A nano enteric-targeted preparation for preventing and treating chicken intestinal bacterial diseases and a preparation method thereof

Abstract

The application relates to a nano enteric-targeting preparation for preventing and treating chicken intestinal bacterial diseases and a preparation method thereof, and belongs to the technical field of biological medicine preparation. The preparation method of the targeting preparation is as follows: polylactic acid-glycolic acid copolymer PLGA is taken as a carrier, drug components are added, and the drug components are mixed with polyethylene glycol PEG300 and Tween 80 to prepare a nano particle suspension liquid; after montmorillonite is added, further addition of acrylic resin II and preparation of an enteric layer are carried out to perform coating; after freeze-drying, probiotics are mixed to prepare a nano enteric-targeting preparation powder. In the application, the problem that drug components have an inhibiting effect on probiotics in the process of compounding microbial and traditional Chinese medicine active ingredients is effectively solved, the solubility of the drug components is improved, the targeting preparation forms a targeting effect on pathogenic bacteria in the intestinal tract, and the effect of synergistically preventing and treating multiple chicken intestinal bacterial diseases is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical preparation technology, specifically to a nano-enteric-coated targeted formulation for the prevention and treatment of intestinal bacterial diseases in chickens and its preparation method. Background Technology

[0002] Poultry intestinal bacterial diseases are a collective term for a group of diseases caused by pathogenic or opportunistic pathogenic bacteria, characterized by inflammation of the intestinal mucosa and digestive and respiratory dysfunction in poultry, including chickens, ducks, geese, and quails. Their core harms include stunted growth, increased feed conversion ratio / feed-to-egg ratio, diarrhea, and increased mortality, severely impacting the economic benefits of poultry farming. Furthermore, some pathogens (such as Salmonella) can be transmitted through the "poultry-meat / egg-human" chain, threatening public health and safety. Common types of pathogens include Salmonella, Escherichia coli, Clostridium perfringens, Campylobacter aerogenes, and Pasteurella multocida.

[0003] Chickens, as the most widely farmed poultry, are highly susceptible to intestinal bacterial diseases due to their physiological structure, high-density, and intensive farming practices. Different intestinal bacterial diseases have their own specificity. For example, pullorum disease (Salmonella) causes white, lime-like feces to stick to the anus of chicks; necrotic enteritis (Clostridium perfringens) causes bloody feces, and necrosis of the adrenal mucosa can be seen upon dissection; and intestinal Escherichia coli disease causes congestion and swelling of the adrenal mucosa, with contents that are mucous or foamy.

[0004] Most intestinal bacterial diseases in chickens are infectious. Salmonella-induced pullorum disease, fowl typhoid, and paratyphoid are highly contagious, with extremely high mortality rates in chicks. Adult chickens can remain asymptomatic carriers and continue transmission. These are zoonotic pathogens; if breeding chickens are infected, their eggs will also be contaminated. Chicks will show symptoms within 1-2 weeks of hatching, making it an inevitably contagious type. Campylobacter jejuni enteritis is also highly contagious, with extremely high infection rates in flocks. It is often a latent infection, with large amounts of bacteria excreted in feces, contaminating the environment and products. Escherichia coli infection and Clostridium perfringens necrotic enteritis are opportunistic infections.

[0005] Traditional Chinese medicine (TCM) preparations, due to their natural origin, have the advantage of being less likely to induce drug resistance compared to chemical antibiotics, and are gradually being used in the prevention and control of intestinal bacterial diseases in chickens. For example, berberine from Coptis chinensis, baicalin from Scutellaria baicalensis, and scutellarin from Pulsatilla chinensis have all been proven to have inhibitory effects on Escherichia coli, Salmonella, and Clostridium perfringens. However, TCM preparations also face challenges in their use, such as complex composition, lack of targeting of intestinal pathogens, easy degradation of drugs in the stomach, low absorption efficiency of drugs in the intestines, rapid metabolism, low bioavailability of drugs, and unsatisfactory prevention and control effects. Summary of the Invention

[0006] Based on the above problems, the purpose of this invention is to provide a nano-enteric-coated targeted formulation for the prevention and treatment of intestinal bacterial diseases in chickens.

[0007] Another objective of this invention is to provide a method for preparing the aforementioned nano-enteric-coated targeted formulation. This solves the technical problems of poor targeting, easy degradation of drug components in the stomach, and low bioavailability of drugs in the intestines in traditional Chinese medicine preparations for the prevention and treatment of intestinal bacterial diseases in chickens.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens is characterized by: using polylactic acid-glycolic acid copolymer (PLGA) as a carrier, adding drug components, mixing with PEG300 and Tween 80 to prepare a nanoparticle suspension, adding montmorillonite, further adding acrylic resin II to prepare an enteric coating layer for coating, freeze-drying, and then mixing with probiotics to prepare a nano-enteric-coated targeted formulation powder.

[0010] Furthermore, the probiotics are derived from Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ) and Lactobacillus acidophilus ( Lactobacillus acidophilus )composition.

[0011] Furthermore, the preparation of the nanoparticle suspension involves dissolving PLGA in anhydrous ethanol to form a PLGA solution, adding the drug component to obtain solution 1, dissolving PEG300 and Tween 80 in deionized water to obtain solution 2, mixing solution 1 and solution 2 to obtain a primary emulsion, and then adding deionized water and stirring to obtain the nanoparticle suspension.

[0012] Furthermore, the PLGA solution is prepared by adding polylactic acid-glycolic acid copolymer (PLGA) to deionized water under stirring at 20-30 rpm, and continuing stirring for 10-15 min. The mass ratio of anhydrous ethanol to PLGA is 6-8:1.

[0013] Furthermore, the drug components are composed of vine tea extract (containing dihydromyricetin ≥30%), coptis extract (containing berberine ≥20%), pulsatilla extract (containing pulsatilla saponins ≥15%), purslane extract (containing flavonoids ≥10%), and licorice extract (containing glycyrrhizic acid ≥20%).

[0014] Furthermore, solution 1 is prepared by mixing vine tea extract, coptis extract, pulsatilla extract, purslane extract, and licorice extract, adding them to a PLGA solution, and stirring at 20-30 rpm for 30-40 minutes.

[0015] Further, solution 1 and solution 2 are mixed and sheared and stirred at 8000~10000 rpm for 8~12 min to obtain a pre-emulsion. Deionized water of 4~5 times its mass is added and stirred at 1000~2000 rpm for 50~60 min to obtain a nanoparticle suspension.

[0016] Furthermore, the mass ratio of deionized water to Tween 80 in solution 2 is 100:0.5~0.8.

[0017] Furthermore, the preparation of the enteric layer involves centrifuging the nanoparticle suspension, taking the precipitate, adding deionized water, adding montmorillonite, stirring to disperse evenly, adjusting the pH value to 5.0~6.0, and adding an acrylic resin II ethanol solution to stir to form an enteric-coated targeted nanoparticle suspension.

[0018] More specifically, the nanoparticle suspension was centrifuged at 8000-10000 rpm for 10-15 min. After centrifugation, the supernatant was removed, and 20-30 times the amount of deionized water was added to the precipitate. The mixture was stirred and dispersed evenly. Montmorillonite was added, and the mixture was stirred at 500-800 rpm for 20-30 min. The pH was adjusted to 5.0-6.0 with 0.1 mol / L hydrochloric acid solution. Then, 2-5% (w / w) acrylic resin II ethanol solution was slowly added, and the mixture was stirred at 500-800 rpm for 30-40 min to obtain an enteric-coated targeted nanoparticle suspension.

[0019] Furthermore, the freeze-drying process involves adding mannitol to an enteric-coated targeted nanoparticle suspension and then freeze-drying it. First, the suspension is pre-frozen at -50 to -45°C for 2 to 3 hours. Then, a vacuum of -0.05 MPa to -0.08 MPa is set, and the temperature is increased to -15 to -10°C at a rate of 5 to 8°C / hour, and held for 120 to 150 minutes. Next, the temperature is increased to 0°C at a rate of 4 to 6°C / hour, and held for 260 to 300 minutes. Finally, the temperature is increased to 25 to 30°C at a rate of 5 to 8°C / hour, and held for 60 to 120 minutes to obtain freeze-dried powder.

[0020] Furthermore, the mass ratio of the enteric-coated targeted nanoparticle suspension to mannitol is 100:5~8.

[0021] Dihydromyricetin exhibits broad-spectrum inhibitory activity against the core pathogens of intestinal bacterial diseases in chickens (Gram-positive bacteria such as Clostridium perfringens, and Gram-negative bacteria such as Escherichia coli and Salmonella). Its target points are concentrated on three key aspects of bacterial cell membrane structure, energy metabolism, and genetic material replication, thus destroying bacteria from both structural and metabolic levels.

[0022] Berberine in Coptis chinensis can block bacterial reproduction and inhibit bacterial virulence factors, thus reducing pathogenicity.

[0023] Pulsatilla saponins in Pulsatilla chinensis can destroy the structure of bacterial spores and inhibit bacterial adhesion and colonization.

[0024] Flavonoids can bind to phospholipids on bacterial cell membranes, disrupting membrane integrity and making it easier for substances such as dihydromyricetin and berberine to enter the bacteria, thus enhancing their inhibitory / killing efficiency.

[0025] Glycyrrhizic acid can alleviate the irritation of the intestinal mucosa by ingredients such as dihydromyricetin and berberine in the formula through its "mucosal protection" and "anti-inflammatory" effects.

[0026] Based on traditional Chinese medicine preparations, probiotics are further added. Bacillus subtilis can secrete antimicrobial peptides to inhibit the growth of pathogenic bacteria, while consuming oxygen in the intestines to create an anaerobic growth environment for Lactobacillus acidophilus. Lactobacillus acidophilus lowers the pH value of the intestines by producing lactic acid, further inhibiting the reproduction of harmful bacteria. The two form a "synergistic colonization" effect, rebuilding the balance of intestinal flora. In addition, it also improves the solubility of traditional Chinese medicine components (such as dihydromyricetin and berberine) and enhances their antibacterial activity.

[0027] However, during the process of combining with traditional Chinese medicine ingredients, the saponins, alkaloids, and other components in the traditional Chinese medicine ingredients can inhibit the activity of probiotics, thus reducing their effectiveness.

[0028] In this invention, after mixing traditional Chinese medicine extracts with PLGA, montmorillonite is added. Montmorillonite has hydrophobic regions within its specific interlayer domains and hydrophilic hydroxyl groups on its surface. In colostrum, montmorillonite undergoes interlayer swelling. The polar groups in some active ingredients of the traditional Chinese medicine interact with the positive charge in the swollen interlayer, embedding and attaching themselves within it. The hydrophilic segments of PLGA bind to the hydrophilic hydroxyl groups on the surface of montmorillonite, while the hydrophobic segments bind to the weakly hydrophobic groups of the active ingredients of the traditional Chinese medicine, forming a ternary anchoring structure. A large number of active ingredients of the traditional Chinese medicine that have not entered the interlayer of montmorillonite are also anchored by PLGA. After coating with an enteric layer, it is then mixed with probiotics. The enteric layer forms a physical barrier between montmorillonite, active ingredients of the traditional Chinese medicine, and probiotics, preventing the inhibition of probiotics by the traditional Chinese medicine components during long-term storage and preventing the adsorption of probiotics by montmorillonite, which would hinder their subsequent colonization in the intestine.

[0029] After the targeted formulation reaches the intestine, probiotics are released simultaneously as the enteric layer dissolves. With the hydrolysis of PLGA, the active ingredients of the traditional Chinese medicine anchored by PLGA are released and preferentially act on free pathogens in the intestinal lumen. At the same time, montmorillonite adsorbs small molecule toxins and pathogens through interlayer charge and nanopores, causing the active ingredients of the traditional Chinese medicine in the interlayer to be passively targeted, forming a local high concentration to kill and eliminate them.

[0030] Furthermore, according to the weight parts, the composition comprises 30-40 parts of vine tea extract, 10-15 parts of coptis extract, 8-12 parts of pulsatilla extract, 6-10 parts of purslane extract, 3-5 parts of licorice extract, 5-8 parts of montmorillonite, 1-3 parts of Bacillus subtilis, 1-3 parts of Lactobacillus acidophilus, 5-8 parts of polylactic-co-glycolic acid copolymer (PLGA, molecular weight 5000-10000), 3-5 parts of acrylic resin II, 3-5 parts of polyethylene glycol 300, and 0.5-0.8 parts of Tween 80.

[0031] In this process, the nanostructures of the components are prepared by emulsification with excipients such as PLGA and PEG300. The anchoring effect of PLGA effectively disperses the active ingredients of traditional Chinese medicine, reduces agglomeration, and thus improves the solubility of the active ingredients of traditional Chinese medicine. The nanoscale interlayer pores and charge environment of montmorillonite embed some of the active ingredients into the interlayer spaces. The interlayer dispersion effect breaks the aggregation of traditional Chinese medicine molecules, improves solubility, and enhances the wettability of the drug, making it easier to be wetted and wrapped by intestinal water. Glycyrrhizic acid in licorice extract has weak surface activity, which helps to improve the solubility of macromolecular drug molecules.

[0032] A method for preparing a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens, characterized by comprising the following steps:

[0033] (1) Formula preparation: According to the weight parts, 30-40 parts of vine tea extract (containing dihydromyricetin ≥30%), 10-15 parts of coptis extract (containing berberine ≥20%), 8-12 parts of pulsatilla extract (containing pulsatilla saponins ≥15%), 6-10 parts of purslane extract (containing flavonoids ≥10%), 3-5 parts of licorice extract (containing glycyrrhizic acid ≥20%), 5-8 parts of montmorillonite (crushed through a 200-mesh sieve), 1-3 parts of Bacillus subtilis, 1-3 parts of Lactobacillus acidophilus, 5-8 parts of polylactic acid-glycolic acid copolymer (PLGA, molecular weight 5000-10000), 3-5 parts of acrylic resin II, 3-5 parts of polyethylene glycol 300, and 0.5-0.8 parts of Tween 80;

[0034] (2) Preparation of nano-suspension:

[0035] Place anhydrous ethanol in a container, add polylactic acid-glycolic acid copolymer (PLGA) while stirring at 20-30 rpm, and continue stirring for 10-15 min to obtain a PLGA solution. The mass ratio of anhydrous ethanol to PLGA is 6-8:1.

[0036] Extracts of vine tea, coptis chinensis, pulsatilla chinensis, purslane, and licorice were mixed and added to a PLGA solution. The mixture was stirred at 20-30 rpm for 30-40 min to obtain solution 1. Deionized water was taken, and PEG300 and Tween 80 were added and stirred to dissolve to obtain solution 2. Solution 1 was slowly added to solution 2, and the mixture was sheared and stirred at 8000-10000 rpm for 8-12 min to obtain a colostrum. 4-5 times the mass of deionized water was added, and the mixture was stirred at 1000-2000 rpm for 50-60 min to obtain a nanoparticle suspension. The mass ratio of deionized water to Tween 80 in solution 2 was 100:0.5-0.8.

[0037] (3) Preparation of enteric layer: The above nanoparticle suspension was centrifuged at 8000-10000 rpm for 10-15 min. After centrifugation, the supernatant was removed, and 20-30 times the amount of deionized water was added to the precipitate. The mixture was stirred and dispersed evenly. Montmorillonite was added, and the mixture was stirred at 500-800 rpm for 20-30 min. The pH was adjusted to 5.0-6.0 with 0.1 mol / L hydrochloric acid solution. Then, 2-5% acrylic resin II ethanol solution was slowly added, and the mixture was stirred at 500-800 rpm for 30-40 min to obtain enteric targeted nanoparticle suspension.

[0038] (4) Freeze-drying and probiotic loading:

[0039] Mannitol was added to an enteric-coated targeted nanoparticle suspension, and the mixture was freeze-dried. First, it was pre-frozen at -50 to -45°C for 2 to 3 hours. Then, a vacuum of -0.05 MPa to -0.08 MPa was set, and the temperature was increased to -15 to -10°C at a rate of 5 to 8°C / hour, and held for 120 to 150 minutes. Next, the temperature was increased to 0°C at a rate of 4 to 6°C / hour, and held for 260 to 300 minutes. Finally, the temperature was increased to 25 to 30°C at a rate of 5 to 8°C / hour, and held for 60 to 120 minutes to obtain a freeze-dried powder. The freeze-dried powder and probiotics were placed in a three-dimensional motion mixer under sterile conditions, and mixed at a speed of 15 to 20 rpm for 30 minutes to obtain the enteric-coated targeted nanoparticle formulation powder. The mass ratio of the enteric-coated targeted nanoparticle suspension to mannitol was 100:5 to 8.

[0040] In terms of intestinal microecological regulation, after the enteric coating dissolves in the intestine, probiotics are released into the intestine. The antibacterial components of traditional Chinese medicine outside the montmorillonite layer first "inhibit the excessive proliferation of harmful bacteria," reducing competition with probiotics and clearing obstacles to probiotic colonization. Subsequently, probiotics (Bacillus subtilis + Lactobacillus acidophilus) consolidate the balance of the flora by competing for nutrients and producing antibacterial substances. At the same time, probiotics can also promote the absorption of drugs in the intestine and enhance the efficacy of drugs. In addition, while montmorillonite adsorbs harmful bacteria, it does not affect the colonization of probiotics (montmorillonite is positively charged and will preferentially adsorb negatively charged harmful bacteria, while Lactobacillus acidophilus and Bacillus subtilis are wrapped with shell / capsule polysaccharides on their surfaces, reducing the exposure of negatively charged groups), further ensuring "microecological stability"—forming a synergistic cycle of "antibacterial inhibition-protection-regulation," avoiding the drawback of traditional antibacterial drugs that "kill bacteria and cause imbalance."

[0041] In this invention, by modifying the nanoparticles with PLGA nanocarriers, the phagocytosis of nanoparticles by intestinal mucosal cells makes it easier for drugs to accumulate at the lesion site, thus enhancing the drug's targeting effect. The nanoparticle structure also improves the solubility of traditional Chinese medicine drugs and enhances the absorption efficiency of drugs in the intestine.

[0042] The ultra-large specific surface area of ​​nanoparticles can significantly reduce the crystallinity of active ingredients in traditional Chinese medicine (such as dihydromyricetin and berberine), increasing their solubility in intestinal fluid. Simultaneously, PLGA, as a biodegradable carrier, slowly hydrolyzes in the intestine, achieving a "slow-release" of the medicinal components, avoiding sudden increases and decreases in blood drug concentration, and prolonging the duration of efficacy. Furthermore, short-chain fatty acids produced by probiotic metabolism can regulate the transport function of intestinal mucosal cells, further promoting the transmembrane absorption of drugs encapsulated by nanoparticles.

[0043] A pharmaceutical powder for preventing and treating intestinal bacterial diseases in chickens is characterized by: using polylactic acid-glycolic acid copolymer (PLGA) as a carrier, adding drug components, mixing with PEG300 and Tween 80 to prepare a nanoparticle suspension, adding montmorillonite, further adding acrylic resin II to prepare an enteric coating layer for coating, freeze-drying, and then mixing with probiotics to form a nano-enteric-coated targeted preparation powder.

[0044] Furthermore, the drug components are composed of vine tea extract (containing dihydromyricetin ≥30%), coptis extract (containing berberine ≥20%), pulsatilla extract (containing pulsatilla saponins ≥15%), purslane extract (containing flavonoids ≥10%), and licorice extract (containing glycyrrhizic acid ≥20%).

[0045] Furthermore, the probiotics are derived from Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ) and Lactobacillus acidophilus ( Lactobacillus acidophilus )composition.

[0046] Furthermore, the preparation of the nanoparticle suspension involves dissolving PLGA in anhydrous ethanol to form a PLGA solution, adding the drug component to obtain solution 1, dissolving PEG300 and Tween 80 in deionized water to obtain solution 2, mixing solution 1 and solution 2 to obtain a primary emulsion, and then adding deionized water and stirring to obtain the nanoparticle suspension.

[0047] Furthermore, the PLGA solution is prepared by adding polylactic acid-glycolic acid copolymer (PLGA) to deionized water under stirring at 20-30 rpm, and continuing to stir for 10-15 min to obtain the PLGA solution. The mass ratio of anhydrous ethanol to PLGA is 6-8:1.

[0048] Furthermore, solution 1 is prepared by mixing vine tea extract, coptis extract, pulsatilla extract, purslane extract, and licorice extract, adding them to a PLGA solution, and stirring at 20-30 rpm for 30-40 minutes.

[0049] Further, solution 1 and solution 2 are mixed and sheared and stirred at 8000~10000 rpm for 8~12 min to obtain a pre-emulsion. Deionized water of 4~5 times its mass is added and stirred at 1000~2000 rpm for 50~60 min to obtain a nanoparticle suspension.

[0050] Furthermore, the mass ratio of deionized water to Tween 80 in solution 2 is 100:0.5~0.8.

[0051] Furthermore, the preparation of the enteric layer involves centrifuging the nanoparticle suspension, taking the precipitate, adding deionized water, adding montmorillonite, stirring to disperse evenly, adjusting the pH value to 5.0~6.0, and adding an acrylic resin II ethanol solution to stir to form an enteric-coated targeted nanoparticle suspension.

[0052] More specifically, the nanoparticle suspension was centrifuged at 8000-10000 rpm for 10-15 min. After centrifugation, the supernatant was removed, and 20-30 times the amount of deionized water was added to the precipitate. The mixture was stirred and dispersed evenly. Montmorillonite was added, and the mixture was stirred at 500-800 rpm for 20-30 min. The pH was adjusted to 5.0-6.0 with 0.1 mol / L hydrochloric acid solution. Then, 2-5% (w / w) acrylic resin II ethanol solution was slowly added, and the mixture was stirred at 500-800 rpm for 30-40 min to obtain an enteric-coated targeted nanoparticle suspension.

[0053] Furthermore, the freeze-drying process involves adding mannitol to an enteric-coated targeted nanoparticle suspension and then freeze-drying it. First, the suspension is pre-frozen at -50 to -45°C for 2 to 3 hours. Then, a vacuum of -0.05 MPa to -0.08 MPa is set, and the temperature is increased to -15 to -10°C at a rate of 5 to 8°C / hour, and held for 120 to 150 minutes. Next, the temperature is increased to 0°C at a rate of 4 to 6°C / hour, and held for 260 to 300 minutes. Finally, the temperature is increased to 25 to 30°C at a rate of 5 to 8°C / hour, and held for 60 to 120 minutes to obtain freeze-dried powder.

[0054] Furthermore, the mass ratio of the enteric-coated targeted nanoparticle suspension to mannitol is 100:5~8.

[0055] Furthermore, according to the weight parts, the composition comprises 30-40 parts of vine tea extract, 10-15 parts of coptis extract, 8-12 parts of pulsatilla extract, 6-10 parts of purslane extract, 3-5 parts of licorice extract, 5-8 parts of montmorillonite, 1-3 parts of Bacillus subtilis, 1-3 parts of Lactobacillus acidophilus, 5-8 parts of polylactic-co-glycolic acid copolymer (PLGA, molecular weight 5000-10000), 3-5 parts of acrylic resin II, 3-5 parts of polyethylene glycol 300, and 0.5-0.8 parts of Tween 80.

[0056] The present invention has the following technical effects:

[0057] This invention effectively solves the problem of drug components inhibiting probiotics during the compounding process of microorganisms and traditional Chinese medicine components, and improves the solubility of drug components. The targeted formulation has a targeted effect on pathogenic bacteria in the intestine, thus improving the synergistic targeted prevention and treatment of various intestinal bacterial diseases in chickens. Detailed Implementation

[0058] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0059] The microorganisms used in this invention were purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC), among which Bacillus subtilis ( Bacillus subtilis The accession number for the sample is GDMCC NO.: 1.131 (live count ≥ 10^6). 9 CFU / g); Lactobacillus acidophilus ( Lactobacillus acidophilus The accession number is GDMCC NO.: 1.1807 (live count ≥ 10^6). 9 CFU / g).

[0060] Vine tea extract (containing dihydromyricetin ≥30%) was purchased from Shaanxi Hefiber Kang Biotechnology Co., Ltd.

[0061] Coptis chinensis extract (containing ≥20% berberine) was purchased from Xi'an Heers Peptide Biotechnology Co., Ltd.

[0062] Pulsatilla chinensis extract (containing ≥15% Pulsatilla chinensis saponins) was purchased from Shaanxi Dongjiang Kangtai Health Industry Co., Ltd.

[0063] Purslane extract (containing ≥10% flavonoids) was purchased from Shanxi Tiangu Biotechnology Co., Ltd.

[0064] Licorice extract (containing ≥20% glycyrrhizic acid) was purchased from Shaanxi Linruisi Rong Biotechnology Co., Ltd.

[0065] Example 1

[0066] A method for preparing a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens includes the following steps:

[0067] (1) Formula preparation: According to the weight parts, the following are the ingredients: 35 parts of vine tea extract (containing dihydromyricetin ≥30%), 12 parts of coptis extract (containing berberine ≥20%), 10 parts of pulsatilla extract (containing pulsatilla saponins ≥15%), 8 parts of purslane extract (containing flavonoids ≥10%), 4 parts of licorice extract (containing glycyrrhizic acid ≥20%), 6 parts of montmorillonite (crushed through a 200-mesh sieve), 2 parts of Bacillus subtilis, 2 parts of Lactobacillus acidophilus, 7 parts of PLGA with a molecular weight of 5000-10000, 4 parts of acrylic resin II, 4 parts of polyethylene glycol 300, and 0.7 parts of Tween 80;

[0068] (2) Preparation of nano-suspension:

[0069] Anhydrous ethanol was placed in a container, and polylactic acid-glycolic acid copolymer (PLGA) was added while stirring at 25 rpm. Stirring was continued for 12 min to obtain a PLGA solution. The mass ratio of anhydrous ethanol to PLGA was 7:1.

[0070] The extracts of vine tea, coptis chinensis, pulsatilla chinensis, purslane, and licorice were mixed and added to a PLGA solution. The mixture was stirred at 25 rpm for 35 min to obtain solution 1. Deionized water was taken, and PEG300 and Tween 80 were added and stirred to dissolve to obtain solution 2. Solution 1 was slowly added to solution 2, and the mixture was sheared and stirred at 9000 rpm for 10 min to obtain a colostrum. 4.5 times the mass of deionized water was added, and the mixture was stirred at 1500 rpm for 55 min to obtain a nanoparticle suspension. The mass ratio of deionized water to Tween 80 in solution 2 was 100:0.7.

[0071] (3) Preparation of enteric layer: The above nanoparticle suspension was centrifuged at 9000 rpm for 12 min. After centrifugation, the supernatant was removed, and 25 times the amount of deionized water was added to the precipitate. The mixture was stirred and dispersed evenly. Montmorillonite was added, and the mixture was stirred at 600 rpm for 25 min. The pH was adjusted to 5.5 with 0.1 mol / L hydrochloric acid solution. Then, 3% acrylic resin II ethanol solution was slowly added, and the mixture was stirred at 600 rpm for 35 min to obtain enteric targeted nanoparticle suspension.

[0072] (4) Freeze-drying and probiotic loading:

[0073] Mannitol was added to an enteric-coated targeted nanoparticle suspension, and the mixture was freeze-dried. First, it was pre-frozen at -50°C for 2.5 hours. Then, a vacuum of -0.06 MPa was set, and the temperature was increased to -12°C at a rate of 6°C / hour and held for 130 minutes. Next, the temperature was increased to 0°C at a rate of 5°C / hour and held for 280 minutes. Finally, the temperature was increased to 28°C at a rate of 6°C / hour and held for 100 minutes to obtain a freeze-dried powder. The freeze-dried powder and probiotics were placed in a three-dimensional motion mixer under sterile conditions and mixed at a speed of 18 rpm for 30 minutes to obtain the enteric-coated targeted nanoparticle formulation powder. The mass ratio of the enteric-coated targeted nanoparticle suspension to mannitol was 100:6.

[0074] Comparative Example 1

[0075] Compared with Example 1, montmorillonite was replaced with diatomaceous earth in the preparation process, and the remaining steps were the same as in Example 1.

[0076] Comparative Example 2

[0077] Compared to Example 1, the carrier PLGA was replaced with polylactic acid PLA, while the other steps remained the same.

[0078] The formulation powders prepared in Example 1, Comparative Example 1, and Comparative Example 2 were placed in a room temperature environment, and the effective content was tested at regular intervals, and the changes in the odor of the formulations were observed. The results are shown in Table 1.

[0079] In vitro experiments:

[0080] (1) Grouping: Set up a single Chinese medicine group, a montmorillonite + Chinese medicine group, and a montmorillonite + Chinese medicine + pathogenic bacteria group.

[0081] In vivo experiments:

[0082] Experimental group: From July 2024 to April 2025, 300 healthy chicks (1 day old) were selected. When they reached 7 days old, 80 of them were orally administered chicken-derived pathogens (Escherichia coli O78, 1×10⁻⁶). 8CFU / animal), and after 24 hours, the selected animals showed "typical intestinal disease symptoms" (diarrhea, loose stools with mucus; lethargy; decreased feed intake).

[0083] The chicks were divided into three groups: a blank control group (60 healthy chicks without infection), an infected control group (40 healthy chicks and 20 sick chicks mixed together), the targeted preparation group of Example 1 (40 healthy chicks and 20 sick chicks mixed together), the targeted preparation group of Comparative Example 1 (40 healthy chicks and 20 sick chicks mixed together), and the targeted preparation group of Comparative Example 2 (40 healthy chicks and 20 sick chicks mixed together). They were raised separately at a temperature of 28-30℃ (suitable temperature for chicks), a humidity of 60%-65%, a light exposure of 16 hours / day, and good ventilation. All groups were fed the same antibiotic-free basal feed and had free access to water.

[0084] Administration: Immediately after the start of rearing, administer the medication once daily via gavage for 5 consecutive days. The targeted preparation group received 2 g / kg via gavage, while the blank control group and the infection control group received an equal volume of physiological saline via gavage. Daily records were kept of the number of deaths, the number of sick chickens (sick but not dead + sick but dead), and changes in symptoms in each group. Mortality rate (number of dead chickens / total number of chickens in each group) and morbidity rate (number of sick chickens / total number of chickens in each group) were calculated. The test period was 8 days. The statistical results at the end of the test are shown in Table 1.

[0085] Table 1: Escherichia coli infection

[0086]

[0087] The same groups were used to test Salmonella and Clostridium perfringens, and the results are shown in Tables 2 and 3.

[0088] Table 2: Salmonella Infections

[0089]

[0090] Table 3: Clostridium perfringens infection

[0091]

[0092] It can be seen that the targeted formulation in Example 1 has a good preventive and therapeutic effect on enteric pathogens such as Escherichia coli, Staphylococcus aureus, and Clostridium perfringens, with low infection and mortality rates in chickens. However, in Comparative Example 1, montmorillonite was replaced with diatomaceous earth. Diatomaceous earth lacks interlayer structure, resulting in the drug being located on the surface and unable to form a passive targeting effect. In Comparative Example 2, the carrier PLGA was replaced with polylactic acid PLA. Since PLA lacks hydrophilic segments, its binding force with montmorillonite decreased, and its degradation was slow, resulting in slow release of the traditional Chinese medicine and insufficient drug concentration, which led to a significant increase in the infection and mortality rates of chickens against various pathogens.

[0093] Example 2

[0094] A method for preparing a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens includes the following steps:

[0095] (1) Formula preparation: According to the weight parts, the following are the ingredients: 30 parts of vine tea extract (containing dihydromyricetin ≥30%), 10 parts of coptis extract (containing berberine ≥20%), 8 parts of pulsatilla extract (containing pulsatilla saponins ≥15%), 6 parts of purslane extract (containing flavonoids ≥10%), 3 parts of licorice extract (containing glycyrrhizic acid ≥20%), 5 parts of montmorillonite (crushed through a 200-mesh sieve), 1 part of Bacillus subtilis, 1 part of Lactobacillus acidophilus, 5 parts of PLGA with a molecular weight of 5000-10000, 3 parts of acrylic resin II, 3 parts of polyethylene glycol 300, and 0.5 parts of Tween 80;

[0096] (2) Preparation of nano-suspension:

[0097] Anhydrous ethanol was placed in a container, and polylactic acid-glycolic acid copolymer (PLGA) was added while stirring at 20 rpm. Stirring was continued for 15 min to obtain a PLGA solution. The mass ratio of anhydrous ethanol to PLGA was 6:1.

[0098] The extracts of vine tea, coptis chinensis, pulsatilla chinensis, purslane, and licorice were mixed and added to a PLGA solution. The mixture was stirred at 20 rpm for 40 min to obtain solution 1. Deionized water was taken, and PEG300 and Tween 80 were added and stirred to dissolve to obtain solution 2. Solution 1 was slowly added to solution 2, and the mixture was sheared and stirred at 8000 rpm for 12 min to obtain a colostrum. 4-5 times the mass of deionized water was added, and the mixture was stirred at 1000 rpm for 60 min to obtain a nanoparticle suspension. The mass ratio of deionized water to Tween 80 in solution 2 was 100:0.5.

[0099] (3) Preparation of enteric layer: The above nanoparticle suspension was centrifuged at 8000 rpm for 15 min. After centrifugation, the supernatant was removed, 20 times the amount of deionized water was added to the precipitate, and the mixture was stirred and dispersed evenly. Montmorillonite was added, and the mixture was stirred at 500 rpm for 30 min. The pH was adjusted to 5.0 with 0.1 mol / L hydrochloric acid solution. Then, 2% acrylic resin II ethanol solution was slowly added, and the mixture was stirred at 500 rpm for 40 min to obtain enteric targeted nanoparticle suspension.

[0100] (4) Freeze-drying and probiotic loading:

[0101] Mannitol was added to an enteric-coated targeted nanoparticle suspension, and the mixture was freeze-dried. First, it was pre-frozen at -45°C for 3 hours. Then, a vacuum of -0.05 MPa was set, and the temperature was increased to -10°C at a rate of 5°C / hour and held for 120 minutes. Next, the temperature was increased to 0°C at a rate of 4°C / hour and held for 260 minutes. Finally, the temperature was increased to 25°C at a rate of 5°C / hour and held for 120 minutes to obtain a freeze-dried powder. The freeze-dried powder and probiotics were placed in a three-dimensional motion mixer under sterile conditions and mixed at 15 rpm for 30 minutes to obtain the enteric-coated targeted nanoparticle powder. The mass ratio of the enteric-coated targeted nanoparticle suspension to mannitol was 100:5.

[0102] Example 3

[0103] A method for preparing a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens includes the following steps:

[0104] (1) Formula preparation: According to the weight parts, the following are the ingredients: 40 parts of vine tea extract (containing dihydromyricetin ≥30%), 15 parts of coptis extract (containing berberine ≥20%), 12 parts of pulsatilla extract (containing pulsatilla saponins ≥15%), 10 parts of purslane extract (containing flavonoids ≥10%), 5 parts of licorice extract (containing glycyrrhizic acid ≥20%), 8 parts of montmorillonite (crushed through a 200-mesh sieve), 3 parts of Bacillus subtilis, 3 parts of Lactobacillus acidophilus, 8 parts of PLGA with a molecular weight of 5000-10000, 5 parts of acrylic resin II, 5 parts of polyethylene glycol 300, and 0.8 parts of Tween 80;

[0105] (2) Preparation of nano-suspension:

[0106] Anhydrous ethanol was placed in a container, and polylactic acid-glycolic acid copolymer (PLGA) was added while stirring at 30 rpm. Stirring was continued for 10 min to obtain a PLGA solution. The mass ratio of anhydrous ethanol to PLGA was 8:1.

[0107] The extracts of vine tea, coptis chinensis, pulsatilla chinensis, purslane, and licorice were mixed and added to a PLGA solution. The mixture was stirred at 30 rpm for 30 min to obtain solution 1. Deionized water was taken, and PEG300 and Tween 80 were added and stirred to dissolve to obtain solution 2. Solution 1 was slowly added to solution 2, and the mixture was sheared and stirred at 10000 rpm for 8 min to obtain a colostrum. Five times the mass of deionized water was added, and the mixture was stirred at 2000 rpm for 50 min to obtain a nanoparticle suspension. The mass ratio of deionized water to Tween 80 in solution 2 was 100:0.8.

[0108] (3) Preparation of enteric layer: The above nanoparticle suspension was centrifuged at 10,000 rpm for 10 min. After centrifugation, the supernatant was removed, 30 times the amount of deionized water was added to the precipitate, and the mixture was stirred and dispersed evenly. Montmorillonite was added, and the mixture was stirred at 800 rpm for 20 min. The pH was adjusted to 6.0 with 0.1 mol / L hydrochloric acid solution. Then, 5% acrylic resin II ethanol solution was slowly added, and the mixture was stirred at 800 rpm for 30 min to obtain enteric targeted nanoparticle suspension.

[0109] (4) Freeze-drying and probiotic loading:

[0110] Mannitol was added to an enteric-coated targeted nanoparticle suspension, and the mixture was freeze-dried. First, it was pre-frozen at -50°C for 2 hours. Then, a vacuum of -0.08 MPa was set, and the temperature was increased to -15°C at 8°C / h and held for 150 minutes. Next, the temperature was increased to 0°C at 6°C / h and held for 300 minutes. Finally, the temperature was increased to 30°C at 8°C / h and held for 60 minutes to obtain a freeze-dried powder. The freeze-dried powder and probiotics were placed in a three-dimensional motion mixer under sterile conditions and mixed at 20 rpm for 30 minutes to obtain the enteric-coated targeted nanoparticle powder. The mass ratio of the enteric-coated targeted nanoparticle suspension to mannitol was 100:8.

[0111] Stability test: Samples from Examples 1, 2, and 3 were placed in high temperature (60℃±2℃), high humidity (relative humidity 75%±5%), and light (illuminance 4500Lux±500Lux) for 1 month after being packaged in the market. Samples were taken and tested at 0, 10, and 30 days. The results are shown in Table 4.

[0112] Taking Example 3 as an example, the initial contents of dihydromyricetin and berberine hydrochloride were calculated:

[0113] Table 4: Product Stability

[0114]

[0115] The results above show that the samples in Examples 1, 2, and 3 of this product, using commercial packaging, all met the stability requirements under extreme conditions, indicating that the active ingredients in the formulation have excellent stability.

Claims

1. A method for preparing a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens, characterized in that: This formulation uses polylactic acid-glycolic acid copolymer (PLGA) as a carrier, adds drug components, and mixes with PEG300 and Tween 80 to prepare a nanoparticle suspension. After adding montmorillonite, acrylic resin II is further added to prepare an enteric coating layer for encapsulation. After freeze-drying, it is mixed with probiotics to prepare a nano-enteric-coated targeted formulation powder. The drug components consist of vine tea extract with a dihydromyricetin content ≥30%, coptis extract with a berberine content ≥20%, pulsatilla extract with a purslane saponin content ≥15%, purslane extract with a flavonoid content ≥10%, and licorice extract with a glycyrrhizic acid content ≥20%. By weight, the components are: vine tea extract 30-40 parts, coptis extract 10-15 parts, pulsatilla extract 8-12 parts, purslane extract 6-10 parts, licorice extract 3-5 parts, montmorillonite 5-8 parts, Bacillus subtilis 1-3 parts, Lactobacillus acidophilus 1-3 parts, and PLGA. 5-8 parts, acrylic resin II 3-5 parts, polyethylene glycol 300 3-5 parts, Tween 80 0.5-0.8 parts.

2. The preparation method of the nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens as described in claim 1, characterized in that: The preparation of the nanoparticle suspension involves dissolving PLGA in anhydrous ethanol to form a PLGA solution, adding the drug component to obtain solution 1, dissolving PEG300 and Tween 80 in deionized water to obtain solution 2, mixing solution 1 and solution 2 to obtain a primary emulsion, and then adding deionized water and stirring to obtain the nanoparticle suspension.

3. The preparation method of the nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens as described in claim 2, characterized in that: The PLGA solution is prepared by adding polylactic acid-glycolic acid copolymer (PLGA) to deionized water while stirring at 20-30 rpm, and continuing to stir for 10-15 minutes. The mass ratio of anhydrous ethanol to PLGA is 6-8:

1.

4. The preparation method of the nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens as described in claim 3, characterized in that: Solution 1 is obtained by mixing vine tea extract, coptis extract, pulsatilla extract, purslane extract, and licorice extract, adding them to a PLGA solution, and stirring at 20-30 rpm for 30-40 minutes.

5. The preparation method of a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens as described in claim 4, characterized in that: Solution 1 and Solution 2 are mixed and sheared and stirred at 8000~10000 rpm for 8~12 min to obtain a primary emulsion. 4~5 times its mass of deionized water is added, and the mixture is stirred at 1000~2000 rpm for 50~60 min to obtain a nanoparticle suspension. The mass ratio of deionized water to Tween 80 in Solution 2 is 100:0.5~0.

8.

6. The preparation method of a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens as described in claim 5, characterized in that: The preparation of the enteric layer involves centrifuging the nanoparticle suspension, collecting the precipitate, adding deionized water, adding montmorillonite, stirring to disperse evenly, adjusting the pH to 5.0-6.0, and then adding an acrylic resin II ethanol solution and stirring to form an enteric-coated targeted nanoparticle suspension.

7. The preparation method of the nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens as described in claim 6, characterized in that: The freeze-drying process involves adding mannitol to an enteric-coated targeted nanoparticle suspension and then freeze-drying it. First, the suspension is pre-frozen at -50 to -45°C for 2 to 3 hours. Then, a vacuum of -0.05 MPa to -0.08 MPa is set, and the temperature is increased to -15 to -10°C at a rate of 5 to 8°C / hour, and held for 120 to 150 minutes. Next, the temperature is increased to 0°C at a rate of 4 to 6°C / hour, and held for 260 to 300 minutes. Finally, the temperature is increased to 25 to 30°C at a rate of 5 to 8°C / hour, and held for 60 to 120 minutes to obtain freeze-dried powder.

8. The preparation method of the nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens as described in claim 7, characterized in that: The mass ratio of the enteric-coated targeted nanoparticle suspension to mannitol is 100:5~8.

9. A method for preparing a nano-enteric-coated targeted formulation for preventing and treating intestinal bacterial diseases in chickens, characterized in that, Includes the following steps: (1) Formula preparation: According to the weight parts, the following are the ingredients: 30-40 parts of vine tea extract with dihydromyricetin ≥30%, 10-15 parts of coptis extract with berberine ≥20%, 8-12 parts of pulveratum extract with pulveratum saponins ≥15%, 6-10 parts of purslane extract with flavonoids ≥10%, 3-5 parts of licorice extract with glycyrrhizic acid ≥20%, 5-8 parts of montmorillonite pulverized through a 200-mesh sieve, 1-3 parts of Bacillus subtilis, 1-3 parts of Lactobacillus acidophilus, 5-8 parts of polylactic acid-glycolic acid copolymer PLGA with a molecular weight of 5000-10000, 3-5 parts of acrylic resin II, 3-5 parts of polyethylene glycol 300, and 0.5-0.8 parts of Tween 80; (2) Preparation of nano-suspension: Place anhydrous ethanol in a container, add polylactic acid-glycolic acid copolymer (PLGA) while stirring at 20-30 rpm, and continue stirring for 10-15 min to obtain a PLGA solution. The mass ratio of anhydrous ethanol to PLGA is 6-8:

1. Extracts of vine tea, coptis chinensis, pulsatilla chinensis, purslane, and licorice were mixed and added to a PLGA solution. The mixture was stirred at 20-30 rpm for 30-40 min to obtain solution 1. Deionized water was taken, and PEG300 and Tween 80 were added and stirred to dissolve to obtain solution 2. Solution 1 was slowly added to solution 2, and the mixture was sheared and stirred at 8000-10000 rpm for 8-12 min to obtain a colostrum. 4-5 times the mass of deionized water was added, and the mixture was stirred at 1000-2000 rpm for 50-60 min to obtain a nanoparticle suspension. The mass ratio of deionized water to Tween 80 in solution 2 was 100:0.5-0.

8. (3) Preparation of enteric layer: The above nanoparticle suspension was centrifuged at 8000-10000 rpm for 10-15 min. After centrifugation, the supernatant was removed, and 20-30 times the amount of deionized water was added to the precipitate. The mixture was stirred and dispersed evenly. Montmorillonite was added, and the mixture was stirred at 500-800 rpm for 20-30 min. The pH was adjusted to 5.0-6.0 with 0.1 mol / L hydrochloric acid solution. Then, 2-5% acrylic resin II ethanol solution was slowly added, and the mixture was stirred at 500-800 rpm for 30-40 min to obtain enteric targeted nanoparticle suspension. (4) Freeze-drying and probiotic loading: Mannitol was added to an enteric-coated targeted nanoparticle suspension, and the mixture was freeze-dried. First, it was pre-frozen at -50 to -45°C for 2 to 3 hours. Then, a vacuum of -0.05 MPa to -0.08 MPa was set, and the temperature was increased to -15 to -10°C at a rate of 5 to 8°C / hour, and held for 120 to 150 minutes. Next, the temperature was increased to 0°C at a rate of 4 to 6°C / hour, and held for 260 to 300 minutes. Finally, the temperature was increased to 25 to 30°C at a rate of 5 to 8°C / hour, and held for 60 to 120 minutes to obtain a freeze-dried powder. The freeze-dried powder and probiotics were placed in a three-dimensional motion mixer under sterile conditions, and mixed at a speed of 15 to 20 rpm for 30 minutes to obtain the enteric-coated targeted nanoparticle formulation powder. The mass ratio of the enteric-coated targeted nanoparticle suspension to mannitol was 100:5 to 8.

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