Attapulgite composite nano-enzyme for resisting porcine viral diarrhea as well as preparation method and application of attapulgite composite nano-enzyme

Abstract

The invention discloses an attapulgite composite nano-enzyme for resisting porcine viral diarrhea as well as a preparation method and application of the attapulgite composite nano-enzyme. According to the composite material, attapulgite is used as a carrier, firstly, an effective ingredient of radix isatidis is extracted through an alcohol-water mixture and loaded on the attapulgite, and then a zinc oxide-cuprous oxide heterojunction nanostructure is constructed on the surface of the attapulgite in situ by utilizing the guiding and reducing effects of the active ingredient of the radix isatidis. The composite nano enzyme obtained by the invention integrates the direct antiviral activity of radix isatidis, the enzyme-like catalytic performance of ZnO-Cu2O heterojunction and the adsorption function of attapulgite, and can efficiently inhibit intestinal pathogens such as porcine epidemic diarrhea virus and the like through the synergistic effect of triple mechanisms of adsorption enrichment, catalytic killing and traditional Chinese medicine inhibition. The material is simple in preparation process, green, safe and free of drug residues, and has wide application prospects in preparation of drugs and feed additives for preventing and treating porcine viral diarrhea.

Description

Technical Field

[0001] This invention belongs to the field of veterinary medical materials technology, specifically relating to a composite nanoenzyme material with anti-swine viral diarrhea function, its preparation method and application. Background Technology

[0002] Porcine viral diarrhea is one of the most common and serious diseases in the pig industry, mainly caused by porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and rotavirus. These viruses attack the intestinal epithelial cells of pigs, leading to atrophy of intestinal villi, impaired digestion and absorption, and causing severe watery diarrhea, dehydration, and high mortality, especially in piglets, resulting in huge economic losses. Animal husbandry environment, 2025, (17): 97-98. Currently, the prevention and control of swine viral diarrhea mainly relies on vaccination and drug treatment. However, the high mutation rate of viruses often leads to unsatisfactory vaccine protection, while chemical antiviral drugs pose risks such as residues, drug resistance, and food safety issues. In farming practice, antibiotics are frequently used to control secondary infections, but long-term use leads to bacterial resistance and drug residues in livestock products, threatening public health and safety.

[0003] Attapulgite is a natural magnesium-aluminate silicate clay mineral with a unique fibrous crystal structure and one-dimensional nanochannels. It possesses a large specific surface area and excellent adsorption properties, enabling it to adsorb pathogenic microorganisms and toxins. Studies have shown that attapulgite can improve intestinal integrity and prevent diarrhea in weaned piglets. Journal of Animal Nutrition, 2024, 36(01):25-32. However, attapulgite alone has limited ability to adsorb and inactivate viruses, making it difficult to effectively control viral diarrhea. Isatis root is a traditional Chinese medicine; its active ingredients, such as indirubin and indigo, have broad-spectrum antiviral effects, but their poor water solubility and low bioavailability limit their application.

[0004] Nanozymes are a class of nanomaterials with enzyme-like activity, capable of mimicking the catalytic function of natural enzymes, and showing great potential in the field of antibacterial and antiviral applications. Practical Clinical Medicine Journal, 2018, 22(15): 1-7+13. Studies have shown that nano-zinc oxide possesses enzyme-like activity and antiviral properties, while cuprous oxide not only exhibits similar catalytic performance but also directly inhibits various viruses. Constructing a heterojunction of these two metal oxides can produce a synergistic catalytic effect, improving antiviral efficiency. However, current technologies lack integrated designs that synergistically integrate traditional Chinese medicine extracts and metal oxide heterojunctions onto mineral carriers, especially multi-mechanism prevention and control products targeting porcine viral diarrhea.

[0005] Therefore, developing a safe and efficient anti-swine viral diarrhea material that integrates multiple functions such as adsorption, catalysis, and traditional Chinese medicine inhibition is of great practical significance and application value. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an attapulgite composite nanoenzyme for combating swine viral diarrhea. This material integrates the triple functions of antiviral properties of traditional Chinese medicine, nanoenzyme catalysis, and clay adsorption, achieving multi-target prevention and control of swine viral diarrhea. It features synergistic effects, multiple mechanisms of action, and is green and safe.

[0007] Another objective of this invention is to provide a green preparation method for the above-mentioned composite nanozyme, which is simple, mild, environmentally friendly, and suitable for large-scale production.

[0008] Another object of the present invention is to provide the application of the above-mentioned composite nanozyme in the preparation of drugs or feed additives for the prevention and / or treatment of swine viral diarrhea.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides an attapulgite composite nanoenzyme for treating swine viral diarrhea. It uses attapulgite as a carrier, loads the effective components of Isatis indigotica on its surface and in its pores, and constructs a zinc oxide-cuprous oxide (ZnO-Cu2O) heterojunction nanostructure in situ.

[0010] The zinc oxide-cuprous oxide heterojunctions are uniformly distributed on the surface of attapulgite in the form of nanoparticles, the particle size of which is less than 5 nm. The main active components of the Isatis root include at least one of indigo, indirubin, adenosine, and polysaccharides.

[0011] This invention provides a method for preparing the above-mentioned composite nanozyme, comprising the following steps: (1) Extraction and adsorption: Mix the Isatis root powder with the alcohol-water mixed solvent, heat and reflux to extract, and filter to obtain the Isatis root extract; add the attapulgite powder to the Isatis root extract, stir to allow the effective components of Isatis root to be fully adsorbed on the attapulgite, and obtain the Isatis root-attapulgite mixed suspension.

[0012] The alcohol-water mixed solvent contains 25% to 70% ethanol by volume; the heating reflux temperature is 50 to 75°C, and the time is 1 to 3 hours. The attapulgite powder undergoes pretreatment before use, which includes acid activation, water washing, drying, and calcination to remove impurities and improve its specific surface area and activity.

[0013] (2) In-situ synthesis and post-processing: Soluble zinc salt and soluble copper salt are added sequentially to the mixed suspension obtained in step (1). After stirring and dissolving, alkaline solution is slowly added dropwise under stirring to adjust the pH to 10-12. The reaction system is heated at 45-95℃ for 2-12 hours. After the reaction is completed, the mixture is cooled naturally. The product is centrifuged, washed and dried to obtain the attapulgite composite nanozyme.

[0014] The soluble zinc salt is zinc nitrate or zinc acetate, and the soluble copper salt is copper nitrate, copper acetate, copper chloride, or copper sulfate; the alkaline solution is sodium hydroxide solution or ammonia water.

[0015] The mass ratio of attapulgite, indigo powder, zinc salt, and copper salt is 1: (5~20): (0.05~0.5): (0.02~0.3).

[0016] The present invention also provides the use of the above-mentioned composite nanozyme in the preparation of drugs or feed additives for the prevention and / or treatment of swine viral diarrhea.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Synergistic Antiviral Mechanisms: This invention integrates the physical adsorption function of attapulgite, the enzyme-like catalytic function of ZnO-Cu2O heterojunctions, and the direct antiviral function of Isatis indigotica. Attapulgite has a strong adsorption capacity, which can adsorb and enrich viral particles in the intestine, creating a local high-concentration environment for subsequent inactivation; the in-situ generated ZnO-Cu2O heterojunctions have excellent peroxidase-like activity, which can catalyze the production of a large amount of highly active ·OH from trace amounts of H2O2 in the intestine, directly oxidizing and destroying the viral protein capsid and nucleic acid, leading to viral inactivation; the loaded Isatis indigotica active ingredients can directly act on the virus, interfering with its adsorption, invasion of host cells, and replication processes. These three mechanisms promote each other and work synergistically, significantly improving antiviral efficiency and reducing the risk of viral resistance.

[0018] 2. Targeted Delivery and Safety: Attapulgite, as a natural mineral carrier, allows active ingredients (the effective components of Isatis indigotica and nanoenzymes) to be more concentrated at the intestinal lesion site, increasing local drug concentration and enhancing efficacy. Simultaneously, the material is made from natural ingredients; Isatis indigotica and attapulgite themselves are safe and non-toxic. The metal oxides exist in a stable nanocrystalline form, minimizing ionic toxicity and eliminating concerns about drug residues, thus meeting the requirements of green aquaculture.

[0019] 3. The preparation process is green and simple: The active ingredients in Isatis root extract serve as natural reducing agents and structure-directing agents, guiding the uniform in-situ growth of ZnO-Cu2O heterojunctions on the carrier surface, reducing the use of additional chemical reagents. The entire process is mild, energy-efficient, environmentally friendly, and easily scaled up for industrial production. Attached Figure Description

[0020] Figure 1 This is a transmission electron microscope (TEM) image of the attapulgite composite nanozyme prepared in Example 1 of the present invention.

[0021] Figure 2 Energy dispersive spectroscopy (EDS) analysis of the attapulgite composite nanozyme prepared in Example 1 of this invention.

[0022] Figure 3 This is a comparison chart showing the virus inhibition rates of Comparative Example 1, Comparative Example 2, and the product of Example 1 of the present invention against porcine epidemic diarrhea virus (PEDV).

[0023] Figure 4 The graph shows the cytotoxicity evaluation of the products of Comparative Example 1, Comparative Example 2 and Example 1 of the present invention on the L929 cell line. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0025] Example 1 (1) Carrier pretreatment: Take 10g of natural attapulgite clay, purify it by water selection, acidify it by stirring at 60℃ for 5 hours with 1mol / L HCl solution, then wash it with deionized water until neutral, dry it at 110℃, calcine it at 350℃ for 2 hours, and grind it into powder for later use.

[0026] (2) Extraction and adsorption of Isatis root: Weigh 5g of Isatis root powder, add 150mL of 25% ethanol aqueous solution, reflux at 75°C for 2 hours, and filter to obtain Isatis root extract. Add 1.0g of the pretreated attapulgite powder to the extract, stir and adsorb at 50°C for 3 hours to obtain a mixed suspension.

[0027] (3) In-situ synthesis of composite nanozymes: 0.05 g of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) and 0.02 g of copper nitrate trihydrate (Cu(NO3)2·3H2O) were added sequentially to the above mixed suspension and stirred until completely dissolved. Under vigorous stirring, 1 mol / L NaOH solution was slowly added dropwise to adjust the pH to 10, and the reaction was carried out at 45°C for 12 hours. After the reaction was completed, the product was naturally cooled, and the product was washed three times each with deionized water and anhydrous ethanol by centrifugation. It was then vacuum dried at 40°C for 24 hours and ground to obtain the attapulgite composite nanozyme.

[0028] Example 2 (1) Carrier pretreatment: Take 10g of natural attapulgite clay, purify it by water selection, acidify it by stirring at 80°C for 1 hour with 1mol / L sulfuric acid solution, then wash it with deionized water until neutral, dry it at 110°C, and then calcine it at 350°C for 2 hours, and grind it for later use.

[0029] (2) Extraction and adsorption of Isatis root: Weigh 10g of Isatis root powder, add 150mL of 45% ethanol aqueous solution, reflux at 65°C for 3 hours, and filter to obtain Isatis root extract. Add 1.0g of the pretreated attapulgite powder to the extract and stir at room temperature for 6 hours for adsorption.

[0030] (3) In-situ synthesis of composite nanozyme: 0.2 g of zinc nitrate hexahydrate and 0.1 g of copper chloride were added sequentially to the above mixed suspension and stirred until completely dissolved. Under vigorous stirring, concentrated ammonia was slowly added dropwise to adjust the pH to 11, and the reaction was carried out at 70°C for 6 hours. After the reaction was completed, the mixture was allowed to cool naturally. The product was washed three times each with deionized water and anhydrous ethanol by centrifugation, dried under vacuum at 60°C for 12 hours, and then ground to obtain the composite nanozyme.

[0031] Example 3 (1) Carrier pretreatment: Take 10g of natural attapulgite clay, purify it by water selection, acidify it by stirring at 60℃ for 4 hours with 2mol / L HCl solution, then wash it with deionized water until neutral, dry it at 110℃, calcine it at 350℃ for 2 hours, and grind it into powder for later use.

[0032] (2) Extraction and adsorption of Isatis root: Weigh 20g of Isatis root powder, add 150mL of 60% ethanol aqueous solution, reflux at 55°C for 4 hours, and filter to obtain Isatis root extract. Add 1.0g of the pretreated attapulgite powder to the extract, and stir at 50°C for 4 hours for adsorption.

[0033] (3) In-situ synthesis of composite nanozyme: 0.5 g of zinc nitrate hexahydrate and 0.3 g of copper sulfate were added sequentially to the above mixed suspension and stirred until completely dissolved. Under vigorous stirring, 2 mol / L NaOH solution was slowly added dropwise to adjust the pH to 12, and the reaction was carried out at 95°C with stirring for 2 hours. After the reaction was completed, the mixture was allowed to cool naturally. The product was washed three times each with deionized water and anhydrous ethanol by centrifugation, dried in an oven at 80°C for 10 hours, and then ground to obtain the composite nanozyme.

[0034] Example 4 (1) Carrier pretreatment: Take 10g of natural attapulgite clay, purify it by water selection, acidify it by stirring at 60℃ for 2 hours with 2mol / L sulfuric acid solution, then wash it with deionized water until neutral, dry it at 110℃, calcine it at 350℃ for 2 hours, and grind it into powder for later use.

[0035] (2) Extraction and adsorption of Isatis root: Weigh 15g of Isatis root powder, add 150mL of 60% ethanol aqueous solution, reflux at 60°C for 2.5 hours, and filter to obtain Isatis root extract. Add 1.0g of the pretreated attapulgite powder to the extract, and stir at 50°C for 5 hours for adsorption.

[0036] (3) In-situ synthesis of composite nanozyme: 0.3 g of zinc acetate and 0.15 g of copper acetate were added sequentially to the above mixed suspension and stirred until completely dissolved. Under vigorous stirring, 1 mol / L NaOH solution was slowly added dropwise to adjust the pH to 10.5, and the reaction was carried out at 80°C with stirring for 4 hours. After the reaction was completed, the mixture was allowed to cool naturally. The product was washed three times each with deionized water and anhydrous ethanol by centrifugation, dried in an oven at 80°C for 10 hours, and then ground to obtain the composite nanozyme.

[0037] Comparative Example 1 Preparation of Isatis root-attapulgite complex: Following steps (1) and (2) of Example 1, attapulgite loaded with the active ingredient of Isatis root was obtained without performing the in-situ synthesis reaction in step (3).

[0038] Comparative Example 2 Preparation of ZnO-Cu2O / attapulgite composite material without Isatis indigotica: The procedure was carried out according to steps (1) and (3) of Example 1, but in step (3), zinc salt and copper salt were added directly to the suspension formed by pretreated attapulgite powder and deionized water, without going through the adsorption process of Isatis indigotica extract in step (2).

[0039] Characterization and performance testing 1. Material Characterization The morphology and composition of the attapulgite composite nanozyme prepared in Example 1 were characterized. Transmission electron microscopy (TEM) images ( Figure 1 The results showed that ultrafine nanoparticles with a diameter of less than 5 nm were successfully loaded onto the surface of the attapulgite support, and the particles were uniformly distributed. Corresponding energy-dispersive X-ray spectroscopy (EDX) analysis... Figure 2 The results indicate the simultaneous presence of characteristic elemental signals such as Si, Mg, Fe, Cu, Zn, and O in the material. Among these, the Cu and Zn signals correspond to the successfully constructed cuprous oxide-zinc oxide heterojunction; Si, Mg, and Fe are inherent elements of the attapulgite support framework. These results collectively confirm the successful fabrication of the target composite structure.

[0040] 2. Evaluation of the antiviral effect and cytotoxicity of attapulgite composite nanozymes Cytotoxicity was determined using the CCK-8 assay, and the inhibitory effect of the material on PEDV was evaluated using in vitro antiviral experiments.

[0041] Antiviral activity test: a) Preventive measures: First, incubate the aseptically treated material with the cells for 2 hours, discard the material, and then add PEDV for infection.

[0042] b) Direct inactivation: The sterile material was first incubated with PEDV virus solution for 2 hours, and then the mixture was added to the cells.

[0043] c) Therapeutic effect: First, infect cells with PEDV for 2 hours, discard the viral fluid, and then add maintenance solution containing the material.

[0044] Control groups were established by adding only cells and maintenance medium, and by adding cells and virus. Control groups without H2O2 were established as b) and c). After culturing these systems for a certain period, the virus inhibition rate was calculated by detecting cytopathic effects and virus titers.

[0045] The results are as follows Figure 3 As shown, in the direct inactivation model, the product of Example 1 of this invention exhibited the strongest antiviral effect (inhibition rate >85%), significantly higher than Comparative Example 1 (Isatis root only, inhibition rate approximately 50%), Comparative Example 2 (nanozyme only, inhibition rate approximately 65%), and the control group without H2O2 (approximately 60%). Similar synergistic effects were also observed in the prevention and treatment models. This indicates that the composite material of this invention achieves a highly efficient antiviral effect through the triple synergistic effect of adsorption, nanozyme catalysis, and traditional Chinese medicine inhibition. Furthermore, different concentrations of the materials from Examples 1-3 and Comparative Examples 1-2 were co-incubated with Vero cells, such as... Figure 4 As shown, the results indicate that all materials exhibit low toxicity to cells within the effective concentration range (cell survival rate >85%), demonstrating good biosafety.

[0046] In summary, the composite nanozyme provided by this invention achieves synergistic effects of adsorption, catalysis, and inhibition by traditional Chinese medicine through its unique structural design, demonstrating high efficiency, safety, and green advantages in the prevention and control of porcine viral diarrhea, and has good application prospects.

Claims

1. An attapulgite composite nanoenzyme for treating swine viral diarrhea, characterized in that, It uses attapulgite as a carrier, on which the active ingredients of Isatis indigotica and the zinc oxide-cuprous oxide heterostructure nanostructure are simultaneously loaded.

2. The composite nanozyme according to claim 1, characterized in that, The zinc oxide-cuprous oxide heterojunction is uniformly distributed on the surface of attapulgite in the form of nanoparticles, and the particle size of the nanoparticles is less than 5 nm.

3. The composite nanozyme according to claim 1, characterized in that, The active ingredients of Isatis root include at least one of indigo, indirubin, adenosine, and polysaccharides.

4. A method for preparing the composite nanozyme according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Extraction and adsorption: Mix the powder of Isatis indigotica with a mixed solvent of alcohol and water, heat and reflux to extract, and filter to obtain the extract of Isatis indigotica; add the powder of attapulgite to the extract of Isatis indigotica, stir to allow the effective components of Isatis indigotica to be adsorbed on the attapulgite, and obtain a mixed suspension. (2) In-situ synthesis: Soluble zinc salt and soluble copper salt are added to the mixed suspension obtained in step (1), and the reaction is carried out under alkaline conditions to generate zinc oxide-cuprous oxide heterostructure nanostructures in situ on the surface of attapulgite; the reaction product is post-processed to obtain composite nanozyme.

5. The method according to claim 4, characterized in that, In step (1), the volume fraction of ethanol in the alcohol-water mixed solvent is 25%~70%; the temperature of the heating and reflux extraction is 50~75℃ and the time is 1~3 hours; the attapulgite powder is pretreated before being added to the Isatis root extract, and the pretreatment includes acid activation, water washing, drying and calcination.

6. The method according to claim 4, characterized in that, In step (2), the soluble zinc salt is zinc nitrate or zinc acetate, and the soluble copper salt is copper nitrate, copper acetate, copper chloride or copper sulfate; the alkaline conditions are adjusted by adding sodium hydroxide solution or ammonia water, and the pH value of the reaction system is controlled at 10~12.

7. The method according to claim 4, characterized in that, In step (2), the temperature of the heating reaction is 45~95℃ and the reaction time is 2~12 hours; the post-treatment includes centrifugation, washing with water and ethanol, and drying.

8. The method according to claim 4, characterized in that, The mass ratio of attapulgite, indigo powder, zinc salt, and copper salt is 1: (5~20): (0.05~0.5): (0.02~0.3).

9. The use of the composite nanozyme as described in any one of claims 1-3 in the preparation of a medicament for the prevention and / or treatment of swine viral diarrhea.

10. The use of the composite nanozyme as described in any one of claims 1-3 in the preparation of a feed additive for the prevention of swine viral diarrhea.

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