Application of luteolin combined with doxycycline in treating duck-origin proteus mirabilis infection

Abstract

The application discloses an application of luteolin combined with doxycycline in a duck-origin proteus mirabilis infection drug, and belongs to the technical field of medical preparations containing organic effective components. An oil-in-water Pickering nanoemulsion stabilized by chitosan nanoparticles is adopted, luteolin is dissolved or dispersed in a dispersed oil phase, doxycycline is loaded on an oil-water interface of the chitosan nanoparticles through hydrogen bond action, crosslinker anion bridging action and polyanion electrostatic complexation, asymmetric double-drug loading and synchronous co-delivery are realized. Through chitosan deacetylation degree control and a sodium alginate outer layer, the nano composition realizes segmented response of low release in the stomach and high release in the intestine in a duck digestive tract. Luteolin is combined with doxycycline at a sub-inhibitory concentration, the minimum inhibitory concentration of doxycycline on a duck-origin proteus mirabilis clinical isolate is significantly reduced, drug resistance is reversed, the doxycycline dosage is reduced, and the waterline drinking water administration in waterfowl breeding is adapted.

Description

Technical Field

[0001] This invention relates to the technical field of pharmaceutical preparations containing organic active ingredients, and more particularly to the application of luteolin combined with doxycycline in the treatment of duck-derived Proteus mirabilis infection. Background Technology

[0002] Proteus mirabilis (PM), a bacterium of duck origin, is a significant Gram-negative opportunistic pathogen affecting waterfowl farming. It can cause various diseases in ducklings, including septicemia, diarrhea, respiratory infections, and serositis, exhibiting high morbidity and mortality rates in intensive farming environments characterized by high temperature and humidity. In recent years, due to the long-term and extensive use of broad-spectrum antibiotics in the aquaculture industry, clinically isolated duck-derived PM strains have shown an increasingly serious trend towards drug resistance, particularly against tetracyclines, β-lactams, and quinolones, posing a significant challenge to clinical treatment.

[0003] Doxycycline (DOX), a second-generation semi-synthetic tetracycline broad-spectrum antibiotic, exerts its antibacterial effect by reversibly binding to the 30S ribosomal subunit of bacteria and inhibiting protein synthesis. It is currently one of the commonly used drugs in veterinary clinical practice for treating duck-derived PM infections. However, with the continuous emergence of drug-resistant strains, the minimum inhibitory concentration (MIC) of DOX alone has increased from 8 μg / mL for standard strains to 32-128 μg / mL for clinical isolates, resulting in a significant decline in clinical efficacy. Continuing to rely solely on increasing DOX dosage to address drug resistance will not only increase drug residues and food safety risks but also accelerate the screening and spread of high-level drug-resistant strains.

[0004] Luteolin (LUT), chemically named 3',4',5,7-tetrahydroxyflavone, is a natural flavonoid widely found in traditional Chinese medicinal herbs such as honeysuckle, chrysanthemum, and perilla. It possesses multiple pharmacological activities, including antibacterial, anti-inflammatory, antioxidant, and immunomodulatory effects. In existing technologies, luteolin has been reported to have certain antibacterial activity, possibly related to affecting bacterial cell membrane integrity, inhibiting biofilm formation, and interfering with bacterial virulence-related processes. Compared to doxycycline, which primarily works by inhibiting bacterial protein synthesis, luteolin exhibits different characteristics. Therefore, from the perspective of complementary mechanisms of action and reducing the pressure of using single-drug antibiotics, combining luteolin and doxycycline for the prevention and treatment of *Proteus mirabilis* infection from ducks has potential application value and is expected to provide a new technical approach to address the problem of drug resistance affecting the efficacy of single-doxycycline treatment. Summary of the Invention

[0005] This invention overcomes the shortcomings of the prior art and provides the application of luteolin combined with doxycycline in the treatment of duck-derived Proteus mirabilis infection.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a nanocomposition loaded with luteolin and doxycycline, comprising: The nanocomposition is an oil-in-water Pickering nanoemulsion, stabilized by chitosan nanoparticles as a particulate emulsifier, comprising a continuous aqueous phase and a dispersed oil phase; the luteolin is dissolved or dispersed in the dispersed oil phase; the doxycycline is loaded onto the oil-water interface of the chitosan nanoparticles through at least one of hydrogen bonding, crosslinking agent anionic bridging, and polyanionic electrostatic complexation.

[0007] In a preferred embodiment of the present invention, the mass ratio of luteolin to doxycycline is 1:10 to 5:1.

[0008] In a preferred embodiment of the present invention, the degree of deacetylation of the chitosan is 85% to 95%, and the molecular weight of the chitosan is 50,000 Da to 200,000 Da.

[0009] In a preferred embodiment of the present invention, the chitosan nanoparticles are formed by chitosan and a crosslinking agent through ionic crosslinking or covalent crosslinking, wherein the crosslinking agent is at least one of sodium tripolyphosphate or genipin, and the mass ratio of chitosan to the crosslinking agent is 3:1 to 6:1.

[0010] In a preferred embodiment of the present invention, the nanocomposition further includes a sodium alginate outer layer, which coats the surface of the chitosan nanoparticles to form a gel barrier under the pH conditions of the stomach.

[0011] In a preferred embodiment of the present invention, the average particle size of the nanocomposition is 200 nm to 500 nm; the cumulative release rate of the nanocomposition in simulated duck gastric fluid is less than 15% after 2 hours and more than 75% after 4 hours in simulated duck intestinal fluid; the pH of the simulated duck gastric fluid is 2.0 and the pH of the simulated duck intestinal fluid is 7.4.

[0012] This invention provides a method for preparing a nanocomposite loaded with luteolin and doxycycline, comprising the following steps: S1. Dissolve chitosan in an acidic aqueous solution and adjust the pH to 4.0–5.5 to obtain an aqueous phase of amino-protonated chitosan. S2. Luteolin is dissolved in medium-chain triglycerides to form a drug-containing oil phase; S3. Dissolve doxycycline hydrochloride in water to prepare a doxycycline aqueous solution; S4. Add the drug-containing oil phase to the chitosan aqueous phase, and emulsify by high-speed shearing to form a chitosan-stable oil-in-water type primary emulsion. Chitosan is adsorbed at the oil-water interface to form chitosan nanoparticles, and luteolin is encapsulated in the dispersed oil phase along with the drug-containing oil phase. S5. Add doxycycline aqueous solution to the colostrum and sonicate. Doxycycline is initially adsorbed onto the oil-water interface layer of chitosan nanoparticles through intermolecular hydrogen bonding to form an emulsion. S6. Add sodium tripolyphosphate and genipin sequentially to the emulsion to crosslink and solidify the chitosan nanoparticles, thereby obtaining a nanocomposite. S7. Add sodium alginate aqueous solution to the nanocomposite solution to form a pH-responsive outer layer on the surface of the nanocomposite droplets.

[0013] In a preferred embodiment of the present invention, in step S6, the phosphate anion of the sodium tripolyphosphate forms an electrostatic bridging structure with the protonated amino group of the doxycycline and the chitosan, thereby reinforcing and anchoring the doxycycline to the oil-water interface layer; the mass ratio of the chitosan to the sodium tripolyphosphate and the genipin is 3:1 to 6:1.

[0014] In a preferred embodiment of the present invention, in step S7, the outer layer of sodium alginate is protonated under the pH conditions of the stomach to form a gel barrier, so that the cumulative release rate of the nanocomposition in the stomach environment is less than 15%; under the pH conditions of the intestine, it is deprotonated and dissolved to restore the pH-responsive release function of the chitosan nanoparticles, so that the cumulative release rate of the nanocomposition in the intestinal environment is greater than 75%.

[0015] This invention provides the application of a nanocomposition loaded with luteolin and doxycycline in a drug for treating duck-derived Proteus mirabilis infection, wherein the luteolin is used in combination with the doxycycline at a sub-inhibitory concentration, the sub-inhibitory concentration being 1 / 16 to 1 / 2 of the minimum inhibitory concentration of luteolin against duck-derived Proteus mirabilis.

[0016] This invention addresses the shortcomings of the prior art and has the following beneficial effects: This invention achieves asymmetric dual-drug loading of luteolin and doxycycline by constructing a stable water-in-oil Pickering nanoemulsion of chitosan nanoparticles, enabling the two drugs to reach the infection site synchronously in a synergistic and effective ratio, thus solving the problem of spatiotemporal asynchrony of conventional drug formulations in vivo.

[0017] This invention utilizes luteolin at a sub-inhibitory concentration in combination with doxycycline. Luteolin disrupts the outer cell membrane and inhibits efflux pump activity, thereby increasing the intracellular accumulation concentration of doxycycline. This results in a general synergistic antibacterial effect against clinical isolates of Proteus mirabilis from ducks. Compared to existing technologies where clinical isolates are generally resistant to drugs, this invention restores and enhances the antibacterial effect while reducing the dosage of doxycycline, thus alleviating antibiotic selection pressure and the risk of drug residues.

[0018] This invention utilizes the inherent pH gradient of the duck digestive tract to achieve a segmented response of gastric stability and intestinal release by regulating the degree of deacetylation of chitosan and protecting the outer layer of sodium alginate with gastric acid. This avoids the problems of premature gastric release and degradation and short intestinal absorption time window of conventional oral preparations, ensuring that the main drug dose reaches the intestinal target site intact.

[0019] This invention also utilizes the antibacterial activity of chitosan itself to form a triple synergistic antibacterial mechanism with luteolin and doxycycline. Furthermore, by leveraging the physical stability of the nanocomposite in drinking water, it adapts to waterline administration, enabling simultaneous administration to a group of waterfowl. This avoids the labor costs and stress damage associated with individual capture and administration, providing a holistic solution for the group control of duck-derived Proteus mirabilis infection. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 This is a diagram illustrating the effect of a preferred embodiment of the present invention, combining doxycycline and luteolin, on 12 strains of Proteus mirabilis. Figure 2 This is a preferred embodiment of the present invention; Figure 3 This is a fitted graph of the Weibull release curve of a preferred embodiment of the present invention; In the diagram: 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0023] Basic principles: This principle presents the in vitro efficacy test results of the combination of luteolin and doxycycline against duck-derived Proteus mirabilis.

[0024] Using 12 clinical isolates of *Proteus mirabilis* from ducks and 1 standard strain as test bacteria, the minimum inhibitory concentrations (MICs) of luteolin and doxycycline were determined by micro-broth dilution method. The MICs of doxycycline against the 12 clinical isolates and standard strain were determined by checkerboard method at luteolin concentrations of 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL. The MIC of luteolin against both the clinical isolates and standard strain was 80 μg / mL. 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL were 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the MIC of luteolin, respectively, all below the MIC of luteolin and thus considered sub-inhibitory concentrations.

[0025] Table 1. Minimum inhibitory concentrations of doxycycline in *Proteus mirabilis* derived from ducks under different concentrations of luteolin.

[0026] Among them, below 0.125 means that the minimum inhibitory concentration is less than 0.125 μg / mL, that is, below the detection limit.

[0027] Table 1 shows that the minimum inhibitory concentration (MIC) of luteolin monotherapy against both the 12 clinical isolates and the standard strain was 80 μg / mL. The MIC of doxycycline monotherapy against the 12 clinical isolates ranged from 32 μg / mL to 128 μg / mL, while the MIC against the standard strain was 8 μg / mL. The MIC of doxycycline against the clinical isolates was 4 to 16 times higher than that against the standard strain, indicating that the clinical isolates had developed varying degrees of resistance to doxycycline.

[0028] When luteolin was used in combination with doxycycline at a sub-inhibitory concentration, the minimum inhibitory concentration (MIC) of doxycycline against each strain decreased, with the degree of decrease increasing with increasing luteolin concentration. At a luteolin concentration of 40 μg / mL, the MIC of doxycycline against 12 clinical isolates decreased to 0.125 μg / mL to 32 μg / mL, with 8 isolates decreasing to below 2 μg / mL and 6 isolates decreasing to below 0.125 μg / mL; the strain with the largest decrease dropped from 128 μg / mL to below 0.125 μg / mL, a decrease of over 1000-fold. At a luteolin concentration of 20 μg / mL, the MIC of doxycycline in 11 of the 12 clinical isolates was lower than that of doxycycline alone; at a luteolin concentration of 5 μg / mL, the MIC of doxycycline in 8 of the 12 clinical isolates still decreased to below 16 μg / mL.

[0029] The partial inhibitory concentration (PIC) was calculated using luteolin at a concentration of 20 μg / mL. The PIC was calculated as the sum of the quotients obtained by dividing the combined luteolin concentration by the minimum inhibitory concentration (MIC) of luteolin alone and the quotients obtained by dividing the combined doxycycline concentration by the MIC of doxycycline alone. Of the 12 clinical isolates, 11 had PICs not greater than 0.5, indicating synergistic effects; 8 of these had PICs not greater than 0.31, indicating strong synergistic effects. One isolate had a PIC of 0.75, indicating an additive effect. The proportion of isolates with PICs not greater than 0.5 was 11 / 12.

[0030] Bactericidal curve experiments showed that, at the same drug concentrations (1 times the minimum inhibitory concentration) in the luteolin monotherapy group, doxycycline monotherapy group, and combination therapy group, after 24 hours of incubation, the bacterial count reduction in the combination therapy group against the 12 clinical isolates was 1.4 to 4.4 Log higher than that in the monotherapy groups. 10 CFU / mL, the bactericidal rate and bactericidal depth of the combined drug group were higher than those of the single drug group.

[0031] The above experimental results confirm that luteolin, at a sub-inhibitory concentration, combined with doxycycline, exhibits a generally synergistic effect against clinical isolates of *Proteus mirabilis* from ducks, reducing the minimum inhibitory concentration of doxycycline by 4 to over 1000 times and enhancing its bactericidal efficacy. This effect provides pharmacodynamic evidence for establishing a combined treatment regimen of luteolin and doxycycline.

[0032] The synergistic effect of luteolin and doxycycline against *Proteus mirabilis* from ducks is based on the following mechanism: luteolin disrupts the bacterial outer cell membrane and increases membrane permeability. *Proteus mirabilis* from ducks is a Gram-negative bacterium, and its outer cell membrane, composed of a lipopolysaccharide and phospholipid bilayer, is the main barrier for antimicrobial drugs to enter the bacterial cell. Luteolin, a hydrophobic flavonoid compound, can insert into the lipid bilayer of the bacterial cell membrane, disrupting the integrity and order of the cell membrane, increasing cell membrane permeability, and making it easier for doxycycline to cross the outer cell membrane and enter the bacterial cell.

[0033] Luteolin inhibits bacterial efflux pump activity. *Proteus mirabilis* from ducks expresses an RND-type efflux pump, which actively pumps tetracycline drugs into the cell, representing one of the main mechanisms of doxycycline resistance in this bacterium. Luteolin competitively binds to the substrate binding site of the efflux pump, inhibiting the active efflux of doxycycline and increasing its intracellular accumulation. In experimental data, the minimum inhibitory concentration (MIC) of doxycycline against clinical isolates ranged from 32 μg / mL to 128 μg / mL. The addition of luteolin decreased the MIC of doxycycline, consistent with this mechanism, indicating that luteolin can reverse doxycycline resistance in *Proteus mirabilis* from ducks.

[0034] Doxycycline binds to the 30S ribosomal subunit of bacteria, inhibiting protein synthesis. Based on luteolin's ability to increase membrane permeability and inhibit efflux pumps, doxycycline accumulates at higher concentrations within the bacterial cell, leading to increased binding to the 30S ribosomal subunit, enhanced inhibition of protein synthesis, and a shift from antibacterial to bactericidal activity.

[0035] The above mechanisms are sequential: luteolin first disrupts the outer cell membrane and inhibits the efflux pump, creating conditions for doxycycline to enter the bacterial cell and accumulate intracellularly. Doxycycline then exerts its inhibitory function on ribosomal protein synthesis. The two drugs act on different antibacterial targets, and the synergistic effect is greater than the sum of the individual effects of each component. The synergistic effect of luteolin combined with doxycycline exhibits pathogen specificity. Existing technologies have disclosed studies on the combined use of luteolin and doxycycline against other pathogens, but the synergistic results are inconsistent. The experimental data of this invention show that luteolin, at a sub-inhibitory concentration, synergistically interacts with doxycycline against 11 out of 12 clinical isolates of *Proteus mirabilis* from ducks, a synergistic ratio of 11 / 12. Furthermore, it is effective against clinical isolates with varying degrees of doxycycline resistance, indicating that this synergistic effect is both universal and pathogen-specific against *Proteus mirabilis* from ducks. This pathogen specificity stems from the fact that the resistance mechanism of *Proteus mirabilis* to doxycycline primarily involves the outer membrane barrier and active efflux pumps, directly corresponding to the target sites of luteolin, which disrupt the outer membrane and inhibit efflux pumps.

[0036] Based on the above experimental results and the principle of synergistic effect, the application of luteolin in combination with doxycycline at a sub-inhibitory concentration was determined. The sub-inhibitory concentration was 1 / 16 to 1 / 2 of the minimum inhibitory concentration (MIC) of luteolin against *Proteus mirabilis* (duck-derived strain), i.e., 5 μg / mL to 40 μg / mL. Within this concentration range, luteolin alone did not produce significant antibacterial activity, but when used in combination with doxycycline, it could reduce the MIC of doxycycline against *Proteus mirabilis* by 4 to more than 1000 times and improve the bactericidal effect.

[0037] The combination therapy regimen reduces the dosage of doxycycline, thereby reducing the pressure of antibiotic selection and the risk of drug residues; luteolin has both anti-inflammatory and antioxidant effects, which can alleviate the inflammatory response and tissue and organ damage caused by duck-derived Proteus mirabilis infection.

[0038] However, when the above-mentioned combination therapy is prepared into a drug for oral administration to ducks, the following drawbacks exist: the saturated solubility of luteolin in water is about 0.5 μg / mL, and the absolute oral bioavailability is less than 5%. After oral administration in conventional dosage forms, the dissolution and absorption of luteolin in the gastrointestinal tract are limited, making it difficult to reach an effective concentration above the sub-inhibitory concentration at the site of infection. The synergistic effect in the combination therapy cannot be fully realized in vivo. Luteolin and doxycycline have significantly different physicochemical properties. Luteolin has an oil-water partition coefficient of about 2.5 and is a hydrophobic flavonoid compound, while doxycycline is a water-soluble tetracycline antibiotic. When two drugs are physically mixed and administered in conventional dosage forms, their dissolution, absorption, distribution, and metabolism in the body are asynchronous, making it difficult for them to reach the infection site simultaneously at the required concentration ratio within the same time window. The pH of the duck's stomach is 2.0 to 3.0, containing highly active pepsin. Conventional oral formulations experience premature drug release and degradation under the acidic conditions of the stomach. The duck's gastrointestinal tract empties quickly, with food taking approximately 3 to 4 hours to pass through the entire digestive tract, resulting in a short absorption window for drugs in the intestines.

[0039] In addition, large-scale waterfowl farming uses waterline drinking water administration for group drug delivery. The preparation needs to be evenly dispersed in the drinking water and remain physically stable during the infusion process of more than 24 hours, without sedimentation, stratification or drug leakage.

[0040] To overcome the aforementioned shortcomings, this invention draws cross-disciplinary inspiration from the mature Pickering emulsion technology in food science and applies it to the field of veterinary antibiotic delivery. This invention co-loads luteolin and doxycycline into an oil-in-water nanocomposite stabilized with chitosan nanoparticles as a particulate emulsifier. Simultaneous co-delivery of the two drugs is achieved through asymmetric dual-drug loading. pH-segmented release in the duck digestive tract is achieved through chitosan deacetylation regulation and a sodium alginate outer layer. Furthermore, the antibacterial activity of chitosan itself forms a triple synergistic antibacterial mechanism involving chitosan, luteolin, and doxycycline. Example 1

[0041] In this embodiment, chitosan nanoparticles were used as particulate emulsifiers to prepare a nanocomposite loaded with luteolin and doxycycline via a two-step emulsification ionic crosslinking method. like Figure 2 As shown, a method for preparing a nanocomposite loaded with luteolin and doxycycline includes: S1. Dissolve chitosan in an acidic aqueous solution and adjust the pH to 4.0–5.5 to obtain an aqueous phase of amino-protonated chitosan. S2. Luteolin is dissolved in medium-chain triglycerides to form a drug-containing oil phase; S3. Dissolve doxycycline hydrochloride in water to prepare a doxycycline aqueous solution; S4. Add the drug-containing oil phase to the chitosan aqueous phase, and emulsify by high-speed shearing to form a chitosan-stable oil-in-water type primary emulsion. Chitosan is adsorbed at the oil-water interface to form chitosan nanoparticles, and luteolin is encapsulated in the dispersed oil phase along with the drug-containing oil phase. S5. Add doxycycline aqueous solution to the colostrum and sonicate. Doxycycline is initially adsorbed onto the oil-water interface layer of chitosan nanoparticles through intermolecular hydrogen bonding to form an emulsion. S6. Add sodium tripolyphosphate and genipin sequentially to the emulsion to crosslink and solidify the chitosan nanoparticles, thereby obtaining a nanocomposite. S7. Add sodium alginate aqueous solution to the nanocomposite solution to form a pH-responsive outer layer on the surface of the nanocomposite droplets.

[0042] Specifically, step S1, preparing the chitosan aqueous phase, includes: Weigh 200 mg of chitosan with a degree of deacetylation of 90% and a molecular weight of 100,000-120,000 Da, dissolve it in 40 mL of 1% (w / v) glacial acetic acid aqueous solution, stir at room temperature for 3 h until completely dissolved, filter through a 0.45 μm filter membrane, and adjust the pH to 5.0 with 1 mol / L sodium hydroxide solution to obtain a chitosan aqueous phase with a concentration of 5 mg / mL and a pH of 5.0.

[0043] The pH of the chitosan aqueous phase is set to 5.0. At this pH, the amino groups of chitosan are protonated, and the molecular chains have sufficient positive charge density to meet the requirements of interfacial adsorption and ionic cross-linking, while avoiding excessively high pH that could lead to chitosan precipitation. The pH of the chitosan aqueous phase can be adjusted within the range of 4.0 to 5.5 to accommodate the solubility characteristics of chitosan with different degrees of deacetylation and molecular weights.

[0044] In step S2, the preparation of the drug-containing oil phase includes: 10 mg of luteolin and 50 mg of soy lecithin were weighed and added to 2 mL of medium-chain triglycerides. The mixture was dissolved using ultrasound in a 40°C water bath to obtain a drug-containing oil phase. The amount of soy lecithin was between 0.1% and 2.0% of the continuous aqueous phase volume. In this example, the mass of soy lecithin was 50 mg, accounting for 0.125% of the 40 mL continuous aqueous phase volume. Soy lecithin is embedded in the interfacial voids between chitosan nanoparticles, reducing the oil-water interfacial tension and assisting the chitosan nanoparticles in spreading fully at the oil-water interface.

[0045] In step S3, the preparation of doxycycline aqueous solution includes: Weigh 10 mg of doxycycline hydrochloride, dissolve it in 2 mL of water (based on doxycycline bases) to obtain a doxycycline aqueous solution with a concentration of 5 mg / mL. Prepare and use immediately and store away from light.

[0046] Furthermore, a two-step emulsification process is performed.

[0047] In step S4, colostrum is prepared and loaded with luteolin, including: The drug-containing oil phase obtained in S2 was added to the chitosan aqueous phase obtained in S1 under ice bath conditions, and homogenized by high-speed shearing at 10000-12000 r / min for 2 min to form an oil-in-water primary emulsion. During emulsification, the chitosan molecular chains migrated to the oil-water interface under hydrophobic drive and mechanical force, and the protonated amino groups provided electrostatic repulsion to inhibit droplet aggregation, forming a primary emulsion stabilized by chitosan nanoparticles; luteolin was encapsulated in the dispersed oil phase along with the drug-containing oil phase; the mass ratio of luteolin to doxycycline was 1:10-5:1, and in this example, the mass ratio of luteolin to doxycycline was 1:1.

[0048] In step S5, doxycycline is loaded, including: Add the doxycycline aqueous solution obtained in S3 to the colostrum obtained in S4, and treat with an ice bath at 150-200W ultrasonic power for 2 minutes. This allows doxycycline to be initially adsorbed onto the oil-water interface layer of chitosan nanoparticles through intermolecular hydrogen bonding. The phenolic hydroxyl, enol hydroxyl, carbonyl, and dimethylamino groups of the doxycycline molecule form a hydrogen bond network with the hydroxyl and amino groups of the chitosan molecular chain, thus pre-anchoring doxycycline to the oil-water interface layer. In S6 and S7, the sodium tripolyphosphate phosphate anion forms an electrostatic bridging structure with the doxycycline cation and the protonated amino group of chitosan, while the sodium alginate carboxyl anion undergoes polyelectrolyte electrostatic complexation with the doxycycline cation, completing the enhanced anchoring of doxycycline.

[0049] Step S6, crosslinking and curing the emulsion, including: A sodium tripolyphosphate aqueous solution (40 mg, dissolved in 5 mL of water) was added to the emulsion obtained in S5. The mass ratio of chitosan to sodium tripolyphosphate was 5:1. The solution was added dropwise at a rate of 1 mL / min and stirred for 30 min, allowing the phosphate groups of sodium tripolyphosphate to undergo ionic crosslinking with the amino groups of chitosan. Subsequently, a genipin aqueous solution (10 mg, dissolved in 2 mL of water) was added, with a chitosan to crosslinking agent mass ratio of 4:1. The mixture was stirred at room temperature in the dark for 2 h, allowing the genipin to undergo covalent crosslinking with the amino groups of chitosan. Ionic crosslinking provides initial mechanical strength to the interfacial layer, while covalent crosslinking provides long-term chemical stability.

[0050] Step S7, coating with sodium alginate, includes: A sodium alginate aqueous solution (100 mg, dissolved in 10 mL of water) was added to the emulsion obtained in S6 and stirred for 30 min, allowing the sodium alginate to adsorb onto the surface of the nanocomposite droplets to form an outer layer. Sodium alginate protonates to form a gel barrier under low pH conditions in the stomach and deprotonates to dissolve under intestinal pH conditions, achieving a segmented response of gastric protection and intestinal release. Simultaneously, sodium alginate enhances the retention of doxycycline at the interface layer through electrostatic complexation between carboxyl anions and doxycycline cations.

[0051] The average particle size and polydispersity index of the nanocomposite were determined by dynamic light scattering. The instrument was a Malvern Zetasizer Nano ZS with a backscattering angle of 173° and a measurement temperature of 25°C. The sample was diluted 100 times with water, and each sample was measured three times. The average particle size was 285±25 nm, and the polydispersity index was 0.18±0.03. The average particle size was in the range of 200 nm to 500 nm, and the polydispersity index was not greater than 0.25, indicating a uniform particle size distribution.

[0052] The Zeta potential was measured using the same instrument in Smoluchowski mode. The sample was diluted 200 times with 1 mmol / L sodium chloride solution, and the Zeta potential was +32.5 ± 3.5 mV. Chitosan nanoparticles endow the emulsion droplets with a strong positive charge. The positive charge is conducive to the electrostatic adsorption of the emulsion droplets with the negatively charged bacterial outer membrane, and to the mucosal adhesion with the negatively charged mucopolysaccharide residues of the intestinal mucus layer. The physical stability of the emulsion is maintained by electrostatic repulsion between droplets.

[0053] Transmission electron microscopy (TEM) was used to observe the morphology of the emulsion. The accelerating voltage was 80 kV, and the sample was negatively stained with 2% phosphotungstic acid. TEM images showed that the emulsion droplets were spherical, and the chitosan nanoparticles were continuously and orderly arranged at the oil-water interface, forming a particle interfacial film with a thickness of 15 nm-30 nm, exhibiting typical Pickering emulsion structure characteristics. Compared with surfactant-stabilized nanoemulsions, the nanocomposite with particle interfacial film stability showed higher anti-agglomeration stability.

[0054] High performance liquid chromatography was used to determine the drug encapsulation efficiency and loading rate.

[0055] Take 1 mL of the freshly prepared nanocomposite and centrifuge it at 4°C and 5000 r / min for 15 min using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa. Collect the filtrate and determine the concentration of free drug. Take an equal amount of nanocomposite, demulsify it with methanol, and extract the total drug amount by ultrasonic extraction.

[0056] The chromatographic conditions for luteolin were: a C18 reversed-phase column, a mobile phase of methanol to 0.2% phosphoric acid aqueous solution (volume ratio 55:45), a detection wavelength of 350 nm, and a flow rate of 1.0 mL / min; the chromatographic conditions for doxycycline were: a C18 reversed-phase column, a mobile phase of acetonitrile to 0.02 mol / L potassium dihydrogen phosphate buffer (volume ratio 25:75), a detection wavelength of 273 nm, and a flow rate of 1.0 mL / min.

[0057] Encapsulation efficiency is calculated by dividing the difference between the total drug amount and the free drug amount by the total drug amount, while loading rate is calculated by dividing the drug amount in the nanoparticles by the sum of the drug amount in the nanoparticles and the total amount of carrier material. The encapsulation efficiency of luteolin is 89.5±3.2%, and the loading rate of doxycycline is 78.3±4.5%. For interfacial loaded drugs, loading rate rather than encapsulation efficiency is more accurate because they are not free in the aqueous phase of the emulsion. The high encapsulation efficiency of luteolin comes from the spontaneous enrichment of its hydrophobic oil core, while the high loading rate of doxycycline comes from the multiple anchoring effects of hydrogen bond network, sodium tripolyphosphate anion bridging, and sodium alginate polyanion complexation. Example 2

[0058] This embodiment investigates the effect of chitosan deacetylation degree on the drug release behavior of the nanocomposite in simulated duck gastric and intestinal fluids.

[0059] Chitosans with deacetylation degrees of 75%, 85%, 90%, and 95%, respectively, and molecular weights standardized to 100,000 ± 10,000 Da, were selected to prepare corresponding nanocomposites according to steps S1 to S6 of Example 1. The sodium alginate outer coating in step S7 was omitted to investigate the effect of deacetylation degree alone. The chitosan concentration, luteolin dosage, doxycycline dosage, oil phase volume, crosslinking agent dosage, and emulsification parameters were kept consistent for all four nanocomposites.

[0060] The release rate was determined using the dialysis bag method. 2 mL of each degree of deacetylation group of the nanocomposite was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, immersed in 50 mL of release medium, and placed in a constant-temperature shaker at 37℃ and 100 r / min. 1 mL samples were taken at 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, and 8 h, and an equal volume of fresh isothermal medium was added. The samples were filtered through a 0.22 μm filter membrane, and the doxycycline concentration was determined by high-performance liquid chromatography (HPLC). The cumulative release rate at each time point was calculated.

[0061] Table 2. Cumulative release rates of nanocomposites prepared from chitosan with different degrees of deacetylation in simulated duck gastric and intestinal fluids.

[0062] Table 2 shows that in simulated duck gastric fluid, as the degree of deacetylation increased from 75% to 95%, the cumulative release rate at 2 hours decreased from 62.3% to 22.4%, and the cumulative release rate at 8 hours decreased from 89.4% to 53.6%. This indicates that the increased amino density improved the interfacial stability of chitosan nanoparticles under strongly acidic conditions, and the continuous leakage of the drug through the stomach decreased with increasing deacetylation. The group with a deacetylation degree of 75% achieved a cumulative release rate of 89.4% at 8 hours in simulated duck gastric fluid, indicating insufficient gastric retention. The underlying principle is that the degree of deacetylation determines the amino density on the chitosan molecular chain. In simulated duck gastric fluid at pH 2.0, the degree of amino protonation increases with increasing amino density, leading to an increased positive charge density on the surface of chitosan nanoparticles, enhanced hydrophilicity, and improved adsorption strength at the oil-water interface. This maintains the structural integrity of the interfacial membrane under strongly acidic conditions, thus inhibiting the release of the drug through diffusion pathways into the stomach.

[0063] Table 2 also shows that in simulated duck intestinal fluid, the 4-hour cumulative release rate increased from 65.7% to 88.3% with increasing degree of deacetylation. The groups with an 8-hour cumulative release rate higher than 92% were those with a degree of deacetylation of 90% and 95%. Within the range of 85% to 95% degree of deacetylation, the 4-hour cumulative release rate of the nanocomposition in simulated duck intestinal fluid was higher than 75%, meeting the requirements for intestinal release. When the degree of deacetylation was lower than 85%, the degree of intestinal deprotonation was insufficient to trigger complete interfacial membrane rupture, and the 4-hour cumulative release rate was lower than 75%. In simulated duck intestinal fluid at pH 7.4, the chitosan amino groups underwent deprotonation, the hydrophilicity of the particles decreased, the three-phase contact angle of the chitosan nanoparticles shifted to a direction greater than 90°, the particles desorbed from the oil-water interface, the interfacial membrane ruptured, and the drug was rapidly released as the emulsion structure was destroyed. The degree of deprotonation increased with increasing degree of deacetylation, and the completeness of the interfacial membrane rupture increased accordingly.

[0064] like Figure 3 As shown, the Weibull equation was used to fit the release curves. The shape parameters of the release curves in the simulated duck gastric fluid ranged from 0.6 to 0.8, while those in the simulated duck intestinal fluid ranged from 1.0 to 1.3. This indicates that gastric release is mainly due to drug diffusion at a low rate, while intestinal release is mainly due to rapid release triggered by interfacial membrane rupture. The simulated intestinal release half-life was 1.2 h to 2.0 h, while the gastric release half-life was greater than 12 h. Example 3

[0065] This embodiment investigates the effect of the sodium alginate outer layer on the gastric stability of the nanocomposite.

[0066] The nanocomposite prepared using the complete steps of Example 1 was used as the experimental group, and the nanocomposite omitting step S7 was used as the control group. Both groups used chitosan with a degree of deacetylation of 90%, and the remaining steps and parameters were the same. A segmented sequential release scheme was adopted. In the first stage, the dialysis bag was immersed in simulated duck gastric fluid for 2 hours. In the second stage, the same dialysis bag was transferred to simulated duck intestinal fluid and the release continued until the 6th hour.

[0067] Table 3. Cumulative release rates of nanocomposites with and without sodium alginate outer layer in simulated duck gastric and intestinal fluids.

[0068] Table 4 shows that in the first stage, the cumulative release rate at 2 hours was 28.7% in the control group and 8.3% in the experimental group, indicating that the sodium alginate outer layer reduced the cumulative release rate in the stomach. In the second stage, the release in the experimental group recovered after being transferred to simulated duck intestinal fluid, with a cumulative release rate of 82.4% at 4 hours after transfer. This was not statistically different from the 85.1% in the control group, indicating that while the sodium alginate outer layer inhibited gastric release, it did not continuously block the total intestinal release.

[0069] The effect of sodium alginate on the outer layer is pH-dependent. In simulated duck gastric fluid at pH 2.0, sodium alginate protonates to form an insoluble gel, creating a physical barrier on the surface of the nanoparticle droplets. This barrier blocks the penetration of gastric acid and pepsin into the emulsion and inhibits premature demulsification of chitosan nanoparticles. Transmission electron microscopy showed that the gel layer thickness was 20 nm to 50 nm after 1 hour of incubation. In simulated duck intestinal fluid at pH 7.4, sodium alginate deprotonates and dissolves, the physical barrier is removed, and the chitosan nanoparticles regain their pH-responsive release function. The release lag caused by the dissolution of sodium alginate is 15 to 30 minutes and does not have a sustained inhibitory effect on the overall release.

[0070] The experimental group, combined with the control group with a degree of deacetylation of 90%, achieved a segmented response release performance with a cumulative release rate of 8.3% in simulated duck gastric juice over 2 hours and 82.4% in simulated duck intestinal juice over 4 hours, meeting the requirements of a cumulative release rate of less than 15% in the stomach and a cumulative release rate of more than 75% in the intestine. Example 4

[0071] This embodiment evaluates the long-term storage stability and waterline administration stability of the nanocomposition.

[0072] In the long-term storage stability evaluation, the two batches of nanocomposites prepared in Example 1 were stored in a light-protected and sealed manner at 4°C and 25°C, respectively. Samples were taken on days 0, 7, 14, 30, 60 and 90 after preparation, and the average particle size, polydispersity index, zeta potential and drug encapsulation efficiency were determined according to the method in Example 1.

[0073] Table 4. Physicochemical properties of the nanocomposition on day 0 and day 90 under storage conditions at 4℃ and 25℃.

[0074] Table 4 shows that under both storage conditions, the average particle size increased slowly with prolonged storage time, and no abrupt increase occurred between adjacent sampling time points. After 90 days of storage at 4℃, the average particle size increased from 285 nm to 302 nm, the polydispersity index remained between 0.19 and 0.21, the zeta potential slowly decreased from +32.5 mV to +30.2 mV, the luteolin encapsulation efficiency decreased from 89.5% to 85.8%, and the doxycycline loading rate decreased from 78.3% to 73.5%. The changes in these indicators were relatively small, indicating that the nanocomposite can maintain shelf-life stability for more than 90 days under 4℃ conditions. After 90 days of storage at 25℃, the average particle size increased to 325 nm, the polydispersity index increased to 0.25, the zeta potential decreased to +27.6 mV, the luteolin encapsulation efficiency decreased to 79.2%, and the doxycycline loading rate decreased to 67.8%, still within the acceptable formulation quality range. Increased storage temperature accelerates the Brownian motion and Austronesian ripening rate of droplets, increases the probability of particle collision and aggregation, and correspondingly increases the particle size increase. The particle size increase from day 30 to day 60 at 25℃ is greater than that at 4℃. The interparticle interface film formed by chitosan nanoparticles at the oil-water interface inhibits droplet aggregation, keeping the particle size increase at a low level during the 90-day observation period. The polydispersity index remains below 0.25, and the particle size distribution does not broaden significantly. The Zeta potential decreases slowly with prolonged storage time, which is due to the slow desorption of some chitosan nanoparticles from the interface and the shift in the deprotonation equilibrium of amino groups. Its absolute value is above 25 mV during the 90-day observation period, and the electrostatic repulsion between droplets is maintained, indicating that the system has not lost its electrostatic stability.

[0075] In the stability evaluation of water-based drug delivery, the nanocomposite prepared in Example 1 was diluted 1:1000 by volume in drinking water with a pH of 7.2, and allowed to stand at 25°C in the dark. Samples were taken at 0h, 4h, 8h, 12h, and 24h after dilution to determine the average particle size and the proportion of free drug. The results are shown in Table 5.

[0076] Table 5. Physical stability of the nanocomposition diluted in drinking water at a volume ratio of 1:1000 within 24 hours.

[0077] When the nanocomposite was diluted 1:1000 by volume in drinking water at pH 7.2, the average particle size showed a continuous and slow increasing trend with the extension of standing time, increasing from an initial 290 nm to 345 nm after 24 hours. The increase was 7.6% from 0 to 8 hours and 10.6% from 8 to 24 hours, with the particle size growth rate slightly accelerating in the later stage of observation. The polydispersity index increased from 0.19 to 0.25, and the particle size distribution remained uniform. The proportion of free drug to total drug loading gradually increased from 3.2% to 7.4%. No visible precipitation or stratification was observed during the 24-hour observation period. Dilution alters the polymer concentration and ionic environment in the system, increasing the interparticle distance. The adsorption equilibrium of chitosan nanoparticles at the interface slowly shifts towards desorption, resulting in a continuous and slow increase in particle size. With prolonged settling time, interfacial film rearrangement and Austronesian ripening continue, slightly accelerating the particle size growth rate. The interparticle interfacial film and the sodium alginate outer layer maintain the integrity of the droplet structure, preventing demulsification and aggregation. Although the proportion of free drug increases slowly with prolonged settling time, it remains at a low level, without precipitation or stratification. These results indicate that the nanocomposite remains physically stable for 24 hours under drinking water dilution conditions, making it suitable for large-scale waterfowl farming waterline drug delivery. Based on the preferred embodiments of the present invention described above, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A nanocomposite loaded with luteolin and doxycycline, characterized in that, include: The nanocomposition is an oil-in-water Pickering nanoemulsion, stabilized by chitosan nanoparticles as a particulate emulsifier, comprising a continuous aqueous phase and a dispersed oil phase; the luteolin is dissolved or dispersed in the dispersed oil phase; the doxycycline is loaded onto the oil-water interface of the chitosan nanoparticles through at least one of hydrogen bonding, crosslinking agent anionic bridging, and polyanionic electrostatic complexation.

2. The nanocomposite of luteolin and doxycycline according to claim 1, characterized in that: The mass ratio of luteolin to doxycycline is 1:10 to 5:

1.

3. The nanocomposite of luteolin and doxycycline according to claim 1, characterized in that: The degree of deacetylation of the chitosan is 85% to 95%, and the molecular weight of the chitosan is 50,000 Da to 200,000 Da.

4. The nanocomposite of luteolin and doxycycline according to claim 1, characterized in that: The chitosan nanoparticles are formed by chitosan and a crosslinking agent through ionic or covalent crosslinking. The crosslinking agent is at least one of sodium tripolyphosphate or genipin, and the mass ratio of chitosan to the crosslinking agent is 3:1 to 6:

1.

5. The nanocomposite of luteolin and doxycycline according to claim 1, characterized in that: The nanocomposition also includes a sodium alginate outer layer, which coats the surface of the chitosan nanoparticles and forms a gel barrier under the pH conditions of the stomach.

6. The nanocomposition loaded with luteolin and doxycycline according to claim 1, characterized in that: The average particle size of the nanocomposition is 200 nm to 500 nm; the cumulative release rate of the nanocomposition in simulated duck gastric fluid is less than 15% after 2 hours and more than 75% after 4 hours in simulated duck intestinal fluid, the pH of the simulated duck gastric fluid is 2.0 and the pH of the simulated duck intestinal fluid is 7.

4.

7. A method for preparing a nanocomposite loaded with luteolin and doxycycline, wherein the nanocomposite loaded with luteolin and doxycycline according to any one of claims 1-6 is characterized in that, Including the following steps: S1. Dissolve chitosan in an acidic aqueous solution and adjust the pH to 4.0–5.5 to obtain an aqueous phase of amino-protonated chitosan. S2. Luteolin is dissolved in medium-chain triglycerides to form a drug-containing oil phase; S3. Dissolve doxycycline hydrochloride in water to prepare a doxycycline aqueous solution; S4. Add the drug-containing oil phase to the chitosan aqueous phase, and emulsify by high-speed shearing to form a chitosan-stable oil-in-water type primary emulsion. Chitosan is adsorbed at the oil-water interface to form chitosan nanoparticles, and luteolin is encapsulated in the dispersed oil phase along with the drug-containing oil phase. S5. Add doxycycline aqueous solution to the colostrum and sonicate. Doxycycline is initially adsorbed onto the oil-water interface layer of chitosan nanoparticles through intermolecular hydrogen bonding to form an emulsion. S6. Add sodium tripolyphosphate and genipin sequentially to the emulsion to crosslink and solidify the chitosan nanoparticles, thereby obtaining a nanocomposite. S7. Add sodium alginate aqueous solution to the nanocomposite solution to form a pH-responsive outer layer on the surface of the nanocomposite droplets.

8. The method for preparing a nanocomposite of luteolin and doxycycline according to claim 7, characterized in that: In step S6, the phosphate anion of sodium tripolyphosphate forms an electrostatic bridging structure with the protonated amino groups of doxycycline and chitosan, thereby anchoring doxycycline to the oil-water interface layer; the mass ratio of chitosan to sodium tripolyphosphate and genipin is 3:1 to 6:

1.

9. The method for preparing a nanocomposite of luteolin and doxycycline according to claim 7, characterized in that: In step S7, the outer layer of sodium alginate is protonated under the pH conditions of the stomach to form a gel barrier, so that the cumulative release rate of the nanocomposition in the stomach environment is less than 15%; under the pH conditions of the intestine, it is deprotonated and dissolved to restore the pH-responsive release function of the chitosan nanoparticles, so that the cumulative release rate of the nanocomposition in the intestinal environment is greater than 75%.

10. The use of a nanocomposition loaded with luteolin and doxycycline in the treatment of duck-derived Proteus mirabilis infection, wherein the nanocomposition loaded with luteolin and doxycycline according to any one of claims 1-6 is characterized in that: The luteolin is used in combination with the doxycycline at a sub-inhibitory concentration, wherein the sub-inhibitory concentration is 1 / 16 to 1 / 2 of the minimum inhibitory concentration of the luteolin against duck-derived Proteus mirabilis.

Images