A traditional Chinese medicine composition, a preparation method therefor and application thereof

Abstract

The present application belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition, a preparation method and application thereof. Specifically, based on the theory of traditional Chinese veterinary medicine of'strengthening the body resistance and eliminating pathogenic factors, and treating the root and branch', aiming at the pathogenesis of 'deficiency in the root and excess in the branch' of low immunity in animals, eight kinds of medicinal materials are scientifically screened to form a traditional Chinese medicine compound with the effects of tonifying qi and nourishing blood, invigorating the spleen and kidney, dredging collaterals and eliminating dampness, and clearing heat and detoxifying. Through the verification of a cyclophosphamide-induced low immunity mouse model, the traditional Chinese medicine composition can significantly improve the mental state of the mice, restore the body weight and thymus / spleen index, repair the pathological damage of the spleen tissue, and adjust the inflammatory factor level in the serum and spleen tissue. In addition, in a Marek's disease virus infection model, the traditional Chinese medicine composition combined with the Marek's disease virus vaccine can significantly reduce the viral load in the peripheral blood, liver and spleen, and reduce the liver enlargement, hemorrhage and microscopic structure damage, showing a good immune synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition, its preparation method, and its application. Background Technology

[0002] The immune system plays a vital role in animal husbandry and production, directly impacting animal health and productivity. Weak or incomplete immunity is a source of susceptibility to or chronic diseases, and the development of immune organs directly affects the strength of the body's immunity, seriously influencing problems in livestock farming.

[0003] With the intensive and large-scale development of modern animal husbandry, animals are often subjected to complex factors such as high-density feeding, environmental stress, unbalanced feed nutrition, and frequent use of antibiotics. This can easily lead to problems such as decreased immunity, impaired intestinal barrier function, metabolic abnormalities, and enhanced chronic inflammatory responses. This state can be viewed as a sub-healthy state in modern animals, characterized by "deficiency of spleen and lung qi and depletion of liver and kidney essence" as the root cause, and "internal damp-heat and meridian obstruction" as the manifestation. Symptoms often include decreased feed intake, slow growth, dull coat, poor disease resistance, and decreased production performance, affecting not only animal health but also reducing farming efficiency. Furthermore, unreasonable farming methods and inappropriate high-dose drug use often suppress the animal's immune system. Long-term use or excessive dosage of some drugs, such as corticosteroids and cyclophosphamide antibiotics, can directly or indirectly lead to immunosuppression in animals, thereby increasing the risk of serious infections. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a traditional Chinese medicine composition, its preparation method, and its application. Specifically, this invention is based on the holistic view and syndrome differentiation principles of traditional Chinese veterinary medicine, which emphasizes "strengthening the body's resistance and eliminating pathogenic factors, treating both the root cause and the symptoms." It integrates theories of pathogenesis such as "prolonged illness entering the collaterals" and "mixed deficiency and excess," constructing a dynamic strengthening plan from multiple dimensions, including regulating qi, nourishing blood, replenishing essence, unblocking collaterals, and resolving dampness. The selected medicinal materials are mostly derived from traditional Chinese medicinal materials that have been used in folk medicine for a long time. Modern pharmacological research has also shown that these traditional Chinese medicinal materials have significant effects in regulating immunity, anti-inflammatory and antioxidant properties, and improving metabolic function, which are highly consistent with the theories of traditional Chinese veterinary medicine, such as "tonifying qi and blood, strengthening the spleen and kidneys, unblocking collaterals and eliminating dampness, and clearing heat and detoxifying." Therefore, based on fully absorbing classical theories and modern scientific research results, this invention aims to provide a safe, effective, and sustainable application plan of traditional Chinese veterinary medicine for immune enhancement in animal husbandry through multi-target and multi-level holistic regulation, serving as an immune enhancer. Furthermore, this invention unexpectedly discovered that its combined use with Marek's disease vaccine shows a good synergistic immune effect. Based on the above research results, this invention is thus completed.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a traditional Chinese medicine composition, said composition being made from the following traditional Chinese medicine raw materials in parts by weight: 67-73 parts of wild ginseng, 67-73 parts of ox-dali, 67-73 parts of five-finger peach, 56-60 parts of chicken blood vine, 50-55 parts of eucommia leaf, 50-55 parts of lotus seed, 50-55 parts of large blood vine, and 67-73 parts of raw coix seed.

[0006] In a second aspect, the present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition, the method comprising: pulverizing and mixing the various traditional Chinese medicine raw materials to obtain a traditional Chinese medicine mixture, and then decocting it to obtain the final product.

[0007] A third aspect of the present invention provides the use of the above-mentioned traditional Chinese medicine composition in any one or two of the following: (a) To prepare products for improving immunodeficiency in animals; (b) To prepare products for the prevention or treatment of Marek's disease.

[0008] The beneficial technical effects of one or more of the above technical solutions are as follows: The above technical solution is based on the traditional Chinese veterinary medicine theory of "strengthening the body's resistance and eliminating pathogens, treating both the root cause and the symptoms." Targeting the pathogenesis of immunodeficiency in animals, which is characterized by "deficiency of the root and excess of the branch," eight medicinal herbs were scientifically selected to form a compound traditional Chinese medicine formula that invigorates qi and blood, strengthens the spleen and kidneys, unblocks the meridians and eliminates dampness, and clears heat and detoxifies. Simultaneously, verification in a cyclophosphamide-induced immunodeficiency mouse model showed that the traditional Chinese medicine composition of this invention can significantly improve the mental state of mice, restore body weight and thymus / spleen index, repair pathological damage to spleen tissue, and restore the levels of inflammatory factors (IL-6, IL-1β) in serum and spleen tissue. Furthermore, in a Marek's disease virus infection model, the traditional Chinese medicine composition of this invention, used in combination with a Marek's disease virus vaccine, can significantly reduce viral load in peripheral blood, liver, and spleen, and alleviate liver enlargement, hemorrhage, and microstructural damage, demonstrating a good synergistic immune effect. The raw materials used are mostly medicinal and edible herbs, making them inexpensive. The extraction and preparation process of traditional Chinese medicine compositions is simple to operate, requires no complicated equipment, is easy to scale up, and meets the needs of green and safe breeding, thus having good practical application value. Attached Figure Description

[0009] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0010] Figure 1This document presents the phenotypic observation and weight changes of mice with cyclophosphamide-induced immunodeficiency treated with the traditional Chinese medicine composition in Example 2 of this invention. A represents the phenotypic results of mice in the blank control group, B represents the phenotypic results of mice in the immunodeficiency model group, C represents the phenotypic results of mice in the single-drug *Spatholobus suberectus* polysaccharide administration group, D represents the phenotypic results of mice in the low-dose group of the traditional Chinese medicine composition, E represents the phenotypic results of mice in the medium-dose group of the traditional Chinese medicine composition, F represents the phenotypic results of mice in the high-dose group of the traditional Chinese medicine composition, and G represents the statistical results of weight changes in each experimental group. This means p < 0.01.

[0011] Figure 2 The images show the apparent results and organ indices of the thymus and spleen in mice with cyclophosphamide-induced immunosuppression treated with the traditional Chinese medicine composition in Example 2 of this invention; where A represents the apparent results of the thymus and spleen in each experimental group, B represents the statistical results of the thymus index in each experimental group, and C represents the statistical results of the spleen index in each experimental group. This means p < 0.05. This means p < 0.01.

[0012] Figure 3 The image shows the spleen pathological changes in mice with cyclophosphamide-induced immunosuppression treated with the traditional Chinese medicine composition in Example 2 of this invention. A represents the spleen pathological changes in the blank control group, B represents the spleen pathological changes in the immunosuppression model group, C represents the spleen pathological changes in the group treated with single-drug *Spatholobus suberectus* polysaccharide, D represents the spleen pathological changes in the low-dose group of the traditional Chinese medicine composition, E represents the spleen pathological changes in the medium-dose group of the traditional Chinese medicine composition, and F represents the spleen pathological changes in the high-dose group of the traditional Chinese medicine composition. The scale bar is 50 μm.

[0013] Figure 4 The changes in inflammatory factors in the serum and spleen tissue of mice with cyclophosphamide-induced immunosuppression are shown in Example 2 of this invention. Among them, A is the expression level of IL-6 in the serum of mice in each experimental group, B is the expression level of IL-1β in the serum of mice in each experimental group, C is the expression level of IL-6 in the spleen tissue of mice in each experimental group, and D is the expression level of IL-1β in the spleen tissue of mice in each experimental group.

[0014] Figure 5 The above are the statistical results of the changes in body weight of the traditional Chinese medicine composition in Marek's disease virus chicken flock in Example 3 of this invention; This means p < 0.05. This means p < 0.01. This means p < 0.0001.

[0015] Figure 6 The results of the changes in viral load of the traditional Chinese medicine composition in Marek's disease virus chicken flock in Example 3 of the present invention are shown; wherein, A is the change in MDV copy number of peripheral lymphocytes of chickens in different experimental groups, B is the change in MDV copy number of liver of chickens in different experimental groups, and C is the change in MDV copy number of spleen of chickens in different experimental groups. This means p < 0.05. This means p < 0.01. This means p < 0.001. This means p < 0.0001.

[0016] Figure 7 The following are the histopathological observation results of the traditional Chinese medicine composition in chickens infected with Marek's disease virus in Example 3 of this invention; wherein, A is the histopathological observation result of the liver tissue of chickens in the blank control group, B is the histopathological observation result of the liver tissue of chickens in the virus-inoculated group, C is the histopathological observation result of the liver tissue of chickens in the vaccine-immunized group, and D is the histopathological observation result of the liver tissue of chickens in the group fed with the traditional Chinese medicine composition; the scale bar is 50 μm. Detailed Implementation

[0017] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0019] In a typical embodiment of the present invention, a traditional Chinese medicine composition is provided, which is made from the following traditional Chinese medicine raw materials in parts by weight: 67-73 parts of wild ginseng, 67-73 parts of ox-dali, 67-73 parts of five-finger peach, 56-60 parts of chicken blood vine, 50-55 parts of eucommia leaf, 50-55 parts of lotus seed, 50-55 parts of large blood vine, and 67-73 parts of raw coix seed.

[0020] This invention uses *Gynostemma pentaphyllum* (dried root of *Portulaca grandiflora*) and *Millettia speciosa* (dried root of *Millettia speciosa*) as principal herbs to replenish qi and blood, strengthen the spleen and lungs, and fortify muscles and bones. *Ficus hirta* (root of *Ficus microcarpa*) tonifies the spleen and stomach and strengthens the exterior; *Spatholobus suberectus* (dried stem of *Vigna angularis*) invigorates blood circulation, replenishes blood, and relaxes muscles and tendons; *Eucommia ulmoides* (dried leaf of *Eucommia ulmoides*) tonifies the liver and kidneys and strengthens muscles and bones. These three herbs serve as assistant herbs, assisting the principal herbs in nourishing blood, clearing the meridians, and strengthening the body's foundation. Lotus seed kernels (dried mature seeds of *Nelumbo nucifera*, processed after removing the lotus seed heart) nourish the heart and kidneys, strengthen the spleen, and stop diarrhea; *Sargentodoxa cuneata* (dried stem of *Sargentodoxa cuneata*) clears heat and detoxifies, invigorates blood, and dispels wind. These two herbs serve as adjuvant herbs, assisting the principal and assistant herbs in nourishing blood and calming the mind, while also clearing damp-heat. Raw Job's tears (the dried, mature seed of the grass *Coix lacryma-jobi*) strengthens the spleen, eliminates dampness, clears heat, and drains pus. As an adjuvant, it harmonizes the other herbs, promoting diuresis without harming the body's vital energy. The entire formula works synergistically to invigorate qi and blood, strengthen the spleen and kidneys, unblock meridians and eliminate dampness, and clear heat and detoxify, effectively addressing the "deficiency of the root and excess of the branch" pathogenesis of weakened immunity in animals. Therefore, it has excellent health-promoting and immune-enhancing effects on poultry and livestock, helping to improve survival rates, growth performance, and ensure the stability of livestock production.

[0021] In another specific embodiment of the present invention, the traditional Chinese medicine composition is made from the following traditional Chinese medicine raw materials in parts by weight: 70 portions of wild ginseng, 70 portions of ox-duck-knee, 70 portions of five-finger peach, 58 portions of chicken blood vine, 52 portions of eucommia leaves, 52 portions of lotus seeds, 52 portions of large blood vine, and 70 portions of raw coix seed.

[0022] In another specific embodiment of the present invention, a method for preparing the above-mentioned traditional Chinese medicine composition is provided, the method comprising: pulverizing and mixing each traditional Chinese medicine raw material to obtain a traditional Chinese medicine mixture, and then decocting it to obtain the final product.

[0023] The specific method of decoction includes: soaking the Chinese herbal medicine mixture in water and boiling it once, filtering it to obtain a first filtrate and a first dregs; adding water to the first dregs and boiling it a second time, filtering it to obtain a second filtrate and a second dregs; adding water to the second dregs and boiling it a third time, filtering it to obtain a third filtrate and a third dregs; and combining and concentrating the first filtrate, the second filtrate, and the third filtrate to obtain the final product.

[0024] In another specific embodiment of the present invention, the mass ratio of the traditional Chinese medicine mixture to water is 1:6-10 (preferably 1:8); the mass ratio of the first medicinal residue to water is 1:4-8 (preferably 1:6); and the mass ratio of the second medicinal residue to water is 1:4-8 (preferably 1:6).

[0025] In another specific embodiment of the present invention, the time for the first boiling and simmering is 1.5-3 hours (preferably 2 hours); the time for the second boiling and simmering is 1-2 hours (preferably 1.5 hours); and the time for the third boiling and simmering is 0.5-1.5 hours (preferably 1 hour).

[0026] In this invention, the concentration specifically involves boiling and concentrating the medicinal liquid until each milliliter of liquid is equivalent to 0.8-1.2g of the original medicinal material (preferably 1g); the preparation method also includes the step of freeze-drying the concentrated medicinal liquid.

[0027] In another specific embodiment of the present invention, the above-mentioned traditional Chinese medicine composition is provided for use in any one or two of the following: (a) To prepare products for improving immunodeficiency in animals; (b) To prepare products for the prevention or treatment of Marek's disease.

[0028] In application (a), the animal includes, but is not limited to, non-human mammals (such as mice, pigs, cattle, etc.) or poultry (such as chickens). The immunodeficiency can be induced by immunosuppressants such as cyclophosphamide.

[0029] In application (b), the treatment can be adjunctive or synergistic, and the product can be used in combination with Marek's disease vaccine to achieve a synergistic effect. The Marek's disease vaccine can be a live Marek's disease gene-deleted vaccine for chickens (SC9-1 strain). Therefore, the traditional Chinese medicine composition can be used as a product to enhance the efficacy of Marek's disease vaccine. Specifically, it synergistically improves the liver tissue's recovery ability and significantly reduces the impact of Marek's disease virus on the liver.

[0030] The product may be a drug. In addition to the above-mentioned traditional Chinese medicine composition as the active ingredient, the drug may also contain pharmaceutically acceptable excipients, including but not limited to one or more of the following: diluents, fillers, disintegrants, lubricants, binders, flow agents, complexing agents, plasticizers, colorants, sweeteners, viscosity enhancers, preservatives, or antioxidants.

[0031] In another specific embodiment of the present invention, the diluent includes, but is not limited to, sorbitol, mannitol, starch, lactose, powdered or microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, etc. The fillers include, but are not limited to, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatinized starch, etc. The disintegrants include, but are not limited to, dry starch, crospovidone, crospovidone sodium carboxymethyl cellulose, sodium carboxymethyl starch, and low-substituted hydroxypropyl cellulose. The lubricant includes, but is not limited to, magnesium stearate, sodium dodecyl sulfate, or micronized silica gel.

[0032] The drug can be formulated into various dosage forms, and the methods of administration include, but are not limited to, oral administration, swallowing, sublingual administration, oral mucosal administration, nasogastric feeding, gastric tube administration, duodenal administration, jejunostomy administration, colostomy administration, rectal administration, enema, suppository insertion, and rectal drip. Among these, oral administration is preferred.

[0033] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0034] Example 1 1.1 Raw materials of traditional Chinese medicine compositions The total amount of medicinal herbs is 494 g, including 70 g of wild ginseng, 70 g of *Smilax glabra*, 70 g of *Ficus hirta*, 58 g of *Spatholobus suberectus*, 52 g of *Eucommia ulmoides* leaves, 52 g of lotus seeds, 52 g of *Spatholobus suberectus*, and 70 g of raw coix seed.

[0035] 1.2 Preparation of Traditional Chinese Medicine Compositions The above-mentioned Chinese herbal medicine composition was pulverized according to the compound formula ratio, added to deionized water at a mass ratio of 1:8, soaked overnight, boiled and decocted for 2 hours, filtered, and the residue was added to deionized water at a mass ratio of 1:6, boiled and decocted for 1.5 hours and 1 hour respectively, filtered, the three decoctions were combined, boiled and concentrated until each milliliter of decoction was equivalent to 1 g of the original medicinal material, and freeze-dried to obtain the Chinese herbal medicine composition.

[0036] Example 2 1.1 Experimental Design Cyclophosphamide (CTX) is a commonly used immunosuppressant in clinical practice. It can induce immunodeficiency by inhibiting antibody production and disrupting the immune barrier, and is a classic model for evaluating immunomodulatory effects.

[0037] Sixty-six male BALB / c mice aged 6-8 weeks and weighing approximately 20 grams were randomly divided into six groups (n=10): blank control group (NC), immunodeficiency model group (MOD), single-drug chicken blood vine polysaccharide administration group (PC), low-dose group of traditional Chinese medicine composition (ZYZHW-L), medium-dose group of traditional Chinese medicine composition (ZYZHW-M), and high-dose group of traditional Chinese medicine composition (ZYZHW-H).

[0038] After 7 days of acclimatization, all mice except the NC group were intraperitoneally injected daily with 0.2 mL of cyclophosphamide (80 mg / kg) from day 8 to 10 to establish an immunocompromised mouse model. The NC group was injected with an equal volume of physiological saline. From day 11 to 28, the ZYZHW-L, ZYZHW-M, and ZYZHW-H groups were administered 200 μL of the traditional Chinese medicine composition by gavage daily at doses of 6.25 mg / kg, 12.5 mg / kg, and 25 mg / kg, respectively; the PC group was administered 200 μL of *Spatholobus suberectus* polysaccharide daily at doses of 75 mg / kg. Mice had free access to water and food during the experiment, and the temperature was maintained at 18–22°C and the humidity at 50–65%.

[0039] 1.2 Sample Collection Mice were weighed and their weight recorded daily during the feeding process. After the feeding and drug administration were completed, all mice were fasted for 12 hours. Blood was collected from the eyeballs and centrifuged at 5000 r / min for 10 min to separate the serum, which was then aliquoted for use. Mice were then sacrificed, and the thymus and spleen were collected, observed for morphology and size, and weighed and recorded. One portion was fixed in 4% paraformaldehyde for HE staining, and the other portion was frozen at -80℃ for subsequent experiments.

[0040] 1.3 Measurement of Inflammatory Factor Indicators in Mice Weigh 0.05g of spleen tissue frozen at -80℃, grind and homogenize it, centrifuge at 12000 r / min for 10 min, collect the supernatant, and use an ELISA kit to measure the levels of inflammatory factors IL-6 and IL-1β in serum and spleen homogenate supernatant.

[0041] 1.4 Preparation and observation of HE staining pathological sections of mouse spleen tissue.

[0042] The preparation steps for hematoxylin and eosin (HE) sections of mouse spleen include the following main steps: First, the spleen is aseptically removed from the mouse peritoneum and quickly fixed in 10% neutral formalin solution for 4-24 hours. Then, a gradient dehydration process is performed using 70%, 80%, 90%, 95%, and 100% ethanol solutions to gradually remove water from the tissue, with each dehydration lasting 30 minutes to 1 hour. Finally, 100% ethanol dehydration is performed twice, 1 hour each time. Next, the dehydrated spleen is cleared twice with xylene for 1 hour each time to remove ethanol and prepare for paraffin embedding. After clearing, the tissue is immersed in molten paraffin at 70-60°C for 2-3 hours, ensuring complete encapsulation. Finally, it is cooled into a paraffin block. Subsequently, the cooled and hardened paraffin block is sliced ​​into 5-7 micrometer thick sections using a microtome, ensuring uniform and undamaged sections. After sectioning, the sections were dewaxed with xylene, then hydrated with a gradient of ethanol (100%, 95%, 85%, 70%), and finally rinsed with water. Next, hematoxylin and eosin (HE) staining was performed: the sections were first stained with hematoxylin for 5-10 minutes, then stained with eosin for 2-5 minutes after differentiation, and finally washed away with running water or PBS to remove excess dye. After staining, the sections were dehydrated with a gradient of ethanol and cleared with xylene. Finally, mounting adhesive was applied to the sections, and covers them with coverslips. Finally, the HE-stained sections were observed under a microscope and photographed.

[0043] 1.5 Experimental Data Analysis Experimental data were processed using Graph Pad Prism 9.0 to represent mean ± standard deviation, and statistical calculations and data analysis were performed using one-way ANOVA.

[0044] 1.6 Results (1) Changes in mouse phenotype and body weight After mice were modeled with cyclophosphamide intervention (e.g.) Figure 1 As shown in Figures A to F, mice in different groups exhibited different mental states. Weight records were kept from the start of the cyclophosphamide model, revealing that the control group showed a steady increase in weight, glossy and smooth fur, and normal behavior. The model group and the treatment group, which received intraperitoneal injections of cyclophosphamide for 3 consecutive days, showed a significant decrease in weight (P<0.01). Simultaneously, the physiological state of the mice was observed. After 3 consecutive days of cyclophosphamide injections, the mice exhibited disheveled and dry fur, wrinkled noses, squinting eyes, lethargy, and slower responses to external stimuli. A few mice displayed agitated and aggressive behavior, and their sleep time decreased, indicating significant physiological discomfort. This suggests that the immunodeficiency model was successfully established.

[0045] After modeling, mice with immunodeficiency were treated with different doses of the traditional Chinese medicine composition and *Spatholobus suberectus* polysaccharide. The results showed that the mice gradually recovered. At the end of the experiment, compared with the model group, all mice showed a significant increase in body weight (P<0.01). The rate of weight gain gradually increased with increasing concentration of the traditional Chinese medicine composition, exhibiting a dose-dependent relationship (e.g., ...). Figure 1 (As shown in G). The mice gradually recovered, their fur became softer, and their activity level gradually increased.

[0046] (2) The traditional Chinese medicine composition can restore the atrophy of the thymus and spleen in CTX-induced model mice. Thymus and spleen of mice were collected 14 days after drug administration and found (e.g. Figure 2 As shown in Figure A), the thymus and spleen of the blank control group were normal in color and size, while the thymus and spleen of the model group mice were smaller. Organ index analysis also showed a significant difference in thymus and spleen between the blank group (NC) and the model group (MOD) (P<0.01). After treatment with single-drug chicken blood vine polysaccharide and different concentrations of traditional Chinese medicine combinations, the thymus and spleen of the mice gradually enlarged, indicating that the thymus and spleen of immunocompromised mice could be restored after drug treatment (e.g., Figure 2 (As shown in B and C).

[0047] (3) The traditional Chinese medicine composition can improve the inflammatory symptoms of spleen tissue in CTX-induced model mice. Further paraffin embedding and HE staining of spleen tissue from each group of mice were performed, and the results showed (e.g.) Figure 3 (As shown in Figures A to F) The spleen tissue structure of mice in the blank control group was intact, with clear and evenly distributed red and white pulp boundaries, numerous splenic nodules, and densely packed lymphocytes within the nodules. In the immunodeficiency model group, the white pulp showed weak staining, and the splenic bodies were slightly atrophied, suggesting that CTX has a damaging effect on the spleen and lymphocytes. Compared with the immunodeficiency model group, the white pulp in the blank control group was more deeply stained and evenly distributed; the boundary between the white and red pulp was clearly visible in the single-drug chicken blood vine polysaccharide treatment group and the traditional Chinese medicine combination group, the number of splenic nodules gradually increased, and the lymphocytes gradually became more densely packed, indicating that the traditional Chinese medicine combination can inhibit CTX-induced spleen damage in mice.

[0048] (4) Regulation of inflammatory factor levels in CTX-induced model mice by traditional Chinese medicine composition Detection of inflammatory factors in the serum and spleen tissue of mice in each group revealed that serum (such as...) Figure 4 (as shown in A to B) and spleen tissue (as shown in A to B) Figure 4The levels of inflammatory factors IL-6 and IL-1β were similar in the groups shown in C to D. Compared with the blank control group, the levels of inflammatory factors in serum and spleen were significantly reduced in the immunodeficiency model group (P<0.01). All drug treatment groups showed a recovery, with the high-dose group of the traditional Chinese medicine composition showing the most significant recovery effect (P<0.01), and the levels of inflammatory factors returning to normal. This indicates that treatment with the traditional Chinese medicine composition can improve the symptoms of immunodeficiency in mice.

[0049] Example 3 Marek's disease (MD) is a lymphoproliferative immunosuppressive infectious disease in chickens caused by Marek's disease virus (MDV). MDV is a widespread immunosuppressive virus in chicken flocks, causing lymphoproliferation, lymphomas, and damage to immune organs. With the increasing scale and density of livestock and poultry farming, leading to more opportunities and types of pathogen infection, and in response to the growing problem of antibiotic resistance due to overuse and misuse, immunomodulators have undoubtedly become a breakthrough in the prevention and control of livestock and poultry diseases, especially immunosuppressive diseases. Immunomodulators, used alone or in combination with MD vaccines, can enhance the body's innate immune response and improve the function of the chicken's immune system.

[0050] 1.1 Experimental Design One hundred 1-day-old broiler chickens were randomly divided into four groups. The virulent MDV strain SDAU1501 was diluted to 1000 PFU / 100 μL using DMEM. Twenty-five broiler chickens were intraperitoneally inoculated with 2000 PFU of SDAU1501 strain at 5 days of age, designated as the inoculation group (SDAU1501). Twenty-five broiler chickens were subcutaneously inoculated with Marek's disease gene-deleted live vaccine (SC9-1 strain) in their necks at 1 day of age, and then intraperitoneally inoculated with 2000 PFU of SDAU1501 strain at 5 days of age, designated as the vaccine immunization group (SC9-1). Twenty-five broiler chickens were subcutaneously inoculated with Marek's disease gene-deleted live vaccine (SC9-1 strain) in their necks at 1 day of age, and fed the optimal traditional Chinese medicine composition solution (25 mg / kg) from Example 2 daily via drinking water from 1 to 5 days of age, and then intraperitoneally inoculated with 2000 PFU / 100 μL. The SDAU1501 strain of PFU was designated as the feeding group of the traditional Chinese medicine composition (ZYZHW+SC9-1); the remaining 25 white-feathered broilers were intraperitoneally inoculated with an equal volume of sterile physiological saline at 5 days of age and designated as the blank control group (CON).

[0051] 1.2 Determination of viral load in peripheral lymphocytes At 3, 7, 14 and 21 days after viral challenge, jugular vein blood was randomly collected from 8 broiler chickens in each group. After separating lymphocytes, total DNA was extracted from the lymphocytes, and MDV replication in peripheral lymphocytes was detected by qPCR.

[0052] 1.3 Determination of viral load in viscera At 3, 7, 14, and 21 days post-infection, eight broiler chickens were randomly selected from each group for necropsy. The liver and spleen tissues were ground, homogenized, and total DNA was extracted from the liver and spleen tissues. The MDV replication status in the liver and spleen was detected by qPCR.

[0053] 1.4 Observation of superficial lesions and histopathological features of liver tissue Twenty-one days after viral challenge, chickens in each group were necropsy-examined, and the liver tissue was cut into pieces approximately 0.5-1 cm in size. 3 Small tissue pieces were immediately fixed in 10% neutral formalin for 12-24 hours. After fixation, the tissue was sequentially dehydrated with a gradient of 70%-100% ethanol and cleared in xylene. The tissue was then embedded in molten paraffin at 60-70°C and cooled to form blocks. The paraffin blocks were cut into 5-7 μm thick sections using a microtome, attached to glass slides, dewaxed in xylene, and hydrated with a gradient of ethanol. The sections were stained with hematoxylin, differentiated by blue staining, and eosin staining, then dehydrated again, cleared, and mounted. The prepared sections were observed and photographed under a microscope.

[0054] 1.5 Experimental Results (1) Changes in body weight of broiler chickens There are differences in body weight changes among different groups of broiler chickens (e.g.) Figure 5 As shown in the figure, the weight of the infected group changed significantly and the rate of weight gain decreased. From the third day onwards, the weight of the infected group was significantly lower than that of the blank control group (p<0.01). The weight of the vaccine group and the group fed with the traditional Chinese medicine composition was higher than that of the infected group. After adding the traditional Chinese medicine composition, the weight gain of the chickens accelerated and was significantly higher than that of the infected group (p<0.05), indicating that the traditional Chinese medicine composition of the present invention has the effect of increasing the growth of white-feathered broilers.

[0055] (2) MDV level of broiler chickens Detection of MDV viral load in chicken flocks at different times revealed (e.g.) Figure 6As shown in Figures A to C, the viral load of MDV in the liver and spleen tissues of the inoculated chicken group was significantly higher than that in the vaccine-immunized group and the group fed with the traditional Chinese medicine composition 3 days after inoculation (p<0.01). The viral load in peripheral lymphocytes was also significantly higher than that in the vaccine-immunized group and the group fed with the traditional Chinese medicine composition 14 days after inoculation (p<0.01). This indicates that the Marek's disease gene-deleted live vaccine (SC9-1 strain) inhibited the replication of MDV in the liver, spleen, and peripheral lymphocytes of the chicken flock. In addition, the viral load of MDV detected in the chicken flock fed with the traditional Chinese medicine composition after vaccination was lower than that in the chicken flock immunized only by vaccination. This indicates that the combined use of the traditional Chinese medicine composition and the Marek's disease gene-deleted live vaccine (SC9-1 strain) better inhibited the replication of MDV in the liver, spleen, and peripheral lymphocytes of the chicken flock.

[0056] (3) Observation of pathological changes in the liver of white-feathered broiler chickens Necropsy of liver tissue from each group of chickens revealed that the livers of the virus-infected group were enlarged, significantly swollen and hemorrhagic, dark brown in color, with blunt, rounded edges, and a brittle, easily broken texture. HE staining showed (e.g.) Figure 7 As shown in Figures A to D), hepatocytes exhibited watery degeneration, fatty degeneration, or vacuolar degeneration; hepatic cords were disorganized or broken; hepatic sinusoids were dilated; and the structure was unclear. In contrast, the liver volume and color of the vaccine-treated group and the group fed with the traditional Chinese medicine composition gradually returned to normal, with a significant reduction in hemorrhage and necrosis. HE sections showed hepatocyte regeneration, reconstruction of hepatic cord structure, reduced inflammatory cell infiltration and hepatic sinusoidal congestion, and gradual clearance of necrotic areas leading to mild fibrosis. The combination of the traditional Chinese medicine composition and the chicken Marek's disease gene-deleted live vaccine (SC9-1 strain) further improved liver tissue recovery and reduced the impact of MDV virus on the liver.

[0057] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, The traditional Chinese medicine composition is made from the following traditional Chinese medicine raw materials in parts by weight: 67-73 parts of wild ginseng, 67-73 parts of ox-dali, 67-73 parts of five-finger peach, 56-60 parts of chicken blood vine, 50-55 parts of eucommia leaf, 50-55 parts of lotus seed, 50-55 parts of large blood vine, and 67-73 parts of raw coix seed.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following traditional Chinese medicine raw materials in parts by weight: 70 portions of wild ginseng, 70 portions of ox-dali, 70 portions of five-finger peach, 58 portions of chicken blood vine, 52 portions of eucommia leaves, 52 portions of lotus seeds, 52 portions of large blood vine, and 70 portions of raw coix seed.

3. The method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that, The preparation method includes: The various Chinese medicinal materials are crushed and mixed to obtain a Chinese medicinal mixture, which is then decocted to obtain the final product.

4. The method for preparing the traditional Chinese medicine composition according to claim 3, characterized in that, The specific method of decoction includes: soaking the Chinese herbal medicine mixture in water and boiling it once, filtering it to obtain a first filtrate and a first dregs; adding water to the first dregs and boiling it a second time, filtering it to obtain a second filtrate and a second dregs; adding water to the second dregs and boiling it a third time, filtering it to obtain a third filtrate and a third dregs; and combining and concentrating the first filtrate, the second filtrate, and the third filtrate to obtain the final product.

5. The method for preparing the traditional Chinese medicine composition according to claim 4, characterized in that, The mass ratio of the herbal mixture to water is 1:6-10, the mass ratio of the first dregs to water is 1:4-8, and the mass ratio of the second dregs to water is 1:4-8.

6. The method for preparing the traditional Chinese medicine composition according to claim 5, characterized in that, The mass ratio of the herbal mixture to water is 1:8, the mass ratio of the first dregs to water is 1:6, and the mass ratio of the second dregs to water is 1:

6.

7. The method for preparing the traditional Chinese medicine composition according to claim 4, characterized in that, The first boiling and simmering time is 1.5-3 hours, the second boiling and simmering time is 1-2 hours, and the third boiling and simmering time is 0.5-1.5 hours.

8. The method for preparing the traditional Chinese medicine composition according to claim 7, characterized in that, The first boiling and simmering time is 2 hours, the second boiling and simmering time is 1.5 hours, and the third boiling and simmering time is 1 hour.

9. The use of the traditional Chinese medicine composition according to claim 1 or 2 in any one or two of the following: (a) To prepare products for improving immunodeficiency in animals; (b) To prepare products for the prevention or treatment of Marek's disease.

10. The application according to claim 9, characterized in that, The product is a medicine.

Images