Dipping agent for cow teats and method of preparation

Abstract

The application discloses a kind of cow teat medicated bath and preparation method, belong to the technical field of cow mastitis prevention and treatment.The application aims at solving the problems of insufficient effect of single antibacterial component, poor water solubility of phloretin and lack of synergistic anti-inflammatory effect in existing medicated bath.The method comprises dissolving chitosan in acetic acid aqueous solution, then adding phloretin, and then reacting with tannic acid aqueous solution to form composite nanoparticles.After synchronous treatment with glycerol glucoside in parallel plate alternating current field, a thickening agent is added.The medicated bath prepared by the method is used for cow teat medicated bath to prevent mastitis.

Description

Technical Field

[0001] This invention relates to the field of prevention and treatment of mastitis in dairy cows, specifically to a medicated teat bath agent for dairy cows and its preparation method. Background Technology

[0002] Mastitis in dairy cows is a common disease that seriously affects milk quality and farming efficiency. Post-milking teat disinfection with medicated baths is a primary method for preventing mastitis.

[0003] Currently, most commercially available dairy cow teat bath solutions use single antibacterial ingredients such as povidone-iodine and chlorhexidine as the main drug. Their mechanisms of action are relatively simple, and long-term use can easily lead to the development of drug resistance in pathogens. At the same time, single antibacterial ingredients lack a direct relief effect on existing inflammatory responses, while mastitis often occurs in conjunction with inflammatory responses. The separation of antibacterial and anti-inflammatory functions necessitates the use of different products, increasing breeding costs and operational complexity.

[0004] In recent years, plant-derived active ingredients have attracted attention due to their multi-target action characteristics. Phloretin, a natural flavonoid extracted from fruits such as apples and pears, possesses both antibacterial and anti-inflammatory activities. However, phloretin has extremely poor water solubility, making it difficult to effectively disperse and absorb when directly added to water-based bath formulations, thus limiting its application in dairy cow teat baths. Although nano-encapsulation technology can improve the dispersibility of poorly soluble drugs, conventional nanoparticle preparation processes often involve organic solvents, high temperatures, or complex emulsification processes, which are not suitable for large-scale preparation of water-based bath formulations.

[0005] Furthermore, the stratum corneum of cow's teat skin is relatively thick, making it difficult for the active ingredients in conventional medicated baths to effectively penetrate deep into the teat sphincter and ducts, resulting in unsatisfactory clearance of latent bacteria in these areas. Improving the dispersibility and stability of active ingredients while enhancing their transdermal penetration has been a long-standing challenge in the development of medicated teat baths for cows.

[0006] Therefore, developing a method for preparing a dairy cow teat bath agent that has synergistic antibacterial and anti-inflammatory effects, good dispersibility of active ingredients, and stable performance is still of great practical significance. Summary of the Invention

[0007] This invention provides a dairy cow teat medicated bath agent and its preparation method, aiming to solve the technical problems of existing dairy cow teat medicated bath agents, such as the easy development of drug resistance due to single antibacterial components, insufficient anti-inflammatory effects, poor water solubility and low bioavailability of phlorizin, and the lack of preparations with synergistic antibacterial and anti-inflammatory effects.

[0008] To achieve these and other advantages according to the present invention, a method for preparing a bovine teat bath is provided, comprising the following steps:

[0009] Step 1: Dissolve chitosan in an aqueous acetic acid solution with a volume fraction of 0.5% to 1.0% to prepare a chitosan solution with a mass-volume concentration of 0.2% to 1.0%.

[0010] Step 2: Under conditions of 20℃ to 30℃ and continuous stirring, add phlorizin powder to the chitosan solution. The amount of phlorizin powder added is 0.1% to 0.5% of the mass of the chitosan solution. Stir for 15 min to 30 min to obtain a chitosan-phlorizin premix (the chitosan molecular chain is rich in amino and hydroxyl groups, which can form intermolecular hydrogen bonds and hydrophobic interactions with the phenolic hydroxyl groups in the phlorizin molecules. Under continuous stirring, the surface of the phlorizin particles is wetted, swollen and gradually depolymerized by chitosan molecules, so that the phlorizin is uniformly dispersed in the chitosan solution as fine particles, forming a stable chitosan-phlorizin premix).

[0011] Step 3: Dissolve tannic acid in deionized water to prepare an aqueous solution of tannic acid with a mass-volume concentration of 0.5% to 2.0%;

[0012] Step 4: At 20℃ to 30℃, stir the tannic acid aqueous solution at a stirring speed of 200 rpm to 400 rpm. Simultaneously, add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 0.5 mL / min to 2.0 mL / min until the mass ratio of chitosan to tannic acid is 1:1 to 1:4. Continue stirring for 20 min to 40 min, then adjust the pH of the reaction system to 5.0 to 6.0 with sodium hydroxide solution, and continue stirring for 10 min to 30 min to obtain tannic acid-chitosan-phloretin composite nanoparticles. The suspension (the composite nanoparticles are a cross-linked network framework structure formed by the self-assembly of tannic acid and chitosan through electrostatic complexation and hydrogen bonding, with phlorizin loaded inside the framework through hydrophobic interactions and intermolecular hydrogen bonds. During the self-assembly process, the chitosan-phlorizin composite aggregates formed in step two undergo structural reorganization after the addition of tannic acid: the electrostatic complexation and multiple hydrogen bonding between tannic acid and chitosan cause the chitosan molecular chains to detach from the surface of phlorizin and re-crosslink with tannic acid to form a dense nanoscale network framework, with phlorizin loaded inside the framework in molecular form).

[0013] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field (the electrode spacing of the parallel plate electric field is 0.5 cm to 2 cm), the electric field strength is 50 V / cm to 200 V / cm, the treatment time is 5 min to 20 min, and at the same time, add 0.5% to 3.0% of the composite nanoparticle suspension by mass of glycerol glucoside; the frequency of the parallel plate AC electric field is preferably 40 Hz to 60 Hz.

[0014] Step 6: Add a thickener to the composite nanoparticle suspension after it has been subjected to an electric field, stir well, and obtain a cow teat bath solution.

[0015] Preferably, in the preparation method of the bovine teat bath agent, in step six, 0.01% to 0.05% of sodium glutamate powder by mass is first added to the composite nanoparticle suspension treated with an electric field, stirred until completely dissolved, allowed to stand for 15 to 30 minutes, and then a thickener is added.

[0016] Preferably, in the preparation method of the bovine teat bath agent, the mass of sodium glutamate powder added is 0.03% of the mass of the composite nanoparticle suspension.

[0017] Preferably, in the preparation method of the bovine teat bath agent, sodium glutamate powder is added to the composite nanoparticle suspension after electric field treatment and stirred until completely dissolved; then the pH value of the system is adjusted back to 4.5 to 4.8 with acetic acid solution (0.1% to 0.5%), and then the pH value of the system is adjusted back to 5.0 to 6.0 with sodium hydroxide solution (1 mol / L). After standing for 15 min to 30 min, a thickener is added.

[0018] Preferably, in the preparation method of the bovine teat bath agent, the thickener is one of sodium alginate, sodium carboxymethyl cellulose and xanthan gum, and the amount of thickener added is 0.1% to 1% of the mass of the composite nanoparticle suspension.

[0019] Preferably, in the preparation method of the bovine teat bath agent, in step one, the mass-volume concentration of the chitosan solution is 0.5%; in step two, the amount of phloretin powder added is 0.3% of the mass of the chitosan solution; in step four, the mass ratio of chitosan to tannic acid is 1:3, and the pH value is 5.5; in step five, 2% by mass of the composite nanoparticle suspension of glycerol glucoside is added.

[0020] Preferably, in the preparation method of the bovine teat bath agent, in step six, a freeze-drying protectant and a portion of sodium glutamate powder are added to the composite nanoparticle suspension after electric field treatment. The amount of freeze-drying protectant added is 5% to 15% of the mass of the composite nanoparticle suspension, and the portion of sodium glutamate powder is 0.005% to 0.01% of the mass of the composite nanoparticle suspension. After mixing evenly, the mixture is freeze-dried to obtain composite nanoparticle freeze-dried powder. Then, the composite nanoparticle freeze-dried powder is reconstituted with deionized water to the original volume, and another portion of sodium glutamate powder is added and stirred until completely dissolved.

[0021] Preferably, in the preparation method of the bovine teat bath agent, in step six, after adding sodium glutamate powder and stirring until completely dissolved, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to the system. The amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride added is 0.5 to 1.0 times the molar amount of sodium glutamate, and the amount of N-hydroxysuccinimide added is 0.5 to 1.0 times the molar amount of sodium glutamate. The reaction is stirred at 20°C to 30°C for 30 to 60 minutes. After the reaction is completed, the system is ultrafiltered to remove unreacted small molecule byproducts, and a thickener is added after standing.

[0022] The present invention also provides a dairy cow teat medicated bath agent, which is prepared by the preparation method of the dairy cow teat medicated bath agent described in any of the above technical solutions.

[0023] The present invention has at least the following beneficial effects:

[0024] I. The present invention, through the synergistic combination of tannic acid, phloretin, and glyceryl glucoside, produces a dairy cow teat bath agent that inhibits LPS-induced TNF-α by 68.3% and IL-6 by 63.7%, exhibiting excellent anti-inflammatory effects.

[0025] II. Based on the combination of tannic acid, phloretin, and glyceryl glucoside, the addition of sodium glutamate in this invention increases the TNF-α inhibition rate to 76.5% and the IL-6 inhibition rate to 71.8%, further enhancing the anti-inflammatory effect.

[0026] III. The tannic acid-chitosan-phlorizin composite nanoparticles prepared by this invention have an average particle size of 156.8 nm, a polydispersity index of 0.12, a zeta potential of +38.5 mV, an encapsulation efficiency of phlorizin of 89.6%, and a drug loading of 6.9%, effectively improving the water solubility and bioavailability of phlorizin.

[0027] IV. The bath preparation obtained by the present invention after EDC / NHS crosslinking modification has an average particle size of 142.3 nm, an encapsulation rate of 94.6%, and a TNF-α inhibition rate of 83.5% after being stored at 4°C for 90 days, and has good long-term storage stability.

[0028] V. The bath preparation of this invention has an erythema score of less than 0.3 and an edema score of less than 0.2 on the teat skin of dairy cows, and its irritation evaluation is non-irritating, indicating good safety in use.

[0029] The mechanism by which monosodium glutamate (MSG) enhances anti-inflammatory activity lies in the fact that the glutamate ions released after MSG dissolves in water are not only important precursors for amino acid metabolism in the body, but also key substrates for glutathione (GSH) synthesis. In the LPS-induced inflammatory microenvironment, after glutamate is taken up by macrophages, it enhances intracellular glutathione synthesis through the glutamine-glutamate metabolic pathway, improving cellular antioxidant defense capabilities and inhibiting the overactivation of the NF-κB signaling pathway, thereby downregulating the transcription and secretion of pro-inflammatory factors such as TNF-α and IL-6. On the other hand, the glutamate ions, with their polar carboxyl and amino groups, can form multiple hydrogen bonds and electrostatic interactions with the amino groups of chitosan and the phenolic hydroxyl groups of tannins on the surface of nanoparticles, promoting the stable adsorption of MSG onto the nanoparticle surface, improving the interfacial charge distribution and structural density of the nanoparticles, and thus enhancing its ability to deliver anti-inflammatory active ingredients. The synergistic effect of these two aspects of metabolic regulation and structural stabilization enables MSG to significantly enhance the anti-inflammatory activity of medicated baths.

[0030] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can implement it based on the description.

[0032] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0033] All raw materials used in the following examples were commercially available analytical grade or pharmaceutical grade products. Chitosan (degree of deacetylation ≥90%, viscosity 50-200 mPa·s), phloretin (purity ≥98%), tannic acid (purity ≥98%), glyceryl glucoside (purity ≥95%), and monosodium glutamate (purity ≥99%) were all purchased from commercial sources. Deionized water was used in the experiments.

[0034] It should be noted that the amounts of each component in the following embodiments are all expressed as mass percentages. In actual implementation, the absolute amount of each component can be determined according to the actual production scale. Taking the formulation ratio of Example 2 as an example, the amounts of each component are as follows: 5.0 g of chitosan (prepared into 1000 mL of chitosan solution with a mass-volume concentration of 0.5%), 3.0 g of phloretin (0.3% of the mass of chitosan solution), 15.0 g of tannic acid (prepared into 1500 mL of tannic acid aqueous solution with a mass-volume concentration of 1.0%, based on a chitosan to tannic acid mass ratio of 1:3), 50.0 g of glyceryl glucoside (2.0% of the mass of composite nanoparticle suspension), and 12.5 g of sodium carboxymethyl cellulose (0.5% of the mass of composite nanoparticle suspension). Phloretin is the key active ingredient in this invention. Phloretin is a natural flavonoid compound extracted from the peels and pomace of fruits such as apples and pears. This application uses commercially available plant-derived phloretin raw materials (purity ≥98%), which are much cheaper than high-purity reagent-grade products, and the raw materials are widely available and in ample supply. Even if we estimate based on the higher price of commercially available plant-derived phloretin raw materials, taking Example 2 as an example, according to the formula ratio of this example (1000 mL of chitosan solution and 1500 mL of tannic acid aqueous solution), the amount of phloretin used is only 3.0 g, accounting for only about 0.12% of the total mass of the entire bath agent (about 2565.5 g), which is extremely low. Even if we estimate based on the higher price of commercially available plant-derived phloretin raw materials, the raw material cost of phloretin in each batch of bath agent is less than 10 yuan, which accounts for a very low proportion of the production cost of the bath agent. The overall cost of the bath agent is comparable to that of similar commercially available products, which has obvious economic feasibility. The technical solution of this invention can achieve significant antibacterial and anti-inflammatory synergistic effects with extremely low dosage of phloretin, effectively preventing mastitis in dairy cows and reducing breeding losses, meeting actual production needs and possessing practicality.

[0035] Performance characterization methods for composite nanoparticles:

[0036] (1) Particle size and Zeta potential determination: The average particle size, polydispersity index (PDI) and Zeta potential of the composite nanoparticles were determined using a dynamic light scattering nanoparticle size analyzer (Malvern Zetasizer NanoZS). For the determination, an appropriate amount of composite nanoparticle suspension was taken, diluted with deionized water to a suitable concentration, and measured at 25℃. Each sample was measured in parallel 3 times, and the average value was taken.

[0037] (2) Encapsulation efficiency determination: The encapsulation efficiency of phlorizin was determined by ultrafiltration centrifugation. 1.0 mL of the composite nanoparticle suspension was placed in an ultrafiltration centrifuge tube (molecular weight cutoff 10 kDa), centrifuged at 4℃ and 12000 rpm for 30 min, and the filtrate was collected. The content of free phlorizin in the filtrate was determined by high performance liquid chromatography (HPLC). Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol-water-glacial acetic acid (45:55:0.5, v / v / v), flow rate: 1.0 mL / min, detection wavelength: 285 nm, column temperature: 30℃. Another volume of the composite nanoparticle suspension was taken, and an appropriate amount of methanol was added to disrupt the nanoparticle structure before determining the total phlorizin content. The encapsulation efficiency was calculated using the following formula:

[0038] Encapsulation rate (%) = (Total phloretin content - Free phloretin content) / Total phloretin content × 100%.

[0039] It should be noted that the "encapsulation rate" mentioned in this application refers to the percentage of phloretin loaded in molecular form inside the cross-linked network backbone of the composite nanoparticles to the total amount of phloretin fed in the application. This is a well-known term in the field, and its determination method adopts the standard ultrafiltration centrifugation method.

[0040] (3) Drug loading determination: The composite nanoparticle suspension was freeze-dried and weighed. The phlorizin content in the freeze-dried powder was determined by HPLC. The drug loading was calculated using the following formula:

[0041] Drug loading (%) = mass of phlorizin in lyophilized powder / total mass of lyophilized powder × 100%.

[0042] (4) In vitro antibacterial activity determination: Referring to the test method for the antibacterial performance of soluble antibacterial (inhibitory) products in the "Disinfection Technical Specifications" (2002 edition), the diameter of the inhibition zone of the medicated bath agent against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), the main pathogens of mastitis in dairy cows, was determined by the agar diffusion method. The activated test strains were adjusted to 1×10⁻⁶ with sterile physiological saline. 8 A 100 μL CFU / mL bacterial suspension was evenly spread onto an MH agar plate. Oxford cups (6 mm inner diameter, 8 mm outer diameter, 10 mm height) were evenly placed on each plate, and 200 μL of the sample to be tested was added to each cup. After incubation at 37℃ for 24 h, the diameter of the inhibition zone was measured. Three replicates were prepared for each sample, and the average value was taken.

[0043] (5) In vitro anti-inflammatory activity assay (TNF-α and IL-6 inhibition rate): A lipopolysaccharide (LPS)-induced mouse macrophage RAW 264.7 inflammation model was used. RAW 264.7 cells were cultured at 1×10⁶ cells per well. 5 Cells were seeded in 24-well plates and cultured for 24 h. A blank control group, an LPS model group, and a drug-treated group were then established. The drug-treated groups were pretreated with the pharmaceutical bath solutions prepared in each example and comparative example (diluted to a final concentration of 100 μg / mL with DMEM medium) for 2 h, followed by stimulation with LPS (final concentration 1 μg / mL) for 24 h. Cell supernatants were collected, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The inhibition rates of TNF-α and IL-6 were calculated using the following formula:

[0044] Inhibition rate (%) = (Inflammatory factor content in LPS model group - Inflammatory factor content in drug treatment group) / (Inflammatory factor content in LPS model group - Inflammatory factor content in blank control group) × 100%.

[0045] Three parallel wells were set for each sample, and the average value was taken.

[0046] (6) Characterization of the dispersion state of chitosan-phlorizin premix: The dispersion state of phlorizin in the chitosan-phlorizin premix obtained in step two was observed using an optical microscope (magnification 400×); at the same time, the average particle size of the phlorizin dispersed particles in the premix was measured using a dynamic light scattering nanoparticle size analyzer (Malvern Zetasizer Nano ZS). For the measurement, an appropriate amount of premix was taken, diluted with deionized water to a suitable concentration, and measured at 25℃. Each sample was measured in parallel 3 times, and the average value was taken.

[0047] Example 1

[0048] A method for preparing a dairy cow teat bath solution includes the following steps:

[0049] Step 1: Dissolve chitosan in a 0.5% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.2%.

[0050] Step 2: Under conditions of 20℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.1% of the mass of the chitosan solution. Stir for 15 min to obtain chitosan-phloretin premix.

[0051] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 0.5%;

[0052] Step 4: At 20℃, stir the tannic acid aqueous solution at a stirring speed of 200 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 0.5 mL / min until the mass ratio of chitosan to tannic acid is 1:1. After stirring continuously for 20 min, adjust the pH value of the reaction system to 5.0 with sodium hydroxide solution, and continue stirring for 10 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0053] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field (frequency 50 Hz), with an electric field strength of 50 V / cm and a treatment time of 5 min. At the same time, add 0.5% of the composite nanoparticle suspension by mass of glycerol glucoside.

[0054] Step 6: Add a thickener (sodium alginate) to the composite nanoparticle suspension after it has been subjected to an electric field. The amount of thickener added is 0.1% of the mass of the composite nanoparticle suspension. Stir well to obtain a dairy cow teat bath solution.

[0055] Example 2

[0056] A method for preparing a dairy cow teat bath solution includes the following steps:

[0057] Step 1: Dissolve chitosan in a 0.5% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.5%.

[0058] Step 2: Under conditions of 25℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.3% of the mass of the chitosan solution. Stir for 20 min to obtain chitosan-phloretin premix.

[0059] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 1.0%;

[0060] Step 4: At 25℃, stir the tannic acid aqueous solution at a stirring speed of 300 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 1.0 mL / min until the mass ratio of chitosan to tannic acid is 1:3. After stirring continuously for 30 min, adjust the pH value of the reaction system to 5.5 with sodium hydroxide solution, and continue stirring for 20 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0061] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field (frequency 50 Hz), with an electric field strength of 100 V / cm and a treatment time of 10 min. At the same time, add 2.0% by weight of glycerol glucoside to the composite nanoparticle suspension.

[0062] Step 6: Add a thickener (sodium carboxymethyl cellulose) to the composite nanoparticle suspension after it has been subjected to an electric field. The amount of thickener added is 0.5% of the mass of the composite nanoparticle suspension. Stir well to obtain a dairy cow teat bath solution.

[0063] Example 3

[0064] A method for preparing a dairy cow teat bath solution includes the following steps:

[0065] Step 1: Dissolve chitosan in a 1.0% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 1.0%.

[0066] Step 2: Under conditions of 30℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.5% of the mass of the chitosan solution. Stir for 30 min to obtain chitosan-phloretin premix.

[0067] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 2.0%;

[0068] Step 4: At 30℃, stir the tannic acid aqueous solution at a stirring speed of 400 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 2.0 mL / min until the mass ratio of chitosan to tannic acid is 1:4. After stirring continuously for 40 min, adjust the pH value of the reaction system to 6.0 with sodium hydroxide solution, and continue stirring for 30 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0069] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field (frequency 50 Hz), with an electric field strength of 200 V / cm and a treatment time of 20 min. At the same time, add 3.0% by weight of glycerol glucoside to the composite nanoparticle suspension.

[0070] Step 6: Add a thickener (xanthan gum) to the composite nanoparticle suspension after it has been subjected to an electric field. The amount of thickener added is 1.0% of the mass of the composite nanoparticle suspension. Stir well to obtain a dairy cow teat bath agent.

[0071] Example 4

[0072] A method for preparing a dairy cow teat bath solution includes the following steps:

[0073] Step 1: Dissolve chitosan in a 0.5% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.5%.

[0074] Step 2: Under conditions of 25℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.3% of the mass of the chitosan solution. Stir for 20 min to obtain chitosan-phloretin premix.

[0075] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 1.0%;

[0076] Step 4: At 25℃, stir the tannic acid aqueous solution at a stirring speed of 300 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 1.0 mL / min until the mass ratio of chitosan to tannic acid is 1:3. After stirring continuously for 30 min, adjust the pH value of the reaction system to 5.5 with sodium hydroxide solution, and continue stirring for 20 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0077] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field (frequency 50 Hz), with an electric field strength of 100 V / cm and a treatment time of 10 min. At the same time, add 2.0% by weight of glycerol glucoside to the composite nanoparticle suspension.

[0078] Step 6: Add 0.03% sodium glutamate powder (by mass of the composite nanoparticle suspension) to the composite nanoparticle suspension after electric field treatment, stir until completely dissolved, let stand for 20 min, then add thickener (sodium carboxymethyl cellulose) at a rate of 0.5% of the composite nanoparticle suspension mass, stir evenly to obtain the dairy cow teat bath agent.

[0079] Example 5

[0080] A method for preparing a dairy cow teat bath solution includes the following steps:

[0081] Step 1: Dissolve chitosan in a 0.5% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.5%.

[0082] Step 2: Under conditions of 25℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.3% of the mass of the chitosan solution. Stir for 20 min to obtain chitosan-phloretin premix.

[0083] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 1.0%;

[0084] Step 4: At 25℃, stir the tannic acid aqueous solution at a stirring speed of 300 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 1.0 mL / min until the mass ratio of chitosan to tannic acid is 1:3. After stirring continuously for 30 min, adjust the pH value of the reaction system to 5.5 with sodium hydroxide solution, and continue stirring for 20 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0085] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field (frequency 50 Hz), with an electric field strength of 100 V / cm and a treatment time of 10 min. At the same time, add 2.0% by weight of glycerol glucoside to the composite nanoparticle suspension.

[0086] Step 6: Add 0.03% (by weight) of sodium glutamate powder to the composite nanoparticle suspension after electric field treatment, and stir until completely dissolved. Then, adjust the pH of the system back to 4.6 with a 0.3% (by volume) acetic acid solution, and then adjust the pH of the system back to 5.5 with a 1 mol / L sodium hydroxide solution. Let it stand for 20 min, and then add a thickener (sodium carboxymethyl cellulose) at a concentration of 0.5% (by weight) of the composite nanoparticle suspension. Stir until homogeneous to obtain the dairy cow teat bath solution.

[0087] It should be noted that in Example 5, the pH value of the system was first adjusted back to 4.6 using a 0.3% acetic acid solution, and then adjusted back to 5.5 using a 1 mol / L sodium hydroxide solution. The mechanism of this back-and-forth pH adjustment is as follows: when the pH value is lowered to 4.6, the acidity of the system increases, the protonation degree of the amino groups on the chitosan molecular chain increases, the positive charge density on the nanoparticle surface increases, the electrostatic repulsion between chitosan segments is enhanced, and the nanoparticle network undergoes reversible relaxation and rearrangement. Subsequently, the pH value is adjusted back to 5.5, and the system is restored to the suitable pH range for electrostatic complexation between chitosan and tannic acid. The protonation degree of the chitosan amino groups decreases, the electrostatic complexation and hydrogen bonding between chitosan and tannic acid are enhanced again, and the nanoparticle network re-densifies and shrinks after rearrangement, so that the previously added monosodium glutamate and the loaded phloretin are more tightly embedded inside the nanoparticles, while reducing the structural defect sites on the nanoparticle surface. Therefore, the pH back-and-forth regulation promoted the secondary densification of the nanoparticle structure, which further improved the encapsulation efficiency (94.3%) and drug loading (7.6%) of Example 5 compared with Example 4, and also increased the TNF-α inhibition rate (80.2%) and IL-6 inhibition rate (75.6%) accordingly.

[0088] Example 6

[0089] A method for preparing a dairy cow teat bath solution includes the following steps:

[0090] Step 1: Dissolve chitosan in a 0.5% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.5%.

[0091] Step 2: Under conditions of 25℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.3% of the mass of the chitosan solution. Stir for 20 min to obtain chitosan-phloretin premix.

[0092] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 1.0%;

[0093] Step 4: At 25℃, stir the tannic acid aqueous solution at a stirring speed of 300 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 1.0 mL / min until the mass ratio of chitosan to tannic acid is 1:3. After stirring continuously for 30 min, adjust the pH value of the reaction system to 5.5 with sodium hydroxide solution, and continue stirring for 20 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0094] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field (frequency 50 Hz), with an electric field strength of 100 V / cm and a treatment time of 10 min. At the same time, add 2.0% by weight of glycerol glucoside to the composite nanoparticle suspension.

[0095] Step Six: Add a freeze-drying protectant and the first part of sodium glutamate powder to the composite nanoparticle suspension after electric field treatment; the freeze-drying protectant is a mixture of trehalose and mannitol in a mass ratio of 1:1, and the amount of freeze-drying protectant added is 10% of the mass of the composite nanoparticle suspension; the amount of the first part of sodium glutamate powder added is 0.008% of the mass of the composite nanoparticle suspension; after mixing evenly, freeze-dry (freezing temperature -40℃, vacuum degree ≤10 Pa, drying time 48 h) to obtain composite nanoparticle freeze-dried powder; then reconstitute the composite nanoparticle freeze-dried powder with deionized water to the original volume, then add the second part of sodium glutamate powder (the amount added is 0.022% of the mass of the composite nanoparticle suspension), stir until completely dissolved, let stand for 20 min, then add a thickener (sodium carboxymethyl cellulose), the amount of thickener added is 0.5% of the mass of the composite nanoparticle suspension, stir evenly, and obtain the dairy cow teat bath agent.

[0096] Example 7

[0097] A method for preparing a dairy cow teat bath solution includes the following steps:

[0098] Step 1: Dissolve chitosan in a 0.5% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.5%.

[0099] Step 2: Under conditions of 25℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.3% of the mass of the chitosan solution. Stir for 20 min to obtain chitosan-phloretin premix.

[0100] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 1.0%;

[0101] Step 4: At 25℃, stir the tannic acid aqueous solution at a stirring speed of 300 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 1.0 mL / min until the mass ratio of chitosan to tannic acid is 1:3. After stirring continuously for 30 min, adjust the pH value of the reaction system to 5.5 with sodium hydroxide solution, and continue stirring for 20 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0102] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field (frequency 50 Hz), with an electric field strength of 100 V / cm and a treatment time of 10 min. At the same time, add 2.0% by weight of glycerol glucoside to the composite nanoparticle suspension.

[0103] Step Six: Add a freeze-drying protectant and the first portion of monosodium glutamate powder to the composite nanoparticle suspension after electric field treatment; the freeze-drying protectant is a mixture of trehalose and mannitol in a 1:1 mass ratio, and the amount of freeze-drying protectant added is 10% of the mass of the composite nanoparticle suspension; the amount of the first portion of monosodium glutamate powder added is 0.008% of the mass of the composite nanoparticle suspension; after mixing evenly, freeze-dry (freezing temperature -40℃, vacuum degree ≤10 Pa, drying time 48 hours). h), to obtain lyophilized composite nanoparticle powder; then, the lyophilized composite nanoparticle powder was reconstituted with deionized water to the original volume, and then the second part of sodium glutamate powder (the amount added was 0.022% of the mass of the composite nanoparticle suspension) was added. After stirring until completely dissolved, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added to the system. The amount of EDC·HCl added was 0.8 times the molar amount of sodium glutamate, and the amount of NHS added was 0.8 times the molar amount of sodium glutamate. The reaction was stirred at 25℃ for 45 min. After the reaction was completed, the system was subjected to ultrafiltration (ultrafiltration membrane molecular weight cutoff 3 kDa, ultrafiltration pressure 0.2 MPa, ultrafiltration 3 times) to remove unreacted small molecule byproducts. After standing for 20 min, a thickener (sodium carboxymethyl cellulose) was added. The amount of thickener added was 0.5% of the mass of the composite nanoparticle suspension. After stirring evenly, a dairy cow teat bath agent was obtained.

[0104] Example 8

[0105] A method for preparing a dairy cow teat bath solution includes the following steps:

[0106] Step 1: Dissolve chitosan in a 0.5% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.5%.

[0107] Step 2: Under conditions of 25℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.3% of the mass of the chitosan solution. Stir for 20 min to obtain chitosan-phloretin premix.

[0108] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 1.0%;

[0109] Step 4: At 25℃, stir the tannic acid aqueous solution at a stirring speed of 300 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 1.0 mL / min until the mass ratio of chitosan to tannic acid is 1:3. After stirring continuously for 30 min, adjust the pH value of the reaction system to 5.5 with sodium hydroxide solution, and continue stirring for 20 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0110] Step 5: The tannic acid-chitosan-phloretin composite nanoparticle suspension was placed on a parallel plate electrode and a dual-frequency modulated AC electric field was applied. The dual-frequency modulated AC electric field was formed by superimposing a first frequency f1 and a second frequency f2, where f1 = 50 Hz and f2 = 80 Hz (f1 ≠ f2 and f1 / f2 = 0.625, which is not an integer). After the two frequencies were superimposed, a composite electric field with periodic modulation of electric field strength was formed. The maximum electric field strength of the composite electric field was 150 V / cm, and the treatment time was 10 min. At the same time, 2.0% of the mass of the composite nanoparticle suspension of glycerol glucoside was added. (After the two frequencies were superimposed, since the frequencies f1 and f2 are different and the ratio is not an integer, the strength of the synthesized electric field is periodically beat-modulated, so that the nanoparticles undergo periodic electrodynamic effects in the electric field, promoting the binding of glycerol glucoside to the nanoparticles).

[0111] Step 6: Add a thickener (sodium carboxymethyl cellulose) to the composite nanoparticle suspension after it has been subjected to an electric field. The amount of thickener added is 0.5% of the mass of the composite nanoparticle suspension. Stir well to obtain a dairy cow teat bath solution.

[0112] Example 9

[0113] A method for preparing a dairy cow teat bath solution includes the following steps:

[0114] Step 1: Dissolve chitosan in a 0.5% (v / v) aqueous acetic acid solution to prepare a chitosan solution with a mass-volume concentration of 0.5%.

[0115] Step 2: Under conditions of 25℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.3% of the mass of the chitosan solution. Stir for 20 min to obtain chitosan-phloretin premix.

[0116] Step 3: Dissolve tannic acid in deionized water to prepare a tannic acid aqueous solution with a mass-volume concentration of 1.0%;

[0117] Step 4: At 25℃, stir the tannic acid aqueous solution at a stirring speed of 300 rpm, and simultaneously add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 1.0 mL / min until the mass ratio of chitosan to tannic acid is 1:3. After stirring continuously for 30 min, adjust the pH value of the reaction system to 5.5 with sodium hydroxide solution, and continue stirring for 20 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension.

[0118] Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension on a parallel plate electrode and apply a dual-frequency modulated AC electric field. The dual-frequency modulated AC electric field is formed by superimposing a first frequency f1 and a second frequency f2, where f1 = 75 Hz and f2 = 90 Hz (f1 ≠ f2 and f1 / f2 = 5 / 6, which is not an integer). After the two frequencies are superimposed, a composite electric field with a periodic modulation of electric field strength is formed. The maximum electric field strength of the composite electric field is 150 V / cm, the treatment time is 10 min, and 2.0% of the mass of the composite nanoparticle suspension of glycerol glucoside is added simultaneously.

[0119] Step Six: Add a lyophilization protectant and the first part of monosodium glutamate (MSG) powder to the composite nanoparticle suspension after electric field treatment. The lyophilization protectant is a mixture of trehalose and mannitol in a 1:1 mass ratio, and the amount of the lyophilization protectant added is 10% of the mass of the composite nanoparticle suspension. The amount of the first part of MSG powder added is 0.008% of the mass of the composite nanoparticle suspension. After mixing evenly, freeze-dry (freezing temperature -40℃, vacuum degree ≤10 Pa, drying time 48 h) to obtain composite nanoparticle lyophilized powder. Then, reconstitute the composite nanoparticle lyophilized powder with deionized water to the original volume, and then add the second part of MSG powder (the amount added is 0.022% of the mass of the composite nanoparticle suspension). Stir until completely dissolved, and then add EDC·HCl and NHS to the system. The amount of EDC·HCl added is 0.8 times the molar amount of MSG, and the amount of NHS added is 0.8 times the molar amount of MSG. Stir the reaction at 25℃ for 45 minutes. min; after the reaction was completed, the system was subjected to ultrafiltration (ultrafiltration membrane molecular weight cutoff 3 kDa, ultrafiltration pressure 0.2 MPa, ultrafiltration 3 times) to remove unreacted small molecule byproducts. After standing for 20 min, a thickener (sodium carboxymethyl cellulose) was added. The amount of thickener added was 0.5% of the mass of the composite nanoparticle suspension. The mixture was stirred evenly to obtain the dairy cow teat bath agent.

[0120] It should be noted that the above embodiments 8 and 9 exemplarily provide two dual-frequency combinations: f1=50 Hz, f2=80 Hz, and f1=75 Hz, f2=90 Hz. According to the technical concept of the present invention, as long as f1 is between 50 Hz and 100 Hz, f2 is between 60 Hz and 120 Hz, and f1≠f2 and f1 / f2 is not an integer, the superposition of the two frequencies can form a composite electric field whose electric field intensity is periodically modulated with time, ensuring that the nanoparticles in each region receive sufficient electric field action within a processing time of 5 min to 20 min. Those skilled in the art, after reading this specification, can select a suitable combination of f1 and f2 within the above frequency range to achieve uniform processing without any creative effort, based on the actual size of the processing cavity and the mobility of the nanoparticles.

[0121] Comparative Example 1

[0122] The same method as in Example 2 was used to prepare the bovine teat bath agent, except that: no phlorizin powder was added in step two, that is, no phlorizin was added to the chitosan solution, and the chitosan solution was directly used as a premix to participate in the subsequent reaction. The remaining steps and parameters were exactly the same as in Example 2.

[0123] Comparative Example 2

[0124] The same method as in Example 2 was used to prepare the bovine teat bath agent, except that tannic acid was not added in steps three and four. That is, the preparation of the tannic acid aqueous solution and the reaction steps with the chitosan-phloretin premix were omitted. The chitosan-phloretin premix was directly used as the complex system in step five. The remaining steps and parameters were exactly the same as in Example 2.

[0125] Comparative Example 3

[0126] The same method as in Example 2 was used to prepare the bovine teat bath agent, except that glycerol glucoside was not added in step five, that is, glycerol glucoside was not added when the composite nanoparticle suspension was treated in the parallel plate AC electric field. The remaining steps and parameters were exactly the same as in Example 2.

[0127] Comparative Example 4

[0128] The dairy cow teat bath agent was prepared using the same method as in Example 2, except that: no phloretin powder was added in step two; and no tannic acid was added in steps three and four, i.e., no phloretin was added to the chitosan solution, and it did not react with tannic acid, but directly entered step five as the chitosan solution; in step five, glycerol glucoside was added and electric field treatment was performed as in Example 2, and the remaining steps and parameters were exactly the same as in Example 2.

[0129] Comparative Example 5

[0130] The dairy cow teat bath agent was prepared using the same method as in Example 2, except that tannic acid was not added in steps three and four; and glycerol glucoside was not added in step five. That is, the chitosan-phloretin premix was directly subjected to electric field treatment under conditions without tannic acid and glycerol glucoside. The remaining steps and parameters were exactly the same as in Example 2.

[0131] Comparative Example 6

[0132] The same method as in Example 2 was used to prepare the bovine teat bath agent, except that: no phloretin powder was added in step two; no glycerol glucoside was added in step five, i.e., the chitosan solution and tannic acid formed a complex, but no phloretin was added, and no glycerol glucoside was added during the electric field treatment. The remaining steps and parameters were exactly the same as in Example 2.

[0133] Comparative Example 7

[0134] The same method as in Example 2 was used to prepare the bovine teat bath agent, except that in step five, no parallel plate AC electric field treatment was applied to the composite nanoparticle suspension. That is, glycerol glucoside was added and stirred for the corresponding time under no electric field conditions. The remaining steps and parameters were exactly the same as in Example 2.

[0135] Comparative Example 8

[0136] The same method as in Example 2 was used to prepare the bovine teat bath agent, except that the nanoparticle preparation process in steps one to four was omitted. Instead, chitosan, phloretin, and tannic acid were dissolved / dispersed in deionized water in the same amounts as in Example 2. The mixture was then stirred and mixed at 25°C for 30 min. Glyceryl glucoside was added under the conditions in step five and subjected to electric field treatment. Finally, a thickener was added under the conditions in step six. All other parameters were the same as in Example 2.

[0137] Comparative Example 9

[0138] The same method as in Example 2 was used to prepare the bovine teat bath agent, except that glycerol glucoside was not added in step five (i.e., glycerol glucoside was not added during the electric field treatment). Instead, glycerol glucoside was reserved for step six and added together with the thickener before the thickener was added and simply stirred and mixed. The remaining steps and parameters were exactly the same as in Example 2.

[0139] Comparative Example 10

[0140] The same method as in Example 2 was used to prepare the bovine teat bath agent, with the only differences being: in step one, chitosan was not used, i.e., the addition of chitosan was omitted, and deionized water was used directly instead of chitosan solution; in step two, there was no chitosan solution, so phlorizin powder was directly dispersed in deionized water to form phlorizin suspension; in steps three and four, the tannic acid aqueous solution was still prepared according to Example 2, but in step four, the phlorizin suspension obtained in step two (without chitosan) was added to the tannic acid aqueous solution, and the remaining steps and parameters were exactly the same as in Example 2.

[0141] Experimental Example 1: Performance Testing of Bathing Agents from Each Example and Comparative Example

[0142] Following the characterization methods described above, the particle size, zeta potential, encapsulation efficiency, drug loading, in vitro antibacterial activity, and in vitro anti-inflammatory activity of the dairy cow teat bath preparations prepared in each example and comparative example were determined. The results are shown in Table 1.

[0143] Table 1. Performance test results of the bath preparations used in each embodiment and comparative example.

[0144] Note: "—" indicates that the sample did not form a nanoparticle system and the corresponding indicators could not be accurately measured; in Comparative Example 1, since phlorizin was not present, the encapsulation efficiency and drug loading could not be measured; in Comparative Example 8, since a nanoparticle system was not formed, its particle size, PDI, Zeta potential, encapsulation efficiency, and drug loading were not applicable (indicated by "—"); in Comparative Example 10, due to the lack of chitosan, the nanoparticles could not be effectively formed, resulting in extremely low encapsulation efficiency and drug loading.

[0145] Meanwhile, the dispersion state of the chitosan-phloretin premix obtained in step two of Example 2 was characterized according to the above characterization method (6). The results showed that after stirring, the average particle size of the chitosan-phloretin complex aggregates in the premix was 12.6 μm (D50), the particle size distribution was uniform, no coarse particles with a particle size greater than 50 μm were observed, and no obvious sedimentation occurred within 30 min of the premix, which showed a uniform and stable dispersion state. As a control, the same mass of phloretin powder was directly added to deionized water and stirred for 20 min. The average particle size of the phloretin particles in the resulting suspension was 85.6 μm (D50), and obvious sedimentation occurred after standing for 10 min. The above results demonstrate that chitosan solution significantly wets, swells, and disperses phlorizin particles through intermolecular hydrogen bonds and hydrophobic interactions. This enables phlorizin to be stably dispersed as fine particles in the chitosan solution, overcoming the technical problems of poor water solubility and difficulty in uniform dispersion of phlorizin. This lays the foundation for subsequent self-assembly with tannic acid to form composite nanoparticles. It should be noted that the 12.6 μm chitosan-phlorizin composite aggregates are intermediate structures in the preparation process. After the addition of tannic acid, they undergo molecular-level deconstruction and reconstruction. The final nanoparticles contain phlorizin dispersed and loaded within the cross-linked network framework in molecular form, rather than being encapsulated as 12.6 μm aggregates.

[0146] Results analysis:

[0147] As shown in Table 1, in Examples 1-3, the TNF-α inhibition rate (68.3%) and IL-6 inhibition rate (63.7%) of Example 2 were significantly higher than those of Example 1 (52.8%, 48.6%) and Example 3 (64.1%, 59.5%), demonstrating that the preferred intermediate value combination used in Example 2 could achieve the best anti-inflammatory effect within a broad range of values. In Example 4, after introducing monosodium glutamate (MSG) based on Example 2, the TNF-α inhibition rate increased from 68.3% to 76.5%, and the IL-6 inhibition rate increased from 63.7% to 71.8%, indicating that the addition of MSG further enhanced the anti-inflammatory effect.

[0148] Regarding anti-inflammatory activity, Example 2 showed inhibition rates of 68.3% and 63.7% for TNF-α and IL-6, respectively. When phlorizin (Comparative Example 1), tannic acid (Comparative Example 2), or glycerol glucoside (Comparative Example 3) were deleted, the inhibition rates of TNF-α decreased to 35.6%, 38.2%, and 40.5%, respectively, and the inhibition rates of IL-6 decreased to 31.2%, 33.5%, and 36.8%, respectively, all showing extremely significant decreases. Comparative Example 4 (containing only glycerol glucoside) showed inhibition rates of only 25.3% and 21.5% for TNF-α and IL-6; Comparative Example 5 (containing only phlorizin) showed 28.6% and 24.3%; and Comparative Example 6 (containing only tannic acid) showed 32.4% and 28.7%. The anti-inflammatory activity of the pairwise deletion groups was significantly reduced compared to the single deletion groups, all less than half of that of Example 2. The above results indicate that the anti-inflammatory effects of tannic acid, phloretin, and glycerol glucoside are not simply additive. The TNF-α inhibition rate (68.3%) of Example 2, which included all three, was much higher than that of the single deletion group (35.6%-40.5%) and the pairwise deletion group (25.3%-32.4%), indicating a significant synergistic anti-inflammatory effect among the three.

[0149] The particle size of Comparative Examples 4-6 (pairwise deletion groups) was significantly increased (185.6-376.4 nm), the PDI was significantly broadened (0.25-0.58), and the Zeta potential was significantly reduced (+12.5 to +25.3 mV), indicating that the nanoparticle structure was severely damaged. Comparative Example 10 (chitosan-free) had a particle size of 412.5 nm and a PDI as high as 0.65, making it almost impossible to form stable nanoparticles. While the nanoparticles of Comparative Examples 1-3 (single deletion groups) could form certain structures, their encapsulation efficiency and drug loading were lower than those of Example 2. This indicates that tannic acid, phlorizin, and glyceroglucoside are all indispensable for the construction and stabilization of the nanoparticle structure.

[0150] In Example 4, after adding monosodium glutamate, the TNF-α inhibition rate increased from 68.3% in Example 2 to 76.5%, and the IL-6 inhibition rate increased from 63.7% to 71.8%, representing increases of 12.0% and 12.7%, respectively. In Example 5, after pH adjustment, the TNF-α inhibition rate further increased to 80.2%, and the IL-6 inhibition rate increased to 75.6%. In Example 7, after EDC / NHS crosslinking, the TNF-α inhibition rate further increased to 84.3%, and the IL-6 inhibition rate increased to 79.8%. In Example 9, after combining a dual-frequency modulated electric field with EDC / NHS crosslinking, the TNF-α inhibition rate reached 87.6%, and the IL-6 inhibition rate reached 83.2%. The above optimized processes made the nanoparticle structure more compact, further enhancing the anti-inflammatory activity.

[0151] In Comparative Example 9, the addition of glycerol glucoside was delayed until step six. The TNF-α inhibition rate (46.8%) and IL-6 inhibition rate (42.5%) decreased by 31.5% and 33.3% respectively compared with Example 2 (68.3% and 63.7%), indicating that glycerol glucoside must be added simultaneously during the electric field treatment to fully exert its synergistic anti-inflammatory effect.

[0152] The structural basis for the synergistic anti-inflammatory effect of glycerol glucoside interacting with nanoparticles in an electric field environment lies in the fact that the glucose group in the glycerol glucoside molecule has multiple adjacent hydroxyl groups, while the glycerol group endows it with strong hydrophilicity and flexible segments. During the parallel plate AC electric field treatment, the glycerol glucoside in the nanoparticle suspension is polarized and oriented under the drive of the electric field. Its glucose group forms bridges with the phenolic hydroxyl groups of tannic acid, the amino groups of chitosan, and the phenolic hydroxyl groups of phlorizin on the nanoparticle surface through multiple hydrogen bonds, allowing the glycerol glucoside to be embedded in the chitosan-tannic acid cross-linked network backbone in the form of molecular bridges. This strengthens the three-dimensional network structure of the nanoparticles and improves their loading stability of phlorizin. At the same time, glycerol glucoside itself has anti-inflammatory activity by downregulating the expression of pro-inflammatory factors. After being embedded in the nanoparticle backbone, it works synergistically with phlorizin and tannic acid at the molecular level to form multiple anti-inflammatory targets, so that the synergistic anti-inflammatory effect can be fully exerted. If glycerol glucoside is added in step six, the nanoparticle network framework is already basically formed and there is no longer an electric field driving it. Glycerol glucoside can only be dispersed in the suspension in a free form and cannot be embedded in the nanoparticle framework to form the above-mentioned bridging structure. Its anti-inflammatory activity and structural enhancement effect cannot be exerted, and thus the anti-inflammatory effect is greatly reduced.

[0153] Comparative Example 10, due to the lack of chitosan, could hardly form nanoparticles (particle size 412.5 nm, PDI 0.65), with an encapsulation efficiency of only 18.7% and inhibition rates of TNF-α and IL-6 of only 22.5% and 19.2%, respectively, exhibiting the worst anti-inflammatory activity among all samples. This indicates that chitosan, as a scaffold material for nanoparticles, is an indispensable basic component for constructing composite nanoparticles.

[0154] As shown in Table 1, regardless of whether a single component was omitted (Comparative Examples 1-3), two components were omitted simultaneously (Comparative Examples 4-6), the electric field treatment conditions were changed (Comparative Examples 7 and 9), the phloretin addition method was changed (Comparative Example 8), or the chitosan backbone was omitted (Comparative Example 10), the TNF-α inhibition rate and IL-6 inhibition rate of the prepared bath preparation were significantly lower than those of Example 2. Among them, Comparative Example 7 (without electric field treatment), which had the best anti-inflammatory effect, had a TNF-α inhibition rate of only 56.2%, a decrease of 17.7% compared to Example 2 (68.3%); and an IL-6 inhibition rate of only 52.1%, a decrease of 18.2% compared to Example 2 (63.7%). This indicates that each preparation step and component composition in the technical solution of the present invention makes an indispensable contribution to the anti-inflammatory effect, and any change in conditions will weaken the anti-inflammatory performance of the final product to varying degrees.

[0155] Experiment Example 2: Stability Test

[0156] The medicated bath preparations prepared in Examples 2, 4, 7, and 9 were sealed and stored in the dark at 4°C. Samples were taken at 0, 30, 60, and 90 days to determine particle size, PDI, encapsulation efficiency, and anti-inflammatory activity (TNF-α inhibition rate) to investigate the storage stability of the medicated bath preparations. The results are shown in Table 2.

[0157] It should be noted that Examples 2, 4, 7, and 9 were selected for stability evaluation in this experiment, while Examples 5 and 6 were not included. The reasons are as follows: Example 5 adds a pH back-and-forth adjustment step to Example 4, aiming to further improve the encapsulation efficiency and anti-inflammatory activity through secondary densification of the nanoparticle structure. It is a continuation and improvement of the process in Example 4. Moreover, the formulation and nanoparticle skeleton composition of Example 5 are consistent with those of Example 4, only the pH adjustment path is different. The two have the same nanoparticle skeleton composition, and the trend of storage stability changes is comparable. Therefore, the stability data of Example 4 is used as the representative for explanation. Example 6 is a separate example of the lyophilization-reconstitution process without the introduction of EDC / NHS crosslinking. The structural stability of its reconstituted system has been further enhanced by crosslinking based on the lyophilization-reconstitution process in Examples 7 and 9. Therefore, Examples 7 and 9 have covered and represent the stability level of the lyophilization-reconstitution system. To avoid experimental duplication and to comprehensively evaluate the storage stability of the bath preparations produced by each major process route, this experiment selected the above four representative examples for 90-day stability tracking.

[0158] Table 2 shows the stability test results (4℃) of the bath preparations used in the examples.

[0159] Results analysis: In Example 2, after 90 days of storage at 4°C, the particle size increased and the encapsulation efficiency decreased, with the TNF-α inhibition rate decreasing from 68.3% to 55.6%. In contrast, in Examples 7 and 9, after EDC / NHS crosslinking, all indicators remained highly stable during the 90-day storage period, indicating that the optimized process significantly enhanced the long-term storage stability of the nanoparticles.

[0160] Experiment Example 3: Irritation Test of Medicated Bath Solution on Cow Teat Skin

[0161] Following the skin irritation test method in the "Disinfection Technical Specifications" (2002 edition), 16 healthy adult dairy cows were selected and randomly divided into 4 groups of 4 cows each. The cows' teats were immersed in the medicated bath solutions prepared in Examples 2, 4, 7, and 9, respectively, twice daily (after milking) for 7 consecutive days. The irritation reactions of the teat skin, such as erythema and edema, were observed and recorded daily, and scored according to the following criteria: no erythema (0 points), mild erythema (1 point), moderate erythema (2 points), severe erythema (3 points); no edema (0 points), mild edema (1 point), moderate edema (2 points), severe edema (3 points). The results are shown in Table 3.

[0162] Table 3 shows the skin irritation test results of the bath products used in the examples.

[0163] Results analysis: All the bath preparations described in the examples did not irritate the teat skin of cows (erythema score <0.5, edema score <0.5), and had good safety.

[0164] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0165] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for preparing a dairy cow teat bath solution, characterized in that, Includes the following steps: Step 1: Dissolve chitosan in an aqueous acetic acid solution with a volume fraction of 0.5% to 1.0% to prepare a chitosan solution with a mass-volume concentration of 0.2% to 1.0%. Step 2: Under conditions of 20℃ to 30℃ and continuous stirring, add phloretin powder to the chitosan solution. The amount of phloretin powder added is 0.1% to 0.5% of the mass of the chitosan solution. Stir for 15 min to 30 min to obtain chitosan-phloretin premix. Step 3: Dissolve tannic acid in deionized water to prepare an aqueous solution of tannic acid with a mass-volume concentration of 0.5% to 2.0%; Step 4: At 20℃ to 30℃, stir the tannic acid aqueous solution at a stirring speed of 200 rpm to 400 rpm. At the same time, add chitosan-phloretin premix to the tannic acid aqueous solution at a dropping rate of 0.5 mL / min to 2.0 mL / min until the mass ratio of chitosan to tannic acid is 1:1 to 1:

4. After stirring for 20 min to 40 min, adjust the pH of the reaction system to 5.0 to 6.0 with sodium hydroxide solution and continue stirring for 10 min to 30 min to obtain a tannic acid-chitosan-phloretin composite nanoparticle suspension. Step 5: Place the tannic acid-chitosan-phloretin composite nanoparticle suspension in a parallel plate AC electric field with an electric field strength of 50V / cm to 200V / cm and a treatment time of 5min to 20min. At the same time, add 0.5% to 3.0% of the composite nanoparticle suspension by mass of glycerol glucoside. Step 6: Add a thickener to the composite nanoparticle suspension after it has been subjected to an electric field, stir well, and obtain a cow teat bath solution.

2. The method for preparing the dairy cow teat bath solution as described in claim 1, characterized in that, In step six, 0.01% to 0.05% of sodium glutamate powder by mass is first added to the composite nanoparticle suspension treated with an electric field, stirred until completely dissolved, allowed to stand for 15 to 30 minutes, and then a thickener is added.

3. The method for preparing the dairy cow teat bath agent as described in claim 2, characterized in that, The mass of sodium glutamate powder added is 0.03% of the mass of the composite nanoparticle suspension.

4. The method for preparing the dairy cow teat bath agent as described in claim 2, characterized in that, Add sodium glutamate powder to the composite nanoparticle suspension after electric field treatment and stir until completely dissolved; then adjust the pH of the system to 4.5 to 4.8 with acetic acid solution, and then adjust the pH of the system to 5.0 to 6.0 with sodium hydroxide solution. Let stand for 15 to 30 minutes, and then add thickener.

5. The method for preparing the dairy cow teat bath solution as described in claim 1, characterized in that, The thickener is one of sodium alginate, sodium carboxymethyl cellulose, and xanthan gum, and the amount of thickener added is 0.1% to 1% of the mass of the composite nanoparticle suspension.

6. The method for preparing the bovine teat bath solution as described in claim 1, characterized in that, In step one, the chitosan solution has a mass-volume concentration of 0.5%; in step two, the amount of phloretin powder added is 0.3% of the mass of the chitosan solution; in step four, the mass ratio of chitosan to tannic acid is 1:3, and the pH value is 5.5; in step five, 2% of the mass of the composite nanoparticle suspension of glycerol glucoside is added.

7. The method for preparing the dairy cow teat bath agent as described in claim 2, characterized in that, In step six, a freeze-drying protectant and a portion of monosodium glutamate powder are added to the composite nanoparticle suspension after electric field treatment. The amount of freeze-drying protectant added is 5% to 15% of the mass of the composite nanoparticle suspension, and the portion of monosodium glutamate powder is 0.005% to 0.01% of the mass of the composite nanoparticle suspension. After mixing evenly, the mixture is freeze-dried to obtain composite nanoparticle freeze-dried powder. Then, the composite nanoparticle freeze-dried powder is reconstituted with deionized water to the original volume, and another portion of monosodium glutamate powder is added and stirred until completely dissolved.

8. The method for preparing the dairy cow teat bath agent as described in claim 7, characterized in that, In step six, after adding sodium glutamate powder and stirring until completely dissolved, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to the system. The amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride added is 0.5 to 1.0 times the molar amount of sodium glutamate, and the amount of N-hydroxysuccinimide added is 0.5 to 1.0 times the molar amount of sodium glutamate. The reaction is stirred at 20°C to 30°C for 30 to 60 minutes. After the reaction is completed, the system is ultrafiltered to remove unreacted small molecule byproducts. After standing, a thickener is added.

9. A dairy cow teat medicated bath agent, characterized in that, It is prepared by the method of any one of claims 1 to 8 for preparing the dairy cow teat bath.

Images