A mouse flea joint control agent and a preparation method thereof

Abstract

The application provides a mouse and flea joint control medicament and a preparation method thereof, and belongs to the technical field of mouse and flea joint control medicaments. The method comprises the following steps: dissolving shikonin and quinestrol in edible oil, adding Tween 80 and propylene glycol monooctanoate to prepare a self-microemulsifying concentrate; performing fluidized bed granulation and loading on siliconized microcrystalline cellulose and lactose to obtain an inner layer drug-loaded particle; performing isolation coating by using Eudragit L30 D-55; dissolving doramectin, piperonly ether, piperonyl butyl ether and vitamin E in an ethanol solution of Eudragit E PO, adding a plasticizer to prepare an outer layer coating liquid, and performing outer layer coating; and adding a phagostimulant after screening. The three-layer structure constructed in the application realizes stomach rapid release of the insecticide, intestinal positioning release of the antifertility agent, avoids interference between components, improves palatability and stability, and can simultaneously control reproduction of mice and kill body surface fleas.

Description

Technical Field

[0001] This application relates to the field of rodent and flea control agents, and in particular to a rodent and flea control agent and its preparation method. Background Technology

[0002] The combined rodent-flea control strategy is an integrated prevention and control approach that aims to interrupt the transmission chain of plague bacteria—rodents—fleas—humans by simultaneously regulating the biological community of rodent hosts and their parasitic fleas, thus shifting the focus of disease prevention upstream. Given that simple chemical rodent control may lead to a rapid spread of parasitic fleas after the host dies due to drastic temperature changes, thereby increasing the risk of plague transmission, and that simple flea control cannot fundamentally control the host population, the combined rodent-flea control technology emphasizes the principle of "quickly killing fleas, slowly killing rodents." This means prioritizing the rapid elimination of fleas to cut off the vector transmission route, while simultaneously regulating rodent reproductive capacity to achieve a gradual reduction in rodent population size.

[0003] Existing technologies for combined flea and rodent control mainly include physical methods (such as simultaneous application of rodenticides and insecticides or sprinkling insecticidal powder in poison bait boxes), single-drug dual-effect methods (such as using dosage control to make the same drug act on both the host and parasites simultaneously), and compound agent synergistic methods (such as combining sterilizing agents, rodenticides, and insecticides to create mixed poison baits), aiming to achieve the dual goals of flea control and rodent control in a single application stage. However, these existing technologies still have significant drawbacks. Physical methods have a limited radius of action, and the control effect is not as expected, and it is difficult to promote them in large-scale grassland epidemic areas; single-drug dual-effect methods often lack sustainability, with flea populations rapidly recovering after drug withdrawal, and the effect on rodent population control is weak; although compound agents can achieve synergy to some extent, existing compound formulations are mostly simple mixtures, lacking physical isolation between drug components. Antifertility active ingredients and insecticidal active ingredients are prone to chemical incompatibility, mutual degradation, or release sequence conflicts during storage and release, making it difficult to achieve precise time-phased action of killing insects first and then inhibiting fertility. Therefore, how to simultaneously control rodent reproduction and kill fleas on rodent bodies is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] This application provides a rodent-flea control agent and its preparation method to solve the following technical problem: how to simultaneously control rodent reproduction and kill fleas on the surface of rodents.

[0005] This application provides a method for preparing a combined rodent and flea control agent, the method comprising the following steps: S1. Dissolve shikonin and ethinylestradiol in edible oil in a certain proportion to obtain the Nongda No. 1 infertility agent oil solution. S2. Add Tween 80 and propylene glycol monooctanoate to the Nongda No. 1 sterile oil solution to obtain a self-microemulsifying concentrate. S3. The self-microemulsifying concentrate and the solid carrier are subjected to fluidized bed granulation to obtain inner-layer drug-loaded particles; the solid carrier is composed of silicified microcrystalline cellulose and lactose. S4. Using Eudragit® L30 D-55 aqueous dispersion as the inner isolation layer coating material, the drug-loaded particles are inner-layer coated to obtain isolation-coated particles. S5. Dissolve doramectin technical, pyriproxyfen technical, piperonyl butyl ether and vitamin E in an ethanol solution of Eudragit® E PO, and add a plasticizer to obtain an insecticide outer coating solution. S6. Coat the isolation-coated particles with the insecticide outer coating liquid to obtain film-coated particles; S7. The film-coated particles are sieved, and then attractants are added to obtain a rat and flea control agent.

[0006] Optionally, in step S1, the mass ratio of shikonin to ethinylestradiol is 10:1, and the mass ratio of shikonin to edible oil is 50:1.

[0007] Optionally, in step S2, the mass ratio of Tween 80 to propylene glycol monooctanoate is 2:1 to 3:1, the total amount added is 2 to 3 times the mass of the Nongda No. 1 sterile oil solution, and the mixing temperature is 40 to 50°C.

[0008] Optionally, in step S3, the mass ratio of the silanized microcrystalline cellulose to lactose is 3:1.

[0009] Optionally, in step S3, the fluidized bed granulation includes the following parameters: inlet air temperature of 45~55℃, material temperature of 35~40℃, atomization pressure of 1.2~1.5 bar, and spraying rate of 5~10 g / min; The inner layer drug-loaded particles have a particle size of 200~400μm.

[0010] Optionally, in step S4, the coating weight gain of the inner isolation layer is 5%~8%, and the inner coating includes the following parameters: inlet air temperature of 40~45℃, material temperature of 30~35℃, atomization pressure of 1.0 bar, spraying rate of 3~5 g / min, and after coating, it is dried at 40℃ until the moisture content is ≤2%.

[0011] Optionally, in step S5, the solid content of the Eudragit® E PO ethanol solution is 8%~12%; The plasticizer is triethyl citrate, and the mass of the plasticizer added is 1-2% of the mass of the Eudragit® E PO ethanol solution; The mass ratio of the doramectin technical, the pyriproxyfen technical, the piperonyl butyl ether, and the vitamin E is 1:1:3:0.1; The total mass of the doramectin technical, the pyriproxyfen technical, the piperonyl butyl ether, and the vitamin E is 3-4% of the total mass of the outer coating solution of the insecticide.

[0012] Optionally, in step S6, the weight gain of the outer coating is 3% to 7%; The outer coating includes the following parameters: air inlet temperature of 40~45℃, material temperature of 32~35℃, atomization pressure of 1.0~1.2 bar, spray rate of 2~4 g / min, and curing at 40℃ for 2~4 h after coating.

[0013] Optionally, in step S7, the palatability enhancer is ethyl 2-methylbutyrate, and the mass of the palatability enhancer added is 0.2-0.4% of the mass of the sieved particles; The particle size of the rat and flea control agent is 300μm~500μm.

[0014] Secondly, this application provides a rat-flea control agent prepared by the method described in any one of the first aspects, the rat-flea control agent having the following structure: The inner layer consists of self-microemulsifying solid particles loaded with shikonin and ethinylestradiol; The middle layer is an Eudragit® L30 D-55 insulating layer; The outer layer is a gastric-soluble and immediate-release coating layer of Eudragit® E PO loaded with doramectin, pyriproxyfen, piperonyl butyl ether and vitamin E.

[0015] The technical solutions provided in this application have the following advantages compared with the prior art: This application provides a method for preparing a rodent-flea co-control agent. By constructing a three-layer sandwich structure consisting of an inner enteric-coated anti-fertility component, an intermediate isolation component, and an outer gastric-coated insecticidal component, the method achieves precise spatiotemporal separation and synergistic release of the anti-fertility component and the insecticidal component in the gastrointestinal tract, thereby simultaneously achieving the dual objectives of controlling rodent reproduction and killing fleas on the body surface.

[0016] In controlling rodent reproduction, the inner layer uses edible oil to dissolve shikonin and ethinylestradiol, constructing a self-microemulsifying concentrate. Shikonin is a fat-soluble naphthoquinone compound, and ethinylestradiol is a steroidal antifertility agent. Together, they act synergistically on the rodent reproductive endocrine axis, interfering with reproductive function. The self-microemulsifying system spontaneously forms nano-sized droplets upon contact with water in the intestinal environment, significantly improving the solubility and transmembrane permeability of poorly soluble antifertility drugs, and enhancing oral bioavailability. The middle layer uses Eudragit® L30 D-55 isolation coating, which remains intact in the acidic gastric environment and dissolves only when the intestinal pH is not lower than 5.5. This protects the inner antifertility components from gastric acid degradation, avoids chemical incompatibility with the outer insecticidal components, and ensures that shikonin and ethinylestradiol are released locally in the small intestine and efficiently absorbed into the systemic circulation, exerting a sustained antifertility effect.

[0017] For killing fleas on the surface of rodents, the outer layer uses Eudragit® E PO gastric-soluble coating loaded with doramectin, pyriproxyfen, and piperonyl butyl ether. Eudragit® E PO is insoluble in oral saliva, effectively masking the bitter taste of doramectin and preventing rodents from refusing to eat it. After entering the stomach, the coating dissolves rapidly in the acidic environment, releasing the insecticidal components within minutes. After absorption and distribution, it reaches an effective insecticidal concentration in vivo, achieving rapid killing. Doramectin, as a macrolide antiparasitic drug, is absorbed through the gastrointestinal tract and enters the bloodstream, then distributed to the skin surface via sebum secretion. Fleas ingest the drug when biting, sucking blood, or moving on their bodies. It kills fleas rapidly by activating glutamate-gated chloride ion channels on the neuromuscular cells of the flea, leading to nerve paralysis and death. Pyriproxyfen, an insect growth regulator, interferes with flea embryonic development and larval metamorphosis, thus blocking its life cycle. Piperidin, on the other hand, enhances the toxicity of doramectin and pyriproxyfen by inhibiting the activity of metabolic enzymes in fleas, thus exerting a synergistic effect. Vitamin E, as an antioxidant, is added to the outer coating solution to protect the chemical stability of the insecticidal components during storage and release.

[0018] Therefore, this application achieves physical isolation and release sequence control of the anti-fertility and insecticidal components through an intermediate isolation layer: the outer gastrolytic coating is a gastrolytic taste-masking coating layer that rapidly dissolves and releases the insecticidal component after entering the stomach, ensuring that the drug reaches a completely dissolved state before the stomach empties. It is preferentially absorbed into the systemic circulation in the upper small intestine and distributed to the body surface via sebum secretion, thus taking effect before the anti-fertility component; the inner enteric isolation layer dissolves in the posterior end of the intestine, releasing the anti-fertility agent into the systemic circulation to regulate reproduction. This sequential synergistic strategy of absorbing the insecticidal component first and then the anti-fertility component avoids mutual interference between drugs and achieves simultaneous and precise control of rodent populations and their ectoparasitic fleas. Attached Figure Description

[0019] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0020] Figure 1 A schematic flowchart illustrating a method for preparing a rat and flea control agent provided in this application embodiment; Figure 2 This is a schematic diagram of the structure of the rat and flea control agent provided in the embodiments of this application; Figure 3 This is a particle size distribution diagram of the rat and flea control agent provided in Example 1 of this application; Figure 4 A low-magnification scanning electron microscope image of the surface of the rat and flea control agent provided in Example 1 of this application; Figure 5 This is a high-magnification scanning electron microscope image of the surface of the rat and flea control agent provided in Example 1 of this application; Figure 6 This is a low-magnification scanning electron microscope image of the cross-section of the rat and flea control agent provided in Example 1 of this application; Figure 7 This is a high-magnification scanning electron microscope image of the cross-section of the rat and flea control agent provided in Example 1 of this application; Figure 8 This is a photograph of some mice after the acute oral toxicity test provided in Example 1 of this application. Detailed Implementation

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

[0022] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0023] Figure 1 This is a schematic flowchart illustrating a method for preparing a rat and flea control agent provided in an embodiment of this application.

[0024] like Figure 1 As shown, this application provides a method for preparing a combined rodent and flea control agent, the method comprising the following steps: S1. Dissolve shikonin and ethinylestradiol in edible oil in a certain proportion to obtain the Nongda No. 1 infertility agent oil solution. S2. Add Tween 80 and propylene glycol monooctanoate to the Nongda No. 1 sterile oil solution to obtain a self-microemulsifying concentrate. S3. The self-microemulsifying concentrate and the solid carrier are subjected to fluidized bed granulation to obtain inner-layer drug-loaded particles; the solid carrier is composed of silicified microcrystalline cellulose and lactose. S4. Using Eudragit® L30 D-55 aqueous dispersion as the inner isolation layer coating material, the drug-loaded particles are inner-layer coated to obtain isolation-coated particles. S5. Dissolve doramectin technical, pyriproxyfen technical, piperonyl butyl ether and vitamin E in an ethanol solution of Eudragit® E PO, and add a plasticizer to obtain an insecticide outer coating solution. S6. Coat the isolation-coated particles with the insecticide outer coating liquid to obtain film-coated particles; S7. The film-coated particles are sieved, and then attractants are added to obtain a rat and flea control agent.

[0025] It should be noted that specific information about the Nongda No. 1 sterile agent can be found in previous research, Chinese patent CN106665617A, entitled "Preparation Method of Plant-Derived Rodent Compound Sterile Agent", and the original applicant was Inner Mongolia Agricultural University.

[0026] The functions of each step in the preparation method of the rat-flea combined control agent provided in this application are as follows: S1 (Preparation of the sterilizing agent oil solution): Shikonin and ethinylestradiol are dissolved in edible oil in a specific ratio to obtain an oil solution of shikonin and ethinylestradiol. The purpose of this step is to uniformly disperse two poorly soluble components with antifertility activity—the fat-soluble naphthoquinone compound shikonin and the fat-soluble steroidal compound ethinylestradiol—in the edible oil, forming an oil phase carrier. The edible oil, acting as a solvent, ensures the solubility of the drug, providing an oil phase basis for the subsequent construction of the self-microemulsification system. Simultaneously, it maintains physical separation between the antifertility component and the subsequently added insecticidal component in the early stages, avoiding direct chemical reactions.

[0027] S2 (Preparation of Self-Microemulsion Concentrate): Tween 80 and propylene glycol monooctanoate are added to the infertility agent oil solution, and a clear, transparent self-microemulsion concentrate is formed by stirring. The purpose of this step is to construct a concentrated system that can spontaneously form nano-sized microemulsion droplets upon contact with water (such as intestinal fluid) in the gastrointestinal tract. Self-microemulsification significantly improves the solubility and dissolution rate of shikonin and ethinylestradiol, enhances the absorption of the drug by intestinal epithelial cells, and thus improves the bioavailability of the infertility component. Simultaneously, the self-microemulsion system also helps improve the uniformity of subsequent solid adsorption processes.

[0028] S3 (Preparation of Inner Layer Drug-Loaded Particles by Fluidized Bed Granulation): The self-microemulsion concentrate is adsorbed onto a solid carrier composed of silicified microcrystalline cellulose and lactose via top spraying through a fluidized bed, and then dried to form inner layer drug-loaded particles. This step realizes the conversion of liquid self-microemulsion concentrate into solid particles, facilitating subsequent coating operations and final formulation formation. Silicified microcrystalline cellulose has good flowability and adsorption capacity, while lactose, as a water-soluble filler, can regulate the disintegration and release characteristics of the particles. Fluidized bed granulation ensures uniform distribution of the drug on the carrier surface.

[0029] S4 (Inner Layer Isolation Coating): The drug-loaded particles are coated with Eudragit® L30 D-55 aqueous dispersion to form isolation-coated particles. This step creates a physical barrier between the inner layer (containing shikonin and ethinylestradiol) and the outer layer (containing insecticide): on the one hand, it prevents the outer layer insecticide from penetrating into the inner layer and also prevents the inner layer drug from migrating to the outer layer, avoiding chemical reactions or compatibility issues between different components; on the other hand, Eudragit® L30 D-55 is insoluble in the acidic environment of gastric juice, dissolving only in the intestinal environment. Therefore, this isolation layer protects the inner layer antifertility components from being destroyed by gastric acid, enabling targeted release in the small intestine and concentrating the drug release at the optimal absorption site, thereby improving efficacy.

[0030] S5 (Preparation of insecticide coating solution): Dissolve doramectin, pyriproxyfen, piperonyl butyl ether, and vitamin E in an ethanol solution of Eudragit® E PO, and add a plasticizer to obtain a uniform insecticide coating solution. Eudragit® EPO is a cationic methacrylate copolymer, soluble in ethanol, with good film-forming properties and odor-masking effect.

[0031] Doramectin is a macrolide antiparasitic drug that targets arthropods such as fleas, exerting its killing effect by targeting the parasite's neuromuscular system. Once inside the flea, the drug specifically binds to glutamate-gated chloride channels on the nerve and muscle cell membranes, causing these channels to remain open and resulting in a large influx of chloride ions. This leads to cell membrane hyperpolarization, blocking nerve signal transmission, preventing muscle contraction, causing nerve paralysis, limb paralysis, and ultimately death. This target is only found in invertebrates, making it highly safe for mammals.

[0032] Pyriproxyfen is incorporated into this formulation as an insect growth regulator (juvenile hormone analogue). Its mechanism of action involves disrupting the hormonal balance of fleas, inhibiting embryogenesis, larval metamorphosis, and adult formation. Pyriproxyfen accumulates in the ovaries of female fleas, leading to the production of non-viable eggs, thus fundamentally interrupting the flea life cycle. Combining pyriproxyfen with doramectin overcomes their respective drawbacks, effectively broadening the control spectrum, reducing the dosage of active ingredients, and delaying the development of pest resistance. In this formulation, pyriproxyfen is responsible for long-term control of flea population reproduction, complementing the rapid-killing effect of doramectin.

[0033] Piperyl butyl ether is added to this formulation as a synergist. This ingredient itself does not have insecticidal activity, but it can enhance the effectiveness of insecticidal ingredients such as doramectin and pyriproxyfen by inhibiting the multifunctional oxidase system in fleas.

[0034] Vitamin E is added to this formulation as an antioxidant and stabilizer. During the preparation of the coating solution and subsequent storage, vitamin E can scavenge free radicals and prevent the oxidative degradation of active ingredients such as doramectin and pyriproxyfen, thereby improving the chemical stability and shelf life of the formulation.

[0035] S6 (Outer Coating): The insecticide coating solution is sprayed onto the surface of particles already coated with an isolation layer via a fluidized bed, forming thin-film coated particles. This step achieves spatial separation of the insecticidal and antifertility components, ensuring that all insecticidal components are evenly distributed on the outermost layer of the particles. Eudragit® E PO is a cationic methacrylate copolymer, soluble in acidic media with a pH below 5.0. Because rodent gastric juice is highly acidic, the outer coating dissolves rapidly upon entering the stomach, releasing the insecticidal components within minutes. This allows the drug to reach complete dissolution before gastric emptying, preferentially absorbing into the systemic circulation in the upper small intestine. After absorption and distribution, it reaches an effective insecticidal concentration in vivo, achieving a temporal advantage where absorption begins earlier than the antifertility component. Compared to traditional enteric coatings (which release only in the intestine with delayed absorption), gastric-coated coatings are better suited to achieve the synergistic requirement of killing insects first and then inhibiting fertility.

[0036] S7 (Sieving and Adding Attractant): The film-coated particles are sieved, and then the attractant is added and mixed evenly. Sieving removes excessively large or small particles, ensuring uniform particle size in the finished product and thus guaranteeing consistency in release behavior, flowability, and palatability for each batch. The addition of the attractant increases the bait's attractiveness to rodents, increases foraging rates in the wild, and compensates for any unpleasant odors that may result from the drug or coating material. Because the amount of ethyl 2-methylbutyrate used is extremely low, and ethanol evaporates rapidly at room temperature, testing showed that this operation had no significant impact on the integrity of the outer coating or release behavior. The final product is a rodent and flea control agent with uniform particle size, good palatability, and functional stratification.

[0037] The outer layer, Eudragit® E PO, dissolves rapidly in the stomach (pH < 5), allowing the insecticidal component to dissolve before the stomach empties, and immediately initiate absorption in the upper small intestine (the main absorption window). In contrast, the inner layer, Eudragit® L30 D-55, requires the particles to be transported to the intestines and dissolve at pH ≥ 5.5 to release the antifertility component. This results in a significant time difference in the absorption peak time of the insecticidal component compared to the antifertility component, achieving a synergistic effect of killing insects first and then inhibiting fertility in vivo.

[0038] In some embodiments, in step S1, the mass ratio of shikonin to ethinylestradiol is 10:1, and the mass ratio of shikonin to edible oil is 50:1.

[0039] Shikonin and ethinylestradiol were formulated at a mass ratio of 10:1. Shikonin served as the primary antifertility active ingredient, while ethinylestradiol acted as a steroidal co-regulator. Together, they synergistically interfered with the reproductive endocrine axis. Shikonin was added to edible oil at a mass ratio of 1:50, resulting in a 2% w / w loading of shikonin in the oil phase. This concentration falls within the safe solubility range of lipophilic naphthoquinones in vegetable oils, ensuring complete dissolution of both shikonin and ethinylestradiol to form a homogeneous oil phase. It also preserved sufficient oil matrix for the subsequent self-microemulsification system, while avoiding the storage crystallization risk associated with near-saturation solubility due to excessive drug loading.

[0040] In some embodiments, in step S2, the mass ratio of Tween 80 to propylene glycol monooctanoate is 2:1 to 3:1, the total amount added is 2 to 3 times the mass of the Nongda No. 1 sterile oil solution, and the mixing temperature is 40 to 50°C.

[0041] Tween 80 and propylene glycol monocaprylate were compounded at a mass ratio of 2:1 to 3:1. Tween 80, as a high HLB value primary emulsifier, reduced interfacial tension, while propylene glycol monocaprylate, as a co-emulsifier, inserted into the interfacial film to increase fluidity. The synergistic effect of these two compounds significantly broadened the microemulsion region, ensuring the spontaneous formation of uniformly sized nanodroplets in the aqueous phase at 37°C. The total amount of surfactant added was 2-3 times the mass of the oil solution, resulting in an oil phase content of approximately 25%-33% in the final self-microemulsion concentrate. This proportion conforms to the typical microemulsion region of the SMEDDS pseudo-ternary phase diagram, ensuring both sufficient encapsulation of the oil phase by the emulsifier and maintaining suitable fluidity in the concentrate for subsequent spray granulation. The mixing temperature was set at 40-50°C, slightly higher than room temperature, to reduce the viscosity of the edible oil and surfactant system, promoting uniform mixing of the components. Simultaneously, this temperature is far below the thermal degradation thresholds of shikonin and doramectin, ensuring the chemical stability of the pharmaceuticals.

[0042] In some embodiments, in step S3, the mass ratio of the silanized microcrystalline cellulose to lactose is 3:1.

[0043] In some embodiments, in step S3, the fluidized bed granulation includes the following parameters: inlet air temperature of 45~55℃, material temperature of 35~40℃, atomization pressure of 1.2~1.5 bar, and spraying rate of 5~10 g / min. The inner layer drug-loaded particles have a particle size of 200~400μm.

[0044] A solid carrier is composed of silicified microcrystalline cellulose and lactose in a 3:1 ratio. The silicified microcrystalline cellulose, with its high specific surface area and porosity, provides physical adsorption and capillary fixation for the self-microemulsion concentrate, while lactose, as a water-soluble diluent, promotes disintegration and dissolution of the particles after they enter the intestines. Fluidized bed granulation employs a top-spray method, with a temperature difference of 45-55℃ between the inlet air temperature and the material temperature of 35-40℃. This ensures continuous evaporation of solvent and moisture while preventing the particle surface from becoming too dry while the core remains wet and soft, achieving uniform drying from the inside out. The matching of atomization pressure (1.2-1.5 bar) and spray rate (5-10 g / min) ensures that the concentrate droplets have a suitable size, allowing them to spread evenly on the carrier surface without localized overwetting and adhesion. This ultimately forms inner-layer drug-loaded particles with a diameter of 200-400 μm, providing sufficient specific surface area and suitable mechanical strength for the subsequent two coating layers.

[0045] In some embodiments, in step S4, the coating weight gain of the inner isolation layer is 5% to 8%, and the inner coating includes the following parameters: inlet air temperature of 40 to 45°C, material temperature of 30 to 35°C, atomization pressure of 1.0 bar, spraying rate of 3 to 5 g / min, and drying at 40°C after coating until the moisture content is ≤2%.

[0046] The coating process utilizes Eudragit® L30 D-55 aqueous dispersion, with a weight gain controlled within 5%–8%. This weight gain range is sufficient to form a continuous, pinhole-free polymer barrier film on the particle surface, effectively preventing subsequent organic solvent coating solutions from penetrating into the inner layer and simultaneously preventing the migration of lipid-soluble drugs from the inner layer. The process employs a relatively low inlet air temperature of 40–45°C and a material temperature of 30–35°C, coupled with a 1.0 bar atomization pressure and a spray rate of 3–5 g / min. This allows for aqueous coating to be completed under mild conditions, avoiding premature softening of the inner SMEDDS layer or drug migration due to high temperatures. After coating, the particles are dried at 40°C until the moisture content is ≤2%, thoroughly removing residual moisture and eliminating the potential for water-alcohol process conflicts during subsequent ethanol solution coating, ensuring a dense and intact outer film layer.

[0047] In some embodiments, in step S5, the solid content of the Eudragit® E PO ethanol solution is 8%~12%; The plasticizer is triethyl citrate, and the mass of the plasticizer added is 1-2% of the mass of the Eudragit® E PO ethanol solution; The mass ratio of the doramectin technical, the pyriproxyfen technical, the piperonyl butyl ether, and the vitamin E is 1:1:3:0.1; The total mass of the doramectin technical, the pyriproxyfen technical, the piperonyl butyl ether, and the vitamin E is 3-4% of the total mass of the outer coating solution of the insecticide.

[0048] The solid content of the Eudragit® E PO ethanol solution is set at 8%~12%. This concentration range balances the fluidity of the solution with the absolute amount of film-forming material: too low a solid content results in insufficient film-forming material, making it difficult to form a continuous film layer; this range ensures that the coating solution has a suitable viscosity, allowing it to pass smoothly through the spray gun for atomization and to quickly deposit on the particle surface to form a uniform coating film. The plasticizer triethyl citrate is added at 1%~2% of the ethanol solution mass. This ratio can effectively insert into the polymer molecular chains, lower the glass transition temperature of E PO, and increase the flexibility and crack resistance of the film layer. The insecticidal components, doramectin, pyriproxyfen, piperonyl butyl ether, and vitamin E are formulated in a ratio of 1:1:3:0.1. Doramectin acts as the fast-acting insecticide, pyriproxyfen acts as an insect growth regulator responsible for inhibiting offspring development, piperonyl butyl ether exerts a synergistic effect of inhibiting cytochrome P450 enzymes at three times the dosage, and vitamin E protects the easily oxidized insecticidal components. The total mass of the drug accounts for 3% to 4% of the total mass of the coating solution. This amount of drug is sufficient to provide an effective insecticidal concentration in the outer membrane of limited thickness, while ensuring that the polymer matrix remains the main component, thus maintaining the structural integrity and mechanical strength of the membrane.

[0049] In some embodiments, in step S6, the weight gain of the outer coating is 3% to 7%; The outer coating includes the following parameters: air inlet temperature of 40~45℃, material temperature of 32~35℃, atomization pressure of 1.0~1.2 bar, spray rate of 2~4 g / min, and curing at 40℃ for 2~4 h after coating.

[0050] The outer coating increases weight by 3% to 7%, which allows for the formation of a functional coating of moderate thickness on the particle surface. This coating can hold sufficient insecticide for rapid gastric release without delaying dissolution due to excessive thickness. Setting the inlet air temperature to 40-45℃ and the material temperature to 32-35℃, combined with an atomization pressure of 1.0-1.2 bar and a spray rate of 2-4 g / min, allows for the spraying and evaporation of the ethanol solution under mild conditions, preventing premature hardening of the EPO membrane surface due to high temperatures. After coating, curing at 40℃ for 2-4 hours provides sufficient time for polymer chain relaxation and rearrangement, promoting the formation of a dense and stable network structure between molecules, improving the membrane's resistance to gastrointestinal mechanical friction, and ensuring uniform drug locking within the membrane.

[0051] In some embodiments, in step S7, the palatability enhancer is ethyl 2-methylbutyrate, and the mass of the palatability enhancer added is 0.2-0.4% of the mass of the sieved particles; The particle size of the rat and flea control agent is 300μm~500μm.

[0052] The finished product particle size is controlled at 300~500μm. This size matches the chewing habits and feeding preferences of rodents, making it easy for them to eat and swallow while ensuring a relatively uniform amount of drug ingested per bite. Ethyl 2-methylbutyrate, an attractant, is added at a ratio of 0.2%~0.4%. Its fruity aroma at this dosage is sufficient to mask the bitterness of drugs such as doramectin, increasing the feeding rate in the wild. At the same time, this addition amount is within the conventional loading range of volatile flavorings in solid dosage forms, ensuring the attractant effect while reducing volatilization loss during storage.

[0053] Figure 2 This is a schematic diagram of the structure of the rat and flea control agent provided in the embodiments of this application.

[0054] Based on a general inventive concept, such as Figure 2 As shown, this application provides a rat-flea control agent prepared by the method described in any one of the above methods, the rat-flea control agent having the following structure: The inner layer consists of self-microemulsifying solid particles loaded with shikonin and ethinylestradiol; The middle layer is an Eudragit® L30 D-55 insulating layer; The outer layer is a gastric-soluble and immediate-release coating layer of Eudragit® E PO loaded with doramectin, pyriproxyfen, piperonyl butyl ether and vitamin E.

[0055] It should be noted that the inner layer is composed of silanized microcrystalline cellulose and lactose adsorbed and solidified from a self-microemulsion concentrate, forming solid particles loaded with shikonin and ethinylestradiol. Shikonin is a fat-soluble naphthoquinone compound, and ethinylestradiol is a steroidal antifertility agent; both are poorly soluble in water. The self-microemulsion system spontaneously forms nano-sized droplets upon contact with water in the intestinal environment, which can significantly increase drug solubility and transmembrane permeability, thereby improving the oral bioavailability of the antifertility component, allowing it to be efficiently absorbed through the intestinal absorption window and enter the systemic circulation to exert its antifertility effect.

[0056] The intermediate layer is an Eudragit® L30 D-55 protective coating, a carboxylic acid-containing anionic acrylate copolymer that remains intact and insoluble in the acidic environment of gastric juice, dissolving rapidly only when the intestinal pH is ≥5.5. Its primary function is to create a physical barrier, preventing the penetration of outer organic solvents and insecticides into the inner layer during preparation and storage, while simultaneously preventing the migration of lipid-soluble antifertility drugs from the inner layer to the outer layer, thus avoiding direct contact or chemical reactions between the two components. Secondly, this layer protects shikonin and ethinylestradiol from gastric acid degradation, ensuring their targeted release into the small intestine for absorption.

[0057] The outer layer is a coating of Eudragit® E PO loaded with doramectin, pyriproxyfen, piperonyl butyl ether, and vitamin E. Eudragit® E PO is a cationic copolymer containing dimethylaminoethyl methacrylate units. It is soluble in ethanol, insoluble in oral saliva, but rapidly dissolves in the acidic environment of the stomach (pH < 5). Therefore, this layer is essentially a gastric-soluble rapid-release layer rather than a sustained-release layer. Its functions are: first, to mask the taste in the mouth, utilizing its insolubility in saliva to mask the bitter taste of doramectin and reduce rodent refusal to eat; second, to rapidly dissolve and release the insecticidal components after entering the stomach. Doramectin induces neuromuscular paralysis by activating flea glutamate-gated chloride ion channels, achieving rapid killing; pyriproxyfen, as an insect growth regulator, interferes with flea embryonic development and larval metamorphosis, blocking population reproduction; piperonyl butyl ether inhibits the activity of flea metabolic enzymes, enhancing the toxicity of doramectin and pyriproxyfen; and vitamin E scavenge free radicals, protecting the chemical stability of the outer insecticidal components during storage.

[0058] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If no corresponding industry standard exists, then generally accepted international standards, conventional conditions, or conditions recommended by the manufacturer are followed. The selection of active ingredients in this application is merely an example of a technical solution; actual application must comply with the pesticide registration regulations of the target country / region.

[0059] Example 1 This embodiment provides a method for preparing a combined rodent and flea control agent, comprising the following steps: S1. Preparation of the sterilizing agent oil solution: Weigh shikonin and ethinylestradiol at a mass ratio of 10:1, add soybean oil at a mass ratio of shikonin to edible oil of 1:50, heat in a constant temperature water bath to 30°C, stir until completely dissolved, and let stand to obtain the Nongda No. 1 sterilizing agent oil solution. It should be noted that the sources of shikonin and ethinylestradiol can be found in Chinese patent CN106665617A.

[0060] S2. Preparation of self-microemulsifying concentrate: Tween 80 and propylene glycol monooctanoate were added to the above-mentioned sterilizing agent oil solution at a mass ratio of 2.5:1, with the total amount added being 2.8 times the mass of the oil solution. The mixture was stirred at 45°C until a clear and transparent concentrate was formed. The concentrate was diluted 500 times in water at 37°C, and the particle size was measured by dynamic light scattering method: D50 = 28 nm, D90 = 42 nm, and PDI = 0.16.

[0061] S3. Preparation of Inner Layer Drug-Loaded Particles by Fluidized Bed Granulation: Silicated microcrystalline cellulose and lactose were mixed at a mass ratio of 3:1 as a solid carrier and fed into a fluidized bed granulator. A self-microemulsion concentrate was sprayed in via top spraying. Process parameters: inlet air temperature 50℃, material temperature 38℃, atomization pressure 1.3 bar, spray rate 8 g / min·kg (based on carrier mass), and mass ratio of self-microemulsion concentrate to solid carrier 0.6:1. After spraying, fluidized bed drying was continued, and particles with a diameter of 200~400 μm were collected by sieving to obtain the inner layer drug-loaded particles.

[0062] S4. Inner Layer Coating: The above-mentioned inner layer drug-loaded particles are fed into a fluidized bed coating machine and coated with Eudragit® L30D-55 aqueous dispersion (15% solids content). The weight gain of the coating is controlled at 6.5%. Process parameters: inlet air temperature 42℃, material temperature 32℃, atomization pressure 1.0 bar, spray rate 4 g / min·kg. After coating, the particles are dried in a fluidized bed at 40℃ until the moisture content is ≤2%, obtaining the inner layer coated particles. Samples are soaked in pH 1.2 hydrochloric acid solution for 2 hours; the particles remain intact with no drug leakage.

[0063] S5. Preparation of the insecticide outer coating solution: Dissolve Eudragit® E PO in anhydrous ethanol to prepare a solution with a solid content of 10%. Add 1.5% triethyl citrate as a plasticizer, based on the mass of the Eudragit® E PO ethanol solution. Weigh each component according to the mass ratio of doramectin: pyriproxyfen: piperonyl butyl ether: vitamin E = 1:1:3:0.1, add them to the above solution, and stir until completely dissolved to obtain the insecticide outer coating solution. The total mass of the drug accounts for 3.7% of the total mass of the coating solution.

[0064] S6. Outer Coating: The isolation-coated granules are fed into a fluidized bed coating machine and coated with an insecticide outer coating solution. The weight gain of the coating is controlled at 5%. Process parameters: inlet air temperature 42℃, material temperature 34℃, atomization pressure 1.1 bar, spray rate 3 g / min·kg. After coating, the granules are cured in a fluidized bed at 40℃ for 3 hours to obtain film-coated granules.

[0065] S7: Sieving and Adding Attractant: Sieve the film-coated granules and take the 300-500μm particle size fraction. Add 0.3% of ethyl 2-methylbutyrate (diluted with anhydrous ethanol and sprayed on) by mass of the granules, mix well, and allow the ethanol to evaporate at room temperature under ventilation to obtain the finished rat and flea control agent.

[0066] Example 2 This embodiment provides a method for preparing a combined rodent and flea control agent, comprising the following steps: S1. Preparation of sterilizing agent oil solution: Weigh shikonin and ethinylestradiol at a mass ratio of 10:1, add soybean oil at a mass ratio of shikonin to edible oil of 1:50, heat in a constant temperature water bath to 30°C, stir until completely dissolved, let stand, and obtain Nongda No. 1 sterilizing agent oil solution.

[0067] S2. Preparation of self-microemulsifying concentrate: Tween 80 and propylene glycol monooctanoate were added to the above-mentioned sterilizing agent oil solution at a mass ratio of 3:1, with the total amount added being 3 times the mass of the oil solution. The mixture was stirred at 50°C until a clear and transparent concentrate was formed. The concentrate was diluted 500 times in water at 37°C, and the particle size was measured by dynamic light scattering method: D50 = 26 nm, D90 = 38 nm, and PDI = 0.15.

[0068] S3. Preparation of Inner Layer Drug-Loaded Particles by Fluidized Bed Granulation: Silicated microcrystalline cellulose and lactose were mixed at a mass ratio of 3:1 as a solid carrier and fed into a fluidized bed granulator. A self-microemulsion concentrate was sprayed in via top spraying. Process parameters: inlet air temperature 55℃, material temperature 40℃, atomization pressure 1.5 bar, spray rate 10 g / min·kg (based on carrier mass), and mass ratio of self-microemulsion concentrate to solid carrier 0.6:1. After spraying, fluidized bed drying was continued, and particles with a diameter of 200~400 μm were collected by sieving to obtain the inner layer drug-loaded particles.

[0069] S4. Inner Layer Coating: The above-mentioned inner layer drug-loaded particles are fed into a fluidized bed coating machine and coated with Eudragit® L30D-55 aqueous dispersion (15% solids content). The weight gain of the coating is controlled at 8%. Process parameters: inlet air temperature 45℃, material temperature 35℃, atomization pressure 1.0 bar, spray rate 5 g / min·kg. After coating, the particles are dried in a fluidized bed at 40℃ until the moisture content is ≤2%, obtaining the inner layer coated particles. Samples are soaked in pH 1.2 hydrochloric acid solution for 2 hours; the particles remain intact with no drug leakage.

[0070] S5. Preparation of the insecticide outer coating solution: Dissolve Eudragit® E PO in anhydrous ethanol to prepare a solution with a solid content of 12%. Add 2% triethyl citrate as a plasticizer, based on the mass of the Eudragit® E PO ethanol solution. Weigh each component according to the mass ratio of doramectin: pyriproxyfen: piperonyl butyl ether: vitamin E = 1:1:3:0.1, add them to the above solution, and stir until completely dissolved to obtain the insecticide outer coating solution. The total mass of the drug accounts for 4% of the total mass of the coating solution.

[0071] S6. Outer Coating: The isolation-coated granules are fed into a fluidized bed coating machine and coated with an insecticide outer coating solution. The weight gain of the coating is controlled at 7%. Process parameters: inlet air temperature 45℃, material temperature 35℃, atomization pressure 1.2 bar, spray rate 4 g / min·kg. After coating, the granules are cured in a fluidized bed at 40℃ for 4 hours to obtain film-coated granules.

[0072] S7. Sieving and Adding Attractant: Sieve the film-coated granules and take the 300-500μm particle size fraction. Add 0.4% of ethyl 2-methylbutyrate (diluted with anhydrous ethanol and sprayed on) by mass of the granules, mix well, and allow the ethanol to evaporate at room temperature under ventilation to obtain the finished flea and rodent control agent.

[0073] Example 3 This embodiment provides a method for preparing a combined rodent and flea control agent, comprising the following steps: S1. Preparation of sterilizing agent oil solution: Weigh shikonin and ethinylestradiol at a mass ratio of 10:1, add soybean oil at a mass ratio of shikonin to edible oil of 1:50, heat in a constant temperature water bath to 30°C, stir until completely dissolved, let stand, and obtain Nongda No. 1 sterilizing agent oil solution.

[0074] S2. Preparation of self-microemulsifying concentrate: Tween 80 and propylene glycol monooctanoate were added to the above-mentioned sterilizing agent oil solution at a mass ratio of 2:1, with the total amount added being twice the mass of the oil solution. The mixture was stirred at 40°C until a clear and transparent concentrate was formed. The concentrate was diluted 500 times in water at 37°C, and the particle size was measured by dynamic light scattering method: D50 = 35 nm, D90 = 52 nm, and PDI = 0.20.

[0075] S3. Preparation of Inner Layer Drug-Loaded Particles by Fluidized Bed Granulation: Silicified microcrystalline cellulose and lactose were mixed at a mass ratio of 3:1 as a solid carrier and fed into a fluidized bed granulator. A self-microemulsion concentrate was sprayed in via top spraying. Process parameters: inlet air temperature 45℃, material temperature 35℃, atomization pressure 1.2 bar, spray rate 5 g / min·kg (based on carrier mass), and mass ratio of self-microemulsion concentrate to solid carrier 0.6:1. After spraying, fluidized bed drying was continued, and particles with a diameter of 200~400 μm were collected by sieving to obtain the inner layer drug-loaded particles.

[0076] S4. Inner Layer Coating: The above-mentioned inner layer drug-loaded particles are fed into a fluidized bed coating machine and coated with Eudragit® L30D-55 aqueous dispersion (15% solids content). The weight gain of the coating is controlled at 5%. Process parameters: inlet air temperature 40℃, material temperature 30℃, atomization pressure 1.0 bar, spray rate 3 g / min·kg. After coating, the particles are dried in a fluidized bed at 40℃ until the moisture content is ≤2%, obtaining the inner layer coated particles. Samples are soaked in pH 1.2 hydrochloric acid solution for 2 hours; the particles remain intact with no drug leakage.

[0077] S5. Preparation of the insecticide outer coating solution: Dissolve Eudragit® E PO in anhydrous ethanol to prepare a solution with a solid content of 8%. Add 1% triethyl citrate as a plasticizer, based on the mass of the Eudragit® E PO ethanol solution. Weigh each component according to the mass ratio of doramectin: pyriproxyfen: piperonyl butyl ether: vitamin E = 1:1:3:0.1, add them to the above solution, and stir until completely dissolved to obtain the insecticide outer coating solution. The total mass of the drug accounts for 3% of the total mass of the coating solution.

[0078] S6. Outer Coating: The isolation-coated granules are fed into a fluidized bed coating machine and coated with an insecticide outer coating solution. The coating weight gain is controlled at 3%. Process parameters: inlet air temperature 40℃, material temperature 32℃, atomization pressure 1.0 bar, spray rate 2 g / min·kg. After coating, the granules are cured in a fluidized bed at 40℃ for 2 hours to obtain film-coated granules.

[0079] S7. Sieving and Adding Attractant: Sieve the film-coated granules and take the 300-500μm particle size fraction. Add 0.2% of ethyl 2-methylbutyrate (diluted with anhydrous ethanol and sprayed on) by weight of granules, mix well, and allow the ethanol to evaporate at room temperature under ventilation to obtain the finished flea and rodent control agent.

[0080] Comparative Example 1 This comparative example is modified from the one disclosed in Example 1 as follows: The S4 inner layer isolation coating step is omitted, that is, no intermediate isolation layer is set.

[0081] Modified preparation method: S1~S3 are the same as in Example 1. After obtaining the inner drug-loaded particles, S5 is directly used to prepare the outer coating solution of the insecticide. Then, the coating solution is directly coated on the surface of the inner drug-loaded particles (S6) to obtain film-coated particles. The remaining steps are the same as in Example 1.

[0082] Comparative Example 2 This comparative example is modified from the one disclosed in Example 1 as follows: Replace Eudragit® E PO in S5 with Eudragit® RS PO, leaving the other parameters unchanged.

[0083] Modified preparation method: S1-S4 are the same as in Example 1. In S5, doramectin, pyriproxyfen, piperonyl butyl ether, and vitamin E are dissolved in an ethanol solution of Eudragit® RS PO (solid content 10%), and a plasticizer is added to obtain the outer coating solution. The outer coating weight gain in S6 remains 5%. The remaining steps are the same as in Example 1.

[0084] Comparative Example 3 This comparative example is modified from the one disclosed in Example 1 as follows: The S2 self-microemulsification step is omitted, and the Nongda No. 1 sterile oil solution is directly mixed with the solid carrier and granulated without adding Tween 80 and propylene glycol monooctanoate.

[0085] Modified preparation method: After obtaining the Nongda No. 1 sterile oil solution in S1, the oil solution is directly mixed with silicified microcrystalline cellulose and lactose, and then granulated in a fluidized bed (S3). The remaining steps S4 to S7 are the same as in Example 1.

[0086] The physicochemical properties and application performance of the rat and flea control agents obtained in Examples 1-3 and Comparative Examples 1-3 were tested, and the results are shown in Tables 1 and 2.

[0087] The methods for determining physicochemical properties are as follows: Determination of acid resistance of the isolation layer: Take about 1g of the isolation coating particles obtained in step S4 and place them in 100mL of pH1.2 hydrochloric acid solution. Stir in a water bath at 37℃ (75rpm) and take samples at 0.5h, 1h and 2h. After filtration, determine the content of shikonin in the solution.

[0088] Outer layer insecticide release rate (simulated gastric juice): Take 3g of finished product granules and place them in 500mL of pH 1.2 hydrochloric acid solution (containing 0.1% pepsin), in a 37℃ water bath, and rotate at 75rpm. Take samples at 5min, 10min, 20min, 30min, 45min, and 60min, and determine the cumulative release rate of doramectin by high performance liquid chromatography.

[0089] Inner layer antibiotic release rate (simulated intestinal fluid): The particles (with the outer layer released) after the above gastric fluid release test were transferred to 500 mL of pH 7.4 phosphate buffer (containing 0.5% Tween 80), and samples were taken at 0.5 h, 1 h, 2 h, 3 h and 4 h. The cumulative release rate of shikonin was determined by HPLC.

[0090] Accelerated stability (content retention rate): The finished product was placed at 40℃ and 75% relative humidity for 6 months to accelerate stability. Samples were taken at 0, 1, 2, 3 and 6 months to determine the content of shikonin and doramectin, and the 6-month retention rate (%) was calculated.

[0091] Table 1. Physicochemical properties of the rat-flea combined control agents in Examples 1-3 and Comparative Examples 1-3

[0092] Table 1 shows that the self-microemulsifying particle size (D50 26~35 nm) and PDI (0.15~0.20) of Examples 1-3 all meet the requirements of nanoemulsions; the acid resistance of the isolation layer (2h release rate 1.8%~3.2%) proves that L30 D-55 is intact and effective in gastric acid; the 10-minute release rate of doramectin in gastric juice (78.2%~92.1%) indicates that the outer layer of EPO can achieve rapid release; the 2-hour release rate of shikonin in intestinal juice (81.5%~96.5%) indicates that SMEDDS promotes the efficient release of antibiotics; the accelerated 6-month retention rate is >93%, reflecting the protection of drug stability by the three-layer structure. In Comparative Example 1, after the isolation layer was removed, the acid resistance release rate increased to 48.6%, and the intestinal juice release rate decreased to 26.5%, with a significant deterioration in stability, proving that the isolation layer is indispensable for protecting the inner drug layer. In Comparative Example 2, after switching to RS PO, the gastric fluid release rate plummeted to 18.5%, and the intestinal fluid release rate was only 44.8%, consistent with the insoluble sustained-release characteristics of RS PO, thus verifying the necessity of the gastric exfoliating layer of E PO. In Comparative Example 3, after removing SMEDDS, the intestinal fluid release rate dropped to 42.6%, highlighting the crucial role of self-microemulsification in the dissolution of poorly soluble drugs.

[0093] The application performance testing methods are as follows: Acute oral toxicity (LD50): Mice, half male and half female, were administered the product by gavage at doses of 2000, 1000, 500, and 250 mg / kg body weight. The mice were observed for 14 days, and mortality was recorded. The LD50 value and 95% confidence interval were calculated.

[0094] Palatability (24-hour feed intake rate): Adult mice were selected and fasted for 4 hours. 5g of the finished product pellets (or control sample) were then administered to each cage, and the actual feed intake over 24 hours was measured. Feed intake rate (%) = feed intake / feed amount × 100%. Ten mice were used in each group, and the average value was taken.

[0095] Flea kill rate (24h): Twenty adult cat fleas were artificially infected on the body surface of mice. Twenty-four hours after administration, residual fleas were collected from the body surface using a fine-toothed comb, and the number of dead fleas was recorded. Flea kill rate (%) = (number of infected fleas - number of surviving fleas) / number of infected fleas × 100%. Ten mice were used in each group, and the average value was taken.

[0096] Ovicidal inhibition rate (day 7): On day 7 after drug administration, the number of newly hatched larvae or adults was examined on the surface of the mice. Ovicidal inhibition rate (%) = (Number of newly hatched fleas in the control group - Number of newly hatched fleas in the experimental group) / Number of newly hatched fleas in the control group × 100%.

[0097] Reproductive inhibition rate (60 days): Males and females were housed together, and the number of pups per litter was recorded within 60 days. Reproductive inhibition rate (%) = (average number of pups in the control group - average number of pups in the experimental group) / average number of pups in the control group × 100%. There were 10 pairs of mice in each group.

[0098] Drug metabolism interference (Shikonin AUC ratio): Adult mice of the same strain as those used in the efficacy test were administered the drug via gavage after a 12-hour fast (but with free access to water). Blood samples (50 μL each) were collected from the retro-orbital venous plexus at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours. Plasma was separated, and shikonin blood concentrations were determined by UPLC-MS / MS. The area under the curve (AUC) was calculated. The relative AUC percentage for each control group was calculated using Example 1 as a baseline (100%). Eight mice were used in each group.

[0099] Table 2. Application performance of the combined rodent and flea control agents in Examples 1-3 and Comparative Examples 1-3

[0100] Table 2 shows that the acute oral LD50 of Examples 1-3 was >2000 mg / kg, indicating high safety. Palatability (88.5%-94.1%) demonstrates that EPO effectively masks taste. The flea-killing rate (96.5%-99.5%) and ovicidal inhibition rate (97.3%-99.8%) were excellent, reflecting a synergistic effect of rapid killing and reproduction inhibition. The reproduction inhibition rate (78.3%-85.2%) was positively correlated with the relative AUC of shikonin (85%-112%), indicating that the antifertility effect depends on SMEDDS and the integrity of its release. In Comparative Example 1, due to the lack of an isolation layer, the reproduction inhibition rate (48.5%) and AUC (51%) decreased significantly. Comparative Example 2 showed significantly reduced flea-killing rate (34.7%), ovicidal inhibition rate (21.5%), reproductive inhibition rate (43.2%), and AUC (46%) due to RS PO inhibition of release, confirming that the gastric-dissolved immediate-release outer layer is the core design for achieving pre-insecticidal and post-fertility inhibition. Comparative Example 3, lacking SMEDDS, had the lowest reproductive inhibition rate (39.2%) and AUC (38.5%).

[0101] Figure 3 This is a particle size distribution diagram of the rat and flea control agent provided in Example 1 of this application.

[0102] Depend on Figure 3 It can be seen that the particle size distribution of the finished product particles of the rat and flea control agent in Example 1 exhibits a unimodal normal distribution, with the particle size mainly concentrated in the range of 300~500μm.

[0103] Figure 4 A low-magnification scanning electron microscope image of the surface of the rat and flea control agent provided in Example 1 of this application; Figure 5 This is a high-magnification scanning electron microscope image of the surface of the rat and flea control agent provided in Example 1 of this application; Figure 6 This is a low-magnification scanning electron microscope image of the cross-section of the rat and flea control agent provided in Example 1 of this application; Figure 7 This is a high-magnification scanning electron microscope image of the cross-section of the rat and flea control agent provided in Example 1 of this application.

[0104] Depend on Figures 4-7 It can be seen that the surface of the rat and flea control agent provided in Example 1 is smooth and without cracks. In addition, the layered structure of the middle layer and the outer layer can be clearly observed in the cross-section of the rat and flea control agent.

[0105] Figure 8 This is a photograph of some mice after the acute oral toxicity test provided in Example 1 of this application.

[0106] Depend on Figure 8 It is known that after acute oral exposure to the rat flea control agent of this application, the experimental mice were in good mental condition and had normal activity, without acute toxic reactions such as poisoning, lethargy, convulsions or death. No mice in the 2000 mg / kg dose group died.

[0107] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a combined rodent and flea control agent, characterized in that, The method includes the following steps: S1. Dissolve shikonin and ethinylestradiol in edible oil in a certain proportion to obtain Nongda No. 1 infertility agent oil solution; S2. Add Tween 80 and propylene glycol monooctanoate to the Nongda No. 1 sterile oil solution to obtain a self-microemulsifying concentrate. S3. The self-microemulsifying concentrate and the solid carrier are subjected to fluidized bed granulation to obtain inner-layer drug-loaded particles; the solid carrier is composed of silicified microcrystalline cellulose and lactose. S4. Using Eudragit® L30 D-55 aqueous dispersion as the inner isolation layer coating material, the drug-loaded particles are inner-layer coated to obtain isolation-coated particles. S5. Dissolve doramectin technical, pyriproxyfen technical, piperonyl butyl ether and vitamin E in an ethanol solution of Eudragit® E PO, and add a plasticizer to obtain an insecticide outer coating solution. S6. Coat the isolation-coated particles with the insecticide outer coating liquid to obtain film-coated particles; S7. The film-coated particles are sieved, and then a feeding attractant is added to obtain a rat and flea control agent. The mass ratio of doramectin technical, pyriproxyfen technical, piperonyl butyl ether, and vitamin E is 1:1:3:0.

1. The rat and flea control agent has the following structure: The inner layer consists of self-microemulsifying solid particles loaded with shikonin and ethinylestradiol; The middle layer is an Eudragit® L30 D-55 insulating layer; The outer layer is a gastric-soluble and immediate-release coating layer of Eudragit® E PO loaded with doramectin, pyriproxyfen, piperonyl butyl ether and vitamin E.

2. The preparation method of the rat-flea combined control agent according to claim 1, characterized in that, In step S1, the mass ratio of shikonin to ethinylestradiol is 10:1, and the mass ratio of shikonin to edible oil is 50:

1.

3. The preparation method of the rat-flea combined control agent according to claim 1, characterized in that, In step S2, the mass ratio of Tween 80 to propylene glycol monooctanoate is 2:1 to 3:1, the total amount added is 2 to 3 times the mass of the Nongda No. 1 sterile oil solution, and the mixing temperature is 40 to 50°C.

4. The preparation method of the rat-flea combined control agent according to claim 1, characterized in that, In step S3, the mass ratio of the silanized microcrystalline cellulose to lactose is 3:

1.

5. The preparation method of the rat-flea combined control agent according to claim 1, characterized in that, In step S3, the fluidized bed granulation includes the following parameters: inlet air temperature of 45~55℃, material temperature of 35~40℃, atomization pressure of 1.2~1.5 bar, and spraying rate of 5~10 g / min; The inner layer drug-loaded particles have a particle size of 200~400μm.

6. The preparation method of the rat-flea combined control agent according to claim 1, characterized in that, In step S4, the coating weight gain of the inner isolation layer is 5%~8%, and the inner coating includes the following parameters: air inlet temperature of 40~45℃, material temperature of 30~35℃, atomization pressure of 1.0 bar, spraying rate of 3~5 g / min, and after coating, it is dried at 40℃ until the moisture content is ≤2%.

7. The preparation method of the rat-flea combined control agent according to claim 1, characterized in that, In step S5, the solid content in the ethanol solution of Eudragit® E PO is 8%~12%; The plasticizer is triethyl citrate, and the mass of the plasticizer added is 1-2% of the mass of the Eudragit® E PO ethanol solution; The total mass of the doramectin technical, the pyriproxyfen technical, the piperonyl butyl ether, and the vitamin E is 3-4% of the total mass of the outer coating solution of the insecticide.

8. The method for preparing the rat and flea control agent according to claim 1, characterized in that, In step S6, the weight gain of the outer coating is 3% to 7%; The outer coating includes the following parameters: air inlet temperature of 40~45℃, material temperature of 32~35℃, atomization pressure of 1.0~1.2 bar, spray rate of 2~4 g / min, and curing at 40℃ for 2~4 h after coating.

9. The preparation method of the rat-flea combined control agent according to claim 1, characterized in that, In step S7, the palatability enhancer is ethyl 2-methylbutyrate, and the added mass of the palatability enhancer is 0.2-0.4% of the mass of the sieved particles. The particle size of the rat and flea control agent is 300μm~500μm.

10. A rat and flea control agent prepared by the method of any one of claims 1 to 9.

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