / mL; the concentration of the zinc ion source solution is 0.55-0.74 mmol / L; the concentration of the eugenol solution is 0.017 mg / mL; and the mass ratio of quercetin-zinc nanozyme to eugenol is 12:1 to 14:1.

[0015] Furthermore, the pH of the system is adjusted using phosphate buffer or Tris-HCl buffer, and the pH is controlled between 6.5 and 8.5. This pH range ensures that the coordination reaction between quercetin and zinc ions proceeds efficiently to form structurally stable nanozyme particles, and also promotes the metal-hydroxyl interaction between eugenol and nanozyme particles to improve loading efficiency, while avoiding degradation of active ingredients or loading failure due to excessively high or low pH.

[0016] Furthermore, the stirring speed is 300-800 rpm. This speed range ensures that the reaction system is mixed evenly, avoids excessive local concentration that could lead to particle agglomeration, and ensures that the coordination reaction and the loading reaction proceed simultaneously and efficiently.

[0017] Compared with the prior art, the present invention has the following technical effects or advantages: 1. Significant advantages of the preparation process: The nanozyme matrix formation and eugenol loading are completed simultaneously in a one-pot process, eliminating the need for step-by-step processing, simplifying the operation, eliminating the need for complex equipment, reducing production costs, and making it suitable for large-scale industrial production; the reaction conditions are mild, effectively avoiding product agglomeration and fluctuations in loading efficiency, and the obtained nanozymes are uniformly dispersed and have stable loading efficiency.

[0018] 2. Highly efficient and synergistic antioxidant effect, blocking oxidative stress damage: The quercetin-zinc nanozyme matrix can mimic the catalytic function of SOD and CAT, effectively clearing excess ROS in the intestines of livestock; when the nanozyme concentration is 20 μg / mL, the simulated SOD-like enzyme activity can reach 12.9 U / mL, and the amount of oxygen produced by CAT-like enzyme catalysis within 10 minutes reaches 23.7 mg / mL. At the same time, the scavenging rate of DPPH and ABTS free radicals can reach more than 85%, blocking the damage of oxidative stress to the intestinal mucosa from the source.

[0019] 3. Highly effective antibacterial and anti-biofilm functions, specifically addressing pathogenic bacterial infections: The loaded eugenol can specifically inhibit the growth of common pathogenic bacteria causing diarrhea in livestock (such as Escherichia coli and Clostridium perfringens), and the clearance rate of pathogenic bacterial biofilms can reach more than 60%, effectively breaking the protective effect of pathogenic bacterial biofilms and solving the problem of poor efficacy of traditional drugs against pathogenic bacteria within biofilms.

[0020] 4. Significant in vivo therapeutic effect, achieving intestinal mucosal repair and diarrhea relief: In vivo experiments at a sheep farm in Tang County, Hebei Province, have verified that this nanoenzyme can significantly alleviate the symptoms of bacterial diarrhea in sheep, improve bloody stools, and repair intestinal mucosal damage. After treatment, the sheep's intestinal mucosa remains intact, without congestion or ulceration.

[0021] 5. Excellent gastrointestinal stability, enabling targeted delivery of active ingredients: The nanozyme can maintain its complete spherical structure in artificial gastric fluid (20 min) and artificial small intestinal fluid (4 h), avoiding premature release of active ingredients; in artificial colonic fluid (4 h), the structure disintegrates, enabling targeted release of eugenol and quercetin-zinc complex, which can accurately act on the lesion site in the intestine.

[0022] 6. High biocompatibility and no residue risk: The raw materials used in the preparation are all natural plant active ingredients or essential trace elements for livestock, with good biocompatibility and no drug residues, meeting the requirements for food safety and green development of the livestock industry.

[0023] 7. Adaptable to the needs of farming scenarios, convenient and efficient drug administration: The drug composition is preferably in oral dosage forms such as drinking water preparations and feed mixing preparations, which can be administered to livestock through free drinking water or feed mixing, solving the problems of cumbersome and stressful administration of traditional injections, and greatly improving the treatment efficiency of large-scale farming. Attached Figure Description

[0024] Figure 1 Scanning electron microscopy (SEM) characterization and particle size distribution of quercetin-zinc nanozymes loaded with eugenol; Figure 2 : SOD-like enzyme activity diagram of quercetin-zinc nanozyme loaded with eugenol; Figure 3 : CAT-like enzyme activity diagram of quercetin-zinc nanozyme loaded with eugenol; Figure 4 : DPPH free radical scavenging rate of quercetin-zinc nanozyme loaded with eugenol; Figure 5 : Graph showing the ABTS free radical scavenging rate of quercetin-zinc nanozyme loaded with eugenol; Figure 6 Plate coating diagram of quercetin-zinc nanozyme loaded with eugenol inhibiting the growth of Escherichia coli and Clostridium perfringens; Figure 7 : Effect of quercetin-zinc nanozyme loaded with eugenol on the anti-biofilm activity of Escherichia coli stained with crystal violet; Figure 8 : Effect of quercetin-zinc nanozyme loaded with eugenol on the anti-biofilm activity of Clostridium perfringens stained with crystal violet; Figure 9 Intestinal diagrams of sheep with diarrhea before and after treatment; Figure 10 Images of bloody stools in sheep with diarrhea before and after treatment; Figure 11 SEM images of quercetin-zinc nanozymes loaded with eugenol after passing through gastric and small intestinal fluids (a) and colonic fluids (b). Detailed Implementation

[0025] The technical solution of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings, thereby making the advantages and effects of the present invention clearer. Those skilled in the art should understand that these specific embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0026] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0027] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0028] Example 1: One-pot preparation method of quercetin-zinc nanozyme loaded with eugenol Preparation of mixed solvent: Take 60 mL of deionized water and 30 mL of ethanol, mix them, and stir well to obtain a water and ethanol mixed solvent with a volume ratio of 2:1. Addition of raw materials: Weigh 10 mg of quercetin, 10 mg of zinc sulfate, and 1.5 mg of eugenol, and add them sequentially to the above mixed solvent. Disperse by sonication for 15 minutes to ensure that the raw materials are fully dissolved. System adjustment: Adjust the pH of the reaction system to 7.0 using 0.1 mol / L phosphate buffer; Reaction conditions: The reaction system was placed in a 37°C constant temperature water bath and magnetically stirred for 4 hours. Post-processing: After the reaction was completed, the precipitate was centrifuged at 8000 rpm for 10 minutes, collected, washed three times alternately with deionized water and ethanol, and dried under vacuum at 60℃ for 4 hours to obtain quercetin-zinc nanozyme loaded with eugenol. The eugenol loading was 13.2%.

[0029] Example 2: One-pot preparation of quercetin-zinc nanozymes loaded with eugenol Preparation of mixed solvent: Take 60 mL of deionized water and 30 mL of ethanol, mix them, and stir well to obtain a water and ethanol mixed solvent with a volume ratio of 2:1. Addition of raw materials: Weigh 10 mg of quercetin, 8.02 mg of zinc sulfate, and 1.5 mg of eugenol, and add them sequentially to the above mixed solvent. Disperse by sonication for 15 minutes to ensure that the raw materials are fully dissolved. System adjustment: Adjust the pH of the reaction system to 7.0 using 0.1 mol / L phosphate buffer; Reaction conditions: The reaction system was placed in a 37°C constant temperature water bath and magnetically stirred for 4 hours. Post-processing: After the reaction was completed, the precipitate was centrifuged at 8000 rpm for 10 minutes, collected, washed three times alternately with deionized water and ethanol, and dried under vacuum at 60℃ for 4 hours to obtain quercetin-zinc nanozyme loaded with eugenol. The eugenol loading was found to be 8.4%.

[0030] Example 3: One-pot preparation of quercetin-zinc nanozymes loaded with eugenol Preparation of mixed solvent: Take 60 mL of deionized water and 30 mL of ethanol, mix them, and stir well to obtain a water and ethanol mixed solvent with a volume ratio of 2:1. Addition of raw materials: Weigh 10 mg of quercetin, 10.69 mg of zinc sulfate, and 1.5 mg of eugenol, and add them sequentially to the above mixed solvent. Disperse by sonication for 15 minutes to ensure that the raw materials are fully dissolved. System adjustment: Adjust the pH of the reaction system to 7.0 using 0.1 mol / L phosphate buffer; Reaction conditions: The reaction system was placed in a 37°C constant temperature water bath and magnetically stirred for 4 hours. Post-processing: After the reaction was completed, the precipitate was centrifuged at 8000 rpm for 10 minutes, collected, washed three times alternately with deionized water and ethanol, and dried under vacuum at 60℃ for 4 hours to obtain quercetin-zinc nanozyme loaded with eugenol. The eugenol loading was 12.5%.

[0031] Example 1: Characterization of quercetin-zinc nanozymes loaded with eugenol SEM characterization: The nanozyme sample prepared in Example 1 was uniformly dispersed on a conductive adhesive, sputtered with gold, and its morphology and size were observed using a scanning electron microscope. The results are as follows: Figure 1 As shown, the nanozyme exhibits a near-spherical structure, is uniformly dispersed, and shows no obvious aggregation.

[0032] Particle size distribution: The particle size distribution of the prepared nanozymes was determined using dynamic light scattering (DLS). The results are as follows: Figure 1 As shown in the illustration, the prepared nanozyme has a particle size of 60-210 nm, which meets the design expectations and is beneficial for retention and action in the intestines of livestock.

[0033] Simulated SOD enzyme activity assay: Using the xanthine oxidase method and pyrogallol auto-oxidation as a model, the scavenging ability of different concentrations of nanozymes for superoxide anions was detected. Results are as follows: Figure 2 As shown, with the increase of nanozyme concentration, the SOD-like enzyme activity gradually increases. When the concentration is 20 μg / mL, the simulated value of SOD-like enzyme activity can reach 12.9 U / mL, indicating that it can efficiently catalyze the conversion of superoxide anions into hydrogen peroxide and exert an antioxidant effect.

[0034] Simulated CAT enzyme activity assay: The dissolved oxygen method was used to detect the ability of the nanozyme to decompose hydrogen peroxide to produce oxygen. Results are as follows: Figure 3 As shown, this nanozyme can rapidly decompose hydrogen peroxide, producing 23.7 mg / mL of oxygen within 10 minutes at a concentration of 20 μg / mL, exhibiting excellent CAT-like catalytic activity.

[0035] DPPH radical scavenging rate detection: A 100 μmol / L DPPH ethanol solution was prepared, and nanozyme samples of different concentrations were added. The reaction was carried out at room temperature in the dark for 30 minutes, and the absorbance at 517 nm was measured. The results are as follows: Figure 4As shown, the scavenging rate of nanozymes against DPPH free radicals increases with increasing concentration. When the concentration is 200 μg / mL, the scavenging rate reaches more than 85%, indicating that it has a strong free radical scavenging ability.

[0036] ABTS free radical scavenging rate detection: ABTS is generated by oxidation of ABTS with potassium persulfate. + Free radical cations were added to nanozyme samples of different concentrations, and the mixture was reacted at room temperature for 10 minutes. The absorbance at 734 nm was then measured. The results are as follows: Figure 5 As shown, the nanozyme achieved a scavenging rate of over 85% for ABTS free radicals at a concentration of 20 μg / mL, further confirming its excellent antioxidant properties.

[0037] Experimental Example 2: Detection of antibacterial and anti-biofilm activity of eugenol-loaded quercetin-zinc nanozymes Plate coating antibacterial test: Common pathogens causing diarrhea in livestock, *Escherichia coli* (K88) and *Clostridium perfringens* (B815403), were selected as test strains. The bacterial suspensions in the logarithmic growth phase were diluted to 10⁻⁶. 4 CFU / mL was used to prepare 100 μL of bacterial culture, which was then evenly spread onto a TSB agar plate and incubated with 20 μg / mL nanozyme at 37°C for 24 hours. The growth of the bacteria was then observed. Results are as follows: Figure 6 As shown, the quercetin-zinc nanozyme loaded with eugenol prepared has a significant antibacterial effect against Escherichia coli and Clostridium perfringens, indicating that it has broad-spectrum and highly efficient antibacterial activity against common pathogens causing diarrhea in livestock.

[0038] Crystal violet staining anti-biofilm experiment: 10 6 CFU / mL of pathogenic bacterial culture and nanozyme were added separately to 96-well plates and incubated at 37°C for 48 hours to form biofilms. The bacterial culture was discarded, the plates were washed three times with PBS, stained with 0.1% crystal violet solution for 15 minutes, and the supernatant was discarded. The morphology of the formed biofilms was then observed. Results are as follows: Figure 7 and 8 As shown, the quercetin-zinc nanozyme loaded with eugenol exhibits a significant scavenging effect on biofilms. This indicates that it can effectively inhibit biofilm formation or disrupt existing biofilms.

[0039] Experimental Example 3: Evaluation of the in vivo therapeutic effect of eugenol-loaded quercetin-zinc nanozyme on diarrhea in sheep. Diarrhea model construction: Sheep from Mazhuang Sheep Farm in Tang County, Hebei Province were selected and acclimatized for 3 days. They were then orally administered Escherichia coli K88 bacterial solution (10 mg / L). 9 A bacterial diarrhea model was constructed using CFU / sheep. Sheep exhibiting diarrhea symptoms after successful modeling were photographed and documented.

[0040] Administration and Testing: Sheep were given the drug once daily with free access to drinking water for 3 consecutive days, while the untreated group had free access to saline solution daily. After treatment, the macroscopic morphology of the sheep's intestines and the state of diarrhea were observed. Sheep in the treatment group showed significant relief from diarrhea, with intact intestinal mucosa and no congestion or ulceration.

[0041] Experimental Example 4: The structural stability of eugenol-loaded quercetin-zinc nanozymes after simulating the gastrointestinal tract. To evaluate the structural stability of the nanozyme in the gastrointestinal environment, artificial gastric juice (pH 1.2), artificial small intestinal juice (pH 6.8), and artificial colonic juice (pH 7.4) were prepared. The nanozyme sample prepared in Example 1 was added to the above simulated digestive solutions and incubated in a constant temperature shaking incubator at 37°C.

[0042] After incubation in artificial gastric fluid for 20 minutes and artificial small intestinal fluid for 4 hours, samples were collected by centrifugation, and their morphology and structure were observed by SEM. Results are as follows: Figure 11 As shown in a, the nanozyme maintains its complete spherical structure in gastric and intestinal fluids, with no significant change in particle size, indicating that it has good structural stability in the gastric and small intestinal environments, which is beneficial for protecting the loaded eugenol and preventing premature release of the active ingredient.

[0043] After incubation in artificial colonic fluid for 4 hours, SEM observation results showed ( Figure 11 (b) The nanozyme particles gradually disintegrate and become irregular in shape, indicating that they can be gradually degraded in the colonic environment, thereby releasing the loaded eugenol and quercetin-zinc complex, which exert antibacterial, antioxidant and intestinal mucosal repair effects.

[0044] This invention successfully constructs a multifunctional quercetin-zinc nanozyme loaded with eugenol by simultaneously achieving the coordination of quercetin and zinc ions and the loading of eugenol in a one-pot process. The preparation method is simple and suitable for large-scale production. The resulting nanozyme possesses highly efficient antioxidant, broad-spectrum antibacterial, anti-biofilm, and intestinal mucosal repair functions, enabling comprehensive treatment of diarrhea-associated enteritis in livestock. Furthermore, it exhibits high biosafety and no drug residues, meeting the needs of green development in the livestock industry. Its veterinary drug composition offers diverse dosage forms, adaptable to large-scale livestock administration scenarios, and is convenient to use, effectively improving treatment efficiency and reducing breeding costs. This invention provides a novel, efficient, and green solution for the treatment of diarrhea-associated enteritis in livestock, possessing significant industrial application value and broad market prospects.

[0045] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0046] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0047] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a quercetin-zinc nanozyme loaded with eugenol for treating enteritis in livestock, characterized in that, include: Quercetin-zinc nanoenzyme particles formed by coordination between quercetin and zinc ions; And eugenol loaded onto the quercetin-zinc nanoenzyme particles via metal-hydroxyl interaction; The quercetin-zinc nanozyme loaded with eugenol is used to treat bacterial enteritis in livestock.

2. The preparation method according to claim 1, characterized in that, The livestock are dairy sheep and meat sheep, and the pathogens causing the bacterial enteritis are Escherichia coli and Clostridium perfringens.

3. The preparation method according to claim 1 or 2, characterized in that, The specific steps of the preparation method are as follows: Step S1: Prepare 90 ml of a deionized water-ethanol mixed solvent with a volume ratio of 2:1; Step S2: Add 10 mg of quercetin, 8.02-10.69 mg of zinc sulfate, and 1.5 mg of eugenol to the mixed solvent in sequence, and sonicate for 15 min to completely dissolve and disperse the raw materials; adjust the pH of the reaction system to 7.0 with 0.1 mol / L phosphate buffer. Step S3: Place the system in a 37°C constant temperature water bath and stir magnetically for 4 hours to simultaneously achieve coordination assembly of quercetin and zinc ions and co-loading of eugenol. After the reaction, centrifuge at 8000 rpm for 10 minutes to collect the precipitate, wash it three times alternately with deionized water and ethanol, and then vacuum dry it at 60°C for 4 hours to obtain quercetin-zinc nanozyme loaded with eugenol.

4. The preparation method according to claim 3, characterized in that, In step S2, the amount of zinc sulfate is 10 mg.

5. The preparation method according to claim 3, characterized in that, The molar ratio of quercetin to zinc ions is 1:1.5-1:2, and the mass ratio of quercetin-zinc nanozyme to eugenol is 12:1-14:

1.

6. The preparation method according to claim 3, characterized in that, The eugenol loading is 8.4%-13.2% of the total mass of the nanozyme.

7. The preparation method according to claim 4, characterized in that, The quercetin-zinc nanozyme particles have a particle size of 60-210 nm.

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