Traditional Chinese medicine nanoparticles for preventing and treating chicken coccidiosis as well as preparation method and application of traditional Chinese medicine nanoparticles

Abstract

The invention provides traditional Chinese medicine nanoparticles for preventing and treating chicken coccidiosis and a preparation method and application thereof, and relates to the technical field of traditional Chinese veterinary medicines. Wherein the medicine component is composed of a first decoction and a second decoction, the first decoction is obtained by decocting astragalus membranaceus and bighead atractylodes rhizome, the second decoction is obtained by decocting antifebrile dichroa and artemisia apiacea, and the first decoction and the second decoction are respectively combined and concentrated to form a unified medicine component according to a preset proportion; the response carrier comprises succinylated chitosan and an azobenzene derivative cross-linked polymer containing azo bonds, and the drug components are embedded in the response carrier to form the traditional Chinese medicine nanoparticles with core-shell structures; the succinylated chitosan is swelled in an acid environment, and azo bonds are structurally changed under the action of azo reductase, so that the traditional Chinese medicine nanoparticles are easier to release medicine components in a chicken coccidiosis related intestinal environment; the invention further provides a preparation method of the traditional Chinese medicine nanoparticles and application of the traditional Chinese medicine nanoparticles in preparation of veterinary medicine preparations or feed additives for preventing and treating chicken coccidiosis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese veterinary medicine technology, and in particular to a traditional Chinese medicine nanoparticle for the prevention and treatment of coccidiosis in chickens, its preparation method, and its application. Background Technology

[0002] Coccidiosis in chickens is a common parasitic disease caused by Eimeria species parasitizing the intestinal epithelial cells of chickens. It is characterized by rapid onset, rapid spread, and wide infection range. Infection can lead to intestinal bleeding, impaired digestion and absorption, stunted growth and development, and decreased feed conversion rate in chickens. In severe cases, it can even cause high mortality rates, posing a persistent threat to the economic benefits and biosecurity of large-scale poultry farming. Therefore, coccidiosis in chickens has always been one of the parasitic diseases that urgently require prevention and control in the poultry farming industry.

[0003] Currently, the main methods for preventing and controlling coccidiosis in chickens include chemical anticoccidial drugs, vaccination, and some traditional Chinese veterinary medicine preparations. Among these, chemical anticoccidial drugs have a relatively fast onset of action and can effectively control infection for a certain period of time. However, long-term or repeated use can easily induce drug resistance in coccidia and may also lead to drug residue risks, adversely affecting the safety of poultry products. Vaccination, on the other hand, has high requirements for breeding management conditions, immunization programs, and environmental control. Moreover, the immunization effect is affected by various factors such as the health status of the flock and the stocking density, and still has certain limitations in practical production applications.

[0004] On the one hand, many traditional Chinese medicine (TCM) prevention and treatment programs typically involve directly decocting multiple herbs together. The release characteristics and stability of the active ingredients in different herbs vary during the decoction process, easily leading to unstable proportions of active ingredients and making it difficult to precisely control the emphasis of each herb in the prevention and treatment process. On the other hand, existing TCM preparations are mostly administered as conventional decoctions or crude extracts. Their release behavior in the intestines lacks a targeted match with the intestinal microenvironment after coccidiosis infection in chickens, making it difficult to achieve effective enrichment or targeted release at the lesion site, thus limiting further improvement in their prevention and treatment efficacy. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the technical problem to be solved by this invention is to propose a traditional Chinese medicine nanoparticle for the prevention and treatment of coccidiosis in chickens, its preparation method, and its application, using the following technical solution: The present invention provides a traditional Chinese medicine nanoparticle for the prevention and treatment of coccidiosis in chickens, characterized in that the above-mentioned traditional Chinese medicine nanoparticle comprises a drug component and a response carrier; The aforementioned drug components include: a first decoction prepared from Astragalus membranaceus and Atractylodes macrocephala, and a second decoction prepared from Dichroa febrifuga and Artemisia annua; The aforementioned response carriers include: succinylated chitosan and cross-linked polymers of azobenzene derivatives containing azo bonds; The aforementioned drug components are encapsulated in the aforementioned response carrier. The aforementioned succinylated chitosan undergoes protonation swelling in an acidic environment. The azo bonds of the aforementioned azobenzene derivative cross-linked polymer can be broken under the action of azo reductase, thereby promoting the release of drug components from the aforementioned traditional Chinese medicine nanoparticles.

[0006] As a further improvement, the first decoction is obtained by decocting Astragalus membranaceus and Atractylodes macrocephala together with water, and the second decoction is obtained by decocting Dichroa febrifuga and Artemisia annua together with water; the first and second decoctions are combined and concentrated to form the above-mentioned drug components.

[0007] As a further improvement, the above-mentioned drug components contain 1g of raw medicinal material. mL -1 .

[0008] As a further improvement, the ratio of the first decoction to the second decoction is as follows: based on the volume of the concentrated decoction, the first decoction is 2 parts and the second decoction is 2.5 to 5 parts.

[0009] As a further improvement, the above-mentioned traditional Chinese medicine nanoparticles have a core-shell configuration, and the above-mentioned azobenzene derivative cross-linked polymer forms an outer shell, the above-mentioned succinylated chitosan forms an inner coating layer, and the above-mentioned drug components are located inside the above-mentioned inner coating layer.

[0010] As a further improvement, the above-mentioned response carrier also includes an ionic crosslinking agent, namely sodium tripolyphosphate, which is used to ionicly crosslink with the above-mentioned succinylated chitosan to form the above-mentioned traditional Chinese medicine nanoparticles.

[0011] Another aspect of the present invention provides a method for preparing traditional Chinese medicine nanoparticles as described above, comprising the following steps: S1: Mix Astragalus membranaceus and Atractylodes macrocephala and decoct with water to obtain the first decoction; mix Dichroa febrifuga and Artemisia annua and decoct with water to obtain the second decoction; combine the first and second decoctions and concentrate them respectively. S2: Succinylated chitosan is dissolved in a weakly acidic aqueous phase, and the above-mentioned drug components are added to obtain a drug-containing aqueous phase; S3: The cross-linked polymer of azobenzene derivative containing azo bonds is dissolved in an organic solvent to obtain an organic phase; S4: Under high-speed shearing and ultrasonic action, the above-mentioned drug-containing aqueous phase is added to the above-mentioned organic phase to form an emulsion, and the above-mentioned emulsion is transferred into an aqueous solution containing sodium tripolyphosphate to form a dispersion of traditional Chinese medicine nanoparticles through ionic cross-linking; S5: After removing the organic solvent, the obtained product is centrifuged, washed and dried to obtain the above-mentioned traditional Chinese medicine nanoparticles.

[0012] As a further improvement, in step S1, the above decoction process includes: soaking the medicinal materials in cold water for 30 minutes, boiling them and then simmering them over low heat for 30 minutes, repeating this process 3 times.

[0013] The present invention further provides the application of the aforementioned traditional Chinese medicine nanoparticles in the preparation of veterinary drug formulations or feed additives for the prevention and treatment of coccidiosis in chickens.

[0014] As a further improvement, the above-mentioned veterinary drug preparation is administered via drinking water, and the equivalent crude drug concentration in the drinking water is 30–60 mg / mL, based on the equivalent crude drug content of the above-mentioned traditional Chinese medicine nanoparticles. -1 .

[0015] Compared with existing technologies, it has the following beneficial effects: Firstly, this invention constructs a traditional Chinese medicine nano-drug delivery system targeting the intestinal lesion microenvironment of chicken coccidiosis by encapsulating the drug components formed from a first decoction and a second decoction in a responsive carrier that simultaneously possesses acid-environment responsiveness and enzyme-enzyme responsiveness. In the responsive carrier, succinylated chitosan undergoes protonation swelling under acidic conditions, and the azobenzene derivative cross-linked polymer containing azo bonds undergoes bond breakage under the action of azo reductase. This makes the nanoparticles more prone to structural changes and promotes drug release in the intestinal microenvironment associated with coccidiosis infection. Through this dual-response mechanism, the drug release behavior is matched with the physiological characteristics of the lesion site in chicken coccidiosis, which is beneficial for improving the specificity and controllability of drug action, thereby enhancing the practical application effect of the traditional Chinese medicine composition in the prevention and treatment of chicken coccidiosis.

[0016] Secondly, this invention involves decocting Astragalus membranaceus and Atractylodes macrocephala, and Dichroa febrifuga and Artemisia annua separately to form a first decoction and a second decoction. After concentration and unification, these decoctions are combined according to volume fractions, allowing for a rational combination of drug components with different effects during the preparation stage. The first decoction focuses on supporting the body's condition and intestinal function, while the second decoction focuses on inhibiting coccidiosis infection. By unifying both decoctions to a concentration equivalent to 1 gram of crude drug per milliliter and limiting the volume ratio range of the first and second decoctions, the proportions of different effects within the drug components can be adjusted while maintaining consistent dosage. This organizational method facilitates the stability and reproducibility of the formulation composition under different infection intensities or application conditions, making the traditional Chinese medicine composition highly feasible in practical prevention and treatment applications.

[0017] Thirdly, the present invention adopts a core-shell configuration of traditional Chinese medicine nanoparticles, and through the ionic cross-linking between sodium tripolyphosphate and succinylated chitosan, stable particle morphology is formed during the preparation of the nanoparticles. The inner coating layer is composed of succinylated chitosan for carrying the drug components, and the outer shell is composed of a cross-linked polymer of azobenzene derivatives containing azo bonds for providing further structural constraints. Through the synergistic effect of ionic cross-linking and polymer cross-linking, the obtained traditional Chinese medicine nanoparticles have good structural stability during processes such as dispersion, centrifugation, washing and drying, and can still undergo responsive changes after entering a specific intestinal microenvironment, which is beneficial to achieving the balance between structural stability and drug release triggerability. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 It is a schematic flow chart of the preparation method of the traditional Chinese medicine nanoparticles of the present invention; Figure 2 It is an experimental control diagram of the influence of the modified Yupingfeng decoction of the pharmaceutical embodiment of the present invention on the cecal morphological structure of Dehua black chickens artificially infected with coccidia. Detailed Embodiments

[0020] For the convenience of those skilled in the art to understand, the structure of the present invention will be further described in detail below by combining the embodiments with experimental data: The present invention provides a traditional Chinese medicine nanoparticle for preventing and treating chicken coccidiosis. The traditional Chinese medicine nanoparticle includes a drug component and a responsive carrier; wherein, the drug component includes: a first decoction prepared from astragalus membranaceus and atractylodes macrocephala, and a second decoction prepared from dichroa febrifuga and artemisia annua; the responsive carrier includes succinylated chitosan and a cross-linked polymer of azobenzene derivatives containing azo bonds; the drug component is embedded in the responsive carrier, the succinylated chitosan undergoes protonation swelling in an acidic environment, and the azo bond of the azobenzene derivative cross-linked polymer can be broken under the action of azoreductase to promote the release of the drug component by the traditional Chinese medicine nanoparticle.

[0021] Specifically, this invention targets the characteristic signals of the intestinal lesion microenvironment in chicken coccidiosis, constructing a dual-trigger drug release pathway based on acidic environment response and enzyme response. After chickens are infected with coccidia, the cecum and rectum are often accompanied by hemorrhage and inflammation, and the local pH value can drop from the normal level of nearly 6.5 to approximately 5.0-6.0. Simultaneously, inflammatory cell infiltration and bacterial proliferation can lead to elevated levels of specific enzymes, such as azoreductase. Based on these lesion signals, this invention utilizes succinylated chitosan to provide protonated swelling properties under acidic conditions, making it easier for nanoparticles to undergo skeletal expansion, increased porosity, or decreased encapsulation strength at the lesion site. Furthermore, it uses a cross-linked polymer containing azo bonds (azobenzene derivatives) to provide the property that azo bonds can be broken by azoreductase, causing the outer cross-linked network to undergo chemical bond breakage and structural loosening under enzymatic action. These two response mechanisms work together within the same nanoparticle system, ensuring the drug component remains relatively stable in the normal intestinal environment and is more easily released in the infection-related microenvironment, thereby improving the specificity and controllability of drug release triggering.

[0022] It should be understood that the above-mentioned pH variation range and enzyme level increase are characterizable features of the intestinal lesion microenvironment associated with coccidiosis infection in chickens, and are used to explain the triggering basis of the response carrier of the present invention. The above values ​​and enzyme levels may fluctuate under different chicken breeds, ages, infection stages and feeding conditions, but the overall characteristics of acidification trend and specific enzyme enrichment trend in lesion sites compared with non-lesion sites remain unchanged.

[0023] In terms of drug composition, this invention employs a two-component decoction system: the first decoction, composed of Astragalus membranaceus and Atractylodes macrocephala, focuses on supporting the body's resistance, invigorating the spleen and replenishing qi, and improving intestinal function; the second decoction, composed of Dichroa febrifuga and Artemisia annua, focuses on inhibiting parasites, clearing heat, and suppressing protozoan reproduction. The two components complement each other: the second decoction directly intervenes in the growth and development of coccidia in the intestine, reducing infection intensity and oocyst excretion; the first decoction provides support against the backdrop of infection stress, decreased food intake, and intestinal mucosal damage, making the intestinal barrier repair and body recovery process more stable, thus ensuring the repeatability and feasibility of the comprehensive prevention and control effect.

[0024] The first decoction was prepared by decocting a mixture of Astragalus membranaceus and Atractylodes macrocephala with water. The second decoction was prepared by decocting a mixture of Dichroa febrifuga and Artemisia annua with water. The decoctions were combined and concentrated to form the medicinal component. The medicinal component contained 1g of raw herbs. mL -1 The ratio of the first decoction to the second decoction should be such that, based on the volume of the concentrated decoction, the first decoction is 2 parts and the second decoction is 2.5 to 5 parts.

[0025] Specifically, this invention uses Astragalus membranaceus and Atractylodes macrocephala as the first medicinal material group, and Dichroa febrifuga and Artemisia annua as the second medicinal material group. These are decocted separately to obtain a first decoction and a second decoction, which are then concentrated and normalized to form drug components by volume fraction. This organizational method allows for the separation and matching of the two medicinal material groups' emphases during the preparation stage, ensuring that the first and second decoctions correspond to the effects of "strengthening the body's resistance" and "insecticidal intervention," respectively, facilitating the subsequent allocation of these two effects in the formulation. Furthermore, by normalizing both decoctions to contain 1g of raw herbs… mL -1 This allows both to maintain consistency in measurement standards, providing a unified basis for subsequent nanoparticle preparation process, including dosage control, equivalent drug dosage measurement, and water administration concentration conversion, thereby reducing the risk of concentration fluctuations due to batch differences.

[0026] It should be understood that the above-mentioned "parts" are units of volume measurement, which are obtained by volume measurement of the concentrated liquid obtained after the first decoction and the second decoction are combined and concentrated to the same concentration of raw medicinal materials. In the embodiments described in this application, the first decoction and the second decoction are both concentrated to a concentration equivalent to 1 gram of raw medicinal materials per milliliter. Based on this, 2 parts of the first decoction and 2.5 to 5 parts of the second decoction are mixed according to the volume ratio to form the drug components.

[0027] In one feasible formulation window, the Chinese herbal raw materials can be composed of 3-6 parts by weight of Astragalus membranaceus, 3-6 parts by weight of Atractylodes macrocephala, 10 parts by weight of Dichroa febrifuga, and 5 parts by weight of Artemisia annua; and in a preferred embodiment, the weight ratio of Astragalus membranaceus to Atractylodes macrocephala is 1:1. This weight window facilitates the organization of the amount of raw materials and batch reproducibility in actual preparation, while not changing the basic technical solution of the present invention, which consists of two decoctions that are concentrated and normalized and mixed according to the proportions.

[0028] It should be understood that the above-mentioned "parts" refer to the weight parts of the medicinal materials, which are used to define the compatibility window of each medicinal material. After the materials are added according to the weight parts and decocted and concentrated to the above-mentioned uniform concentration, they are then mixed according to the volume parts, so that the material compatibility and the mixing ratio remain consistent under the same implementation path.

[0029] Regarding the mixing ratio of the first decoction and the second decoction, this invention limits the first decoction to 2 parts and the second decoction to 2.5 to 5 parts, reflecting the idea of ​​"relatively stable support dosage and adjustable insecticidal intensity": the first decoction, as the basic support dosage, remains fixed, which is beneficial to maintain continuous support for the body's condition and intestinal function under different infection pressures; the second decoction, as the insecticidal dosage, is adjusted within the range of 2.5 to 5 parts, so that the insecticidal intensity can be adapted to different infection intensities, different breeding conditions, or different age stages, without changing the overall framework of the drug components consisting of two types of decoctions.

[0030] To verify the efficacy of the drug components consisting of the first and second decoctions in preventing and treating coccidiosis in chickens, a chicken coccidiosis infection model was established through artificial infection with coccidia, and a control experiment was conducted. The experimental chickens were healthy Dehua Black Chickens of uniform condition, tested negative for coccidiosis, and were acclimatized before the experiment began. No antibiotics or anticoccidial drugs were added to the feed during the experiment, and water was freely available. The experimental chickens were randomly divided into six groups of 20 birds each, with the grouping and treatment as follows.

[0031]

[0032] Table 1 - Grouping and Treatment of Experimental Chickens During the administration period, the medication was administered via drinking water, diluted at a ratio of 1:20 (medicine to water by volume). Throughout the experiment, the chickens' mental state, feed intake, water intake, and fecal characteristics were recorded, as well as mortality. After the experiment, samples were collected and tested to evaluate the effects of the drug components on the severity of coccidiosis infection, the degree of intestinal lesions, and the chickens' recovery.

[0033] The extent of cecal epithelial cell shedding and the degree of cecal lesions were evaluated using a scoring system. The scoring criteria for cecal epithelial cell shedding are summarized below: A score of 0 indicates that the mucosal epithelium is intact and the cell morphology is normal, with no obvious inflammatory changes observed; Scoring 1 to 2 indicates varying degrees of necrosis and shedding of epithelial cells, nuclear pyknosis, deep staining, and fragmentation, but no obvious inflammatory changes. A score of 3 indicates multiple epithelial cell loss, exposure of the lamina propria, and an increase in the number and irregular arrangement of lymphocytes. A score of 4 indicates extensive loss of epithelial cells, with visible edema of the lamina propria, loose connective tissue, and increased cell count. The scoring criteria for cecal lesions are summarized below: A score of 0 indicates no visible lesions; A score of 1 indicates that there are a few scattered petechiae on the cecal wall, but the intestinal wall is not thickened and the contents are normal; A score of 2 indicates numerous lesions with significant blood in the contents and slight thickening of the intestinal wall; A score of 3 indicates a large amount of blood or intestinal core in the cecum, with significant thickening of the intestinal wall and little or no fecal matter. A score of 4 indicates that the cecum is filled with a large amount of blood or intestinal contents and is swollen. The intestinal contents may or may not contain fecal matter, or the chicken may die.

[0034] The above scoring criteria are used to evaluate the comparability of the degree of intestinal damage in different treatment groups, providing a basis for subsequent comprehensive evaluation.

[0035] The results of cecal epithelial cell shedding score and cecal submucosal lymphocyte density are as follows.

[0036]

[0037] Table 2 - Effects of Modified Yupingfeng Decoction on Cecal Lesions and Morphological Structure of Dehua Black Chickens Artificially Infected with Coccidia Combined with the above table and Figure 2 It can be seen that the cecal epithelial cell exfoliation score of the infection control group was relatively high, indicating that the intestinal mucosal structure was significantly affected after infection; the scores of the drug administration groups decreased to varying degrees, and the scores of the low-dose and high-dose modified Yupingfeng groups were lower than those of the corresponding groups using dichroa and artemisia annua alone, indicating that under the conditions of this example, combining astragalus membranaceus and atractylodes macrocephala on the basis of the coccidia-inhibiting effects of dichroa and artemisia annua helped to reduce the degree of cecal mucosal epithelial cell exfoliation. The density of lymphocytes in the submucosa of the cecum showed an increasing trend in the infection control group, and the values of the drug administration groups decreased or remained at a relatively low level, indicating that traditional Chinese medicine intervention had a certain regulatory effect on the tissue responses related to infection.

[0038] To evaluate the growth recovery after infection, the body weight before infection, body weight 7 days after infection, average daily gain, and relative weight gain rate were recorded, and the results are as follows.

[0039]

[0040] Table 3 - Effects of Modified Yupingfeng Decoction on the Weight Gain Rate of Dehua Black Chickens Artificially Infected with Coccidia As can be seen from the above table, the relative weight gain rate of the infection control group was lower than that of the blank control group, indicating that infection had an adverse effect on growth; the relative weight gain rate of the drug administration groups was higher than that of the infection control group, indicating that traditional Chinese medicine intervention helped to improve the growth recovery under the background of infection. The relative weight gain rate of the low-dose modified Yupingfeng group was higher than that of the low-dose dichroa and artemisia annua group, indicating that under the conditions of this example, combining astragalus membranaceus and atractylodes macrocephala could provide support for growth recovery on the basis of coccidia inhibition.

[0041] To comprehensively evaluate the prevention and treatment effects, the survival rate, cecal lesion score, average number of oocysts per gram of feces, and oocyst value were statistically analyzed, and the anti-coccidial index ACI was calculated, and the results are as follows.

[0042]

[0043] Table 4 - Effects of Modified Yupingfeng Decoction on ACI of Dehua Black Chickens Artificially Infected with Coccidia As can be seen from the above table, the infected control group was at a relatively high level in terms of cecal lesion score and average oocyst count per gram of feces, and had a lower ACI, indicating that the infection caused obvious comprehensive damage; the cecal lesion score and average oocyst count per gram of feces in the medicated group decreased, and the ACI increased, indicating that the traditional Chinese medicine intervention could improve the comprehensive damage level related to the infection. Under the same administration background, the cecal lesion score and average oocyst count per gram of feces in the low-dose Yupingfeng modified formula group were lower than those in the low-dose Dichroa febrifuga and Artemisia annua group, and the ACI was higher, indicating that the addition of Astragalus membranaceus and Atractylodes macrocephala helped to further improve the comprehensive prevention and treatment effect under the conditions of this example. A similar trend was also shown when the high-dose Yupingfeng modified formula group was compared with the high-dose Dichroa febrifuga and Artemisia annua group, indicating that this compatibility system had a certain stability under different dose conditions.

[0044] To evaluate the changes in the immune status of the body, the levels of cytokines and intestinal secretory immunoglobulin SIgA were detected, and the results are as follows.

[0045]

[0046] Table 5 - Effects of Yupingfeng modified formula on serum cytokines and intestinal SIgA antibodies in Dehua black chickens artificially infected with coccidia As can be seen from the above table, the values of the infected control group in terms of IL-2 and SIgA were relatively low, while the values of IL-6, IFN-γ and TNF-α were relatively high, indicating obvious changes in the immune response and inflammatory response under the infection background; the levels of IL-2 and SIgA in the medicated group increased, and the values of the inflammation-related factors decreased relatively, indicating that the traditional Chinese medicine intervention had a certain regulatory effect on the immune status related to the infection. Among them, the Yupingfeng modified formula group had higher values in terms of SIgA and other indicators than the group using only Dichroa febrifuga and Artemisia annua, indicating that the addition of Astragalus membranaceus and Atractylodes macrocephala helped to improve the indicators related to intestinal mucosal immunity, enabling the composition system to take into account the support of the immune status while suppressing parasites.

[0047] To evaluate the changes in the oxidative stress status related to the infection, indicators such as malondialdehyde (MDA), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and catalase (CAT) were detected, and the results are shown in the following table.

[0048]

[0049] Table 6 - Effects of Yupingfeng modified formula on the antioxidant function of Dehua black chickens artificially infected with coccidia As shown in the table above, the MDA value was higher in the infection control group, while the values ​​of GSH-Px, SOD, T-AOC, and CAT were lower, indicating enhanced oxidative stress and decreased antioxidant defense capacity under the background of infection. The MDA value decreased and antioxidant-related indicators rebounded in the treatment group, suggesting that traditional Chinese medicine intervention has a certain ameliorative effect on infection-related oxidative stress. Among them, the modified Yupingfeng group showed higher levels of multiple antioxidant indicators than the single-use Changshan Qinghao group, indicating that the addition of Huangqi and Baizhu helps to improve the body's antioxidant-related indicators, enabling the composition system to both suppress insects and buffer and restore the body's stress state.

[0050] Combining cecal tissue morphology indicators, weight gain rate indicators, oocyst excretion-related indicators, and ACI comprehensive evaluation indicators, it can be seen that the drug components of this invention inhibit coccidia reproduction and reduce infection intensity through the second medicinal material group, while simultaneously providing support for the body's condition and intestinal function through the first medicinal material group, thereby improving the degree of intestinal damage, oocyst excretion level, and comprehensive evaluation indicators under the background of infection. The formulation preparation method of separate decoction and combined administration, as well as the limitations on drinking water administration, dilution ratio, and dosage of the two decoctions, ensures the operability and consistency of the preparation and application processes, facilitating their widespread application in the prevention and treatment of chicken coccidiosis.

[0051] In the above embodiments, the traditional Chinese medicine nanoparticles have a core-shell configuration, and the azobenzene derivative cross-linked polymer forms the outer shell, while succinylated chitosan forms the inner coating layer, with the drug components located inside the inner coating layer.

[0052] Specifically, this invention preferably employs a core-shell configuration to enhance the "gating" effect of drug release triggering and system stability. The inner coating layer is formed of succinylated chitosan, with the drug component located inside, ensuring that the drug component is separated from the external environment by at least one responsive polymer coating. Succinylated chitosan contains carboxyl-related substituents, which can maintain a relatively dense and stable coating state under normal intestinal conditions; however, in disease-related acidic environments (e.g., pH approximately 5.0–6.0), these substituents undergo protonation, leading to changes in the electrostatic interactions and hydrophilic / hydrophobic balance between polymer chains. This causes the inner coating layer to swell, increase porosity, or partially disintegrate, thereby reducing the restriction on the drug component and promoting diffusion release.

[0053] The outer shell is formed from a cross-linked polymer of azobenzene derivatives containing azo bonds, which provides further mechanical constraint and chemical barrier to the outer surface of the nanoparticles through a cross-linked network. In the disease microenvironment, when the level of azo reductase increases, the azo bonds break under the action of the enzyme, reducing the effective cross-linking density of the cross-linked network, and causing the outer shell to become structurally loose or even locally ruptured. This change in the outer shell is superimposed on the swelling effect of the inner coating layer in an acidic environment: the loosening of the outer shell provides a release channel for the swelling of the inner coating layer and drug diffusion, while the swelling of the inner coating layer promotes the outward migration of drug components. This forms a dual-response drug release pathway, enabling the nanoparticles to stably carry drug components in non-disease environments, while release is more easily triggered in disease microenvironments.

[0054] It should be understood that the "inner coating layer / outer shell" description of the core-shell configuration is used to clarify the spatial division of labor between the two types of responsive materials: the inner layer is mainly pH-responsive, and the outer layer is mainly enzyme-responsive, thereby achieving a synergistic triggering of "acid swelling + enzyme cleavage". This configuration also facilitates the formation of a layered encapsulation system through emulsification and cross-linking processes during preparation, improving batch stability.

[0055] Furthermore, the response carrier also includes an ionic crosslinking agent, namely sodium tripolyphosphate, which is used to ionicly crosslink with succinylated chitosan to form traditional Chinese medicine nanoparticles.

[0056] Specifically, succinylated chitosan possesses ionizable groups in the aqueous phase. Sodium tripolyphosphate, as a polyanionic crosslinking agent, can undergo ionic crosslinking with the charged groups on the chitosan chains, thereby achieving ionic gelation under aqueous conditions. This ionic crosslinking process can solidify and stabilize the inner coating layer at the nanoscale, enabling the drug-containing aqueous phase to maintain a granular structure even under external shearing, ultrasonic, and emulsifying disturbances, rather than simply forming a solution or flocculation.

[0057] In this invention, sodium tripolyphosphate not only promotes granulation but also improves the stability and separability of the resulting dispersion system: the particles formed through ionic crosslinking have more defined solid-phase boundaries, enabling them to be effectively collected during subsequent centrifugation and washing; simultaneously, the crosslinking network limits the leakage of drug components during preparation to a certain extent, which is beneficial for improving encapsulation efficiency and reducing the loss of active ingredients. The ionic crosslinking of sodium tripolyphosphate with succinylated chitosan also provides a responsive basis for subsequent "swelling under acidic conditions": when entering an acidic environment, the change in the ionization state of the chitosan chains can weaken the effective effect of the crosslinking network, making the particles more prone to swelling, thereby achieving pH-triggered structural loosening and drug release promotion.

[0058] like Figure 1 As shown, another aspect of the present invention provides a method for preparing traditional Chinese medicine nanoparticles, comprising the following steps: S1: Mix Astragalus membranaceus and Atractylodes macrocephala and decoct with water to obtain the first decoction. Mix Dichroa febrifuga and Artemisia annua and decoct with water to obtain the second decoction. Combine the first and second decoctions and concentrate them.

[0059] Specifically, step S1 involves decocting the herbs separately according to the "first herb group" and "second herb group," thus separating the two types of effects from the source. The first decoction corresponds to the aqueous decoction of Astragalus membranaceus and Atractylodes macrocephala, while the second decoction corresponds to the aqueous decoction of Dichroa febrifuga and Artemisia annua. To ensure the operability and consistency of the decoction acquisition process, in one embodiment, the decoction includes: soaking the herbs in cold water for 30 minutes, boiling, and then simmering for 30 minutes, repeating this process three times. This method allows the herbs to be fully soaked and promotes the release of active ingredients, while reducing fluctuations in decoction concentration caused by variations in decoction time. The three decoctions from the same herb group are collected and combined, then concentrated and normalized to the target concentration of raw herbs, for example, 1g. mL -1 This allows the two decoctions to maintain consistent measurement, facilitating the subsequent formation of drug components by volume fraction and providing a controllable raw material basis for the nanoparticle preparation process.

[0060] Furthermore, without altering the basic process described above, step S1 can be further enriched using adsorption and concentration methods to organize the decoction components. For example, macroporous resin adsorption can be used to enrich the active ingredients in the second decoction that are more focused on insect inhibition, and to enrich the polysaccharide components in the first decoction that are more focused on immunomodulation, thereby increasing the relative proportion of the target components in the subsequent encapsulation system and reducing the impact of non-target impurities on granulation and stability. The above enrichment steps are optional implementation methods and can be configured according to batch differences in raw materials and the scale of preparation.

[0061] S2: Succinylated chitosan is dissolved in a weakly acidic aqueous phase, and the drug component is added to obtain a drug-containing aqueous phase.

[0062] Specifically, in step S2, succinylated chitosan is placed in a weakly acidic aqueous phase to ensure uniform dispersion, thus forming the inner coating layer of the subsequent nanoparticles. The drug component obtained in step S1 and formulated according to the specified ratio is then added to this phase, allowing the drug component to be uniformly distributed within the succinylated chitosan system, resulting in a drug-containing aqueous phase. Since the drug component contains multiple water-soluble or dispersible components, the aqueous phase system is more conducive to achieving uniform mixing and reducing the risk of phase separation. By introducing the drug component in the aqueous phase stage, it can be simultaneously embedded within the particles during subsequent emulsification and crosslinking processes, forming a spatial distribution basis where the drug component is located inside the inner coating layer.

[0063] S3: The cross-linked polymer of azobenzene derivative containing azo bonds is dissolved in an organic solvent to obtain an organic phase.

[0064] Specifically, in step S3, the azobenzene derivative crosslinked polymer containing azo bonds is dissolved in an organic solvent to obtain the organic phase of the outer shell material. The organic solvent can be a solvent system that is compatible with the emulsification-solvent evaporation process and is easily removed subsequently, such as dichloromethane. The purpose of placing the outer shell material in the organic phase is to make it tend to distribute in the oil phase or oil-water interface during emulsification, thereby facilitating the formation of the nanoparticle outer shell during subsequent solvent evaporation and structural solidification, and providing a spatial basis for the fracture response triggered by azoreductase.

[0065] S4: The drug-containing aqueous phase is added to the organic phase to form an emulsion under high-speed shearing and ultrasonic action; then, under stirring conditions, the emulsion is added dropwise or slowly to an aqueous solution containing sodium tripolyphosphate, so that succinylated chitosan and sodium tripolyphosphate undergo ionic cross-linking, and under the synergistic effect of emulsification and ionic cross-linking, a dispersion system containing the traditional Chinese medicine nanoparticles is formed, and a traditional Chinese medicine nanoparticle dispersion is obtained.

[0066] Specifically, in step S4, the drug-containing aqueous phase is first added to the organic phase under high-speed shearing and ultrasonication, causing the aqueous phase to form fine droplets and dissolve uniformly in the organic phase, resulting in a primary emulsion system with more uniform particle size. High-speed shearing is used to provide macroscopic dispersion and initial refinement, while ultrasonication is used to further reduce droplet size and agglomeration, making the subsequent granulation process more stable. Subsequently, the emulsion is transferred to an aqueous solution containing sodium tripolyphosphate. During the phase transfer process, sodium tripolyphosphate undergoes ionic cross-linking with succinylated chitosan, causing the succinylated chitosan within the aqueous droplets or at its interface to form a cross-linked network and solidify, thereby obtaining a dispersion of traditional Chinese medicine nanoparticles with a stable particle morphology.

[0067] S5: After removing the organic solvent, the obtained product is centrifuged, washed and dried to obtain traditional Chinese medicine nanoparticles.

[0068] Specifically, step S5 removes the organic solvent from the dispersion system obtained in step S4, reducing the residual organic phase and promoting further solidification or stabilization of the outer shell material on the particle surface. Subsequently, the herbal nanoparticles are separated from the dispersion by centrifugation, and unencapsulated free components, unreacted crosslinking agents, and other impurities are removed by washing. Finally, the nanoparticles are dried to obtain the herbal nanoparticles. Freeze-drying can be selected based on storage and application requirements to obtain herbal nanoparticle powder that is easy to store, transport, and subsequently reconstitute, thus clearly distinguishing it from the "dispersion" form and facilitating dosage and concentration measurement in veterinary drug formulations or feed additive applications.

[0069] In one embodiment, centrifugation and washing are used to remove uncrosslinked free components and residual solvents, and drying is performed by freeze drying or vacuum drying, so that the traditional Chinese medicine nanoparticles are transformed from a dispersion system into a storable solid powder form.

[0070] This invention also provides the application of traditional Chinese medicine nanoparticles in the preparation of veterinary drug formulations or feed additives for the prevention and treatment of coccidiosis in chickens. The veterinary drug formulation is administered via drinking water, and the concentration of the traditional Chinese medicine nanoparticles in the drinking water is 30–60 mg, based on the equivalent raw drug amount. mL -1 .

[0071] To ensure a comparable metrological standard between the novel nanoparticle formulation and traditional decoction systems, this invention employs an equivalent crude drug content measurement method to determine the dosage intensity in drinking water. Specifically, the equivalent crude drug content of the drug components embedded in the herbal nanoparticles is used as a conversion benchmark to calculate the equivalent crude drug concentration per unit volume of drinking water. In one embodiment, the equivalent crude drug concentration in the drinking water is 30–60 mg. mL -1 Correspondingly, the dosage of traditional Chinese medicine nanoparticles per unit volume of drinking water can be calculated and determined based on the drug loading of the nanoparticles. Through this conversion method, different batches of traditional Chinese medicine nanoparticles can still be calibrated using the equivalent raw drug dosage caliber even when the drug loading fluctuates, ensuring the repeatability and feasibility of the dosage.

[0072] Furthermore, in application, the concentration range can be selected by combining factors such as daily water consumption, age, weight, and infection pressure in the breeding scenario; and the intensity of the insect-suppressing direction can be graded and configured by combining the ratio window of the first decoction and the second decoction, so that the prevention and control strategy has a certain degree of adjustability while maintaining a consistent framework.

[0073] It should be understood that the above embodiments are used to illustrate the traditional Chinese medicine nanoparticles of the present invention, their preparation methods and applications; without departing from the technical framework of "the drug components are composed of a first decoction and a second decoction, the response carrier includes a cross-linked polymer of succinylated chitosan and azobenzene derivatives containing azo bonds, and the release is promoted through acid swelling and azo reductase cleavage", the source of raw materials, solvent system, shear and ultrasonic parameters, cross-linking agent addition method, centrifugation and washing conditions, drying method and specific execution details of drug administration can be adjusted according to the production scale and process conditions, and all should fall within the protection scope of the present invention.

Claims

1. A type of traditional Chinese medicine nanoparticle for the prevention and treatment of coccidiosis in chickens, characterized in that, The traditional Chinese medicine nanoparticles include drug components and a response carrier; The drug components include: a first decoction prepared from Astragalus membranaceus and Atractylodes macrocephala, and a second decoction prepared from Dichroa febrifuga and Artemisia annua; The response carrier comprises: succinylated chitosan and a cross-linked polymer of azobenzene derivatives containing azo bonds; The drug component is encapsulated by the response carrier, the succinylated chitosan undergoes protonation swelling in an acidic environment, and the azo bonds of the azobenzene derivative cross-linked polymer can be broken under the action of azo reductase to promote the release of the drug component from the traditional Chinese medicine nanoparticles.

2. The traditional Chinese medicine nanoparticles according to claim 1, characterized in that, The first decoction is obtained by decocting a mixture of Astragalus membranaceus and Atractylodes macrocephala with water, and the second decoction is obtained by decocting a mixture of Dichroa febrifuga and Artemisia annua with water. The first and second decoctions are combined and concentrated to form the drug components.

3. The traditional Chinese medicine nanoparticles according to claim 2, characterized in that, The drug component contains 1g of crude drug. mL -1 .

4. The traditional Chinese medicine nanoparticles according to claim 3, characterized in that, The ratio of the first decoction to the second decoction is as follows: based on the volume of the concentrated decoction, the first decoction is 2 parts and the second decoction is 2.5 to 5 parts.

5. The traditional Chinese medicine nanoparticles according to claim 1, characterized in that, The traditional Chinese medicine nanoparticles have a core-shell configuration, and the azobenzene derivative crosslinked polymer forms the outer shell, the succinylated chitosan forms the inner coating layer, and the drug component is located inside the inner coating layer.

6. The traditional Chinese medicine nanoparticles according to claim 5, characterized in that, The response carrier further includes an ionic crosslinking agent, which is sodium tripolyphosphate, used to ionicly crosslink with the succinylated chitosan to form the traditional Chinese medicine nanoparticles.

7. A method for preparing traditional Chinese medicine nanoparticles as described in any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Mix Astragalus membranaceus and Atractylodes macrocephala and decoct with water to obtain the first decoction; mix Dichroa febrifuga and Artemisia annua and decoct with water to obtain the second decoction; combine the first and second decoctions and concentrate them respectively. S2: Succinylated chitosan is dissolved in a weakly acidic aqueous phase, and the drug component is added to obtain a drug-containing aqueous phase; S3: The cross-linked polymer of azobenzene derivative containing azo bonds is dissolved in an organic solvent to obtain an organic phase; S4: The drug-containing aqueous phase is added to the organic phase under high-speed shearing and ultrasonic action to form an emulsion, and the emulsion is transferred into an aqueous solution containing sodium tripolyphosphate to form a dispersion of traditional Chinese medicine nanoparticles through ionic cross-linking; S5: After removing the organic solvent, the obtained product is centrifuged, washed and dried to obtain the traditional Chinese medicine nanoparticles.

8. The preparation method according to claim 7, characterized in that, In step S1, the decoction process includes: soaking the medicinal materials in cold water for 30 minutes, boiling them, and then simmering them over low heat for 30 minutes, repeating this process 3 times.

9. The use of the traditional Chinese medicine nanoparticles according to any one of claims 1 to 6 in the preparation of veterinary drug preparations or feed additives for the prevention and treatment of coccidiosis in chickens.

10. The application according to claim 9, characterized in that, The veterinary drug preparation is administered via drinking water, and the equivalent crude drug concentration in the drinking water is 30–60 mg / mL, based on the equivalent crude drug content of the traditional Chinese medicine nanoparticles. -1 .

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