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

Abstract

The application discloses a traditional Chinese medicine extract composition and a preparation method and application thereof. The traditional Chinese medicine extract composition is prepared from the following raw materials in parts by weight: Sargentodoxa 30-40 parts, Herba Euphorbiae Humifusae 20-25 parts, Dwarf Tea 15-20 parts, Crataegus Pinnatifida Bunge 10-15 parts and Potentilla 5-12 parts. The preparation method comprises the following steps: crushing the medicinal materials, adding water and heating to extract, adding water to re-extract the residues, and then concentrating and drying the combined extract to obtain the traditional Chinese medicine extract composition. The traditional Chinese medicine extract composition has obvious inhibitory effect on Clostridium perfringens of chickens, and can be used for preventing and treating chicken necrotic enteritis, repairing necrotic intestinal mucosa, reducing intestinal inflammation, improving the production performance of broilers, and can be used for the egg incubation period of broiler breeders.

Description

Technical Field

[0001] This invention belongs to the field of veterinary traditional Chinese medicine preparations and livestock and poultry intestinal health regulation technology. Specifically, it relates to a traditional Chinese medicine extract composition and its preparation method and application, especially to a traditional Chinese medicine extract composition for targeted prevention and treatment of necrotizing enteritis in chickens, its preparation process, in vitro antibacterial verification, animal challenge model verification, and large-scale breeding application. Background Technology

[0002] Necrotic enteritis in chickens is one of the most common and costly bacterial intestinal diseases in large-scale chicken farming. It is mainly caused by the overgrowth of Clostridium perfringens type A (Clostridium perfringens) and the secretion of toxins such as NetB. Typical pathological manifestations include congestion and hemorrhage of the small intestinal mucosa, coagulative necrosis, large-area mucosal shedding, intestinal wall ulceration, and even perforation. Clinical symptoms include black, tarry, watery feces, decreased feed intake, dirty and ruffled feathers, growth inhibition, increased feed conversion ratio, and acute death in severe cases.

[0003] Currently, the livestock industry mainly relies on antibiotics such as colistin and amoxicillin for disease control. However, long-term overuse can lead to problems such as drug resistance, drug residues in livestock and poultry products, disruption of intestinal flora, and frequent secondary enteritis, which is no longer in line with the national policy of antibiotic-free farming.

[0004] Most of the existing publicly disclosed traditional Chinese medicine patents are broad-spectrum antidiarrheal formulas, which have the following shortcomings: the ingredients are complicated and the targets are not clear; they only stop diarrhea, do not inhibit bacteria, and are difficult to repair necrotic and sloughed intestinal mucosa; there are too many cold and cooling drugs, which are not suitable for broiler breeders during the egg-laying period and may affect the egg production rate and hatchability; there is a lack of standardized antibacterial data, animal challenge models and clinical data, which are not innovative and convincing enough.

[0005] Therefore, there is an urgent need for a green and safe Chinese herbal extract composition that is simple in formulation, has a clear target, can inhibit Clostridium perfringens and repair intestinal mucosa, and is suitable for use in broiler breeders throughout the entire process. Summary of the Invention

[0006] This invention aims to overcome the problems of existing traditional Chinese medicine compound formulas, such as complex ingredients, poor targeting, ineffective repair of necrotic intestinal mucosa, and limited use in breeder hens during the egg-laying period. It provides a traditional Chinese medicine extract composition, its preparation method, and its application. This composition should have the effects of inhibiting Clostridium perfringens in chickens, reducing intestinal inflammation, promoting wound healing and repair of necrotic intestinal mucosa, and blocking the absorption of enterotoxins. It can be safely used in broilers, pullets, laying hens, and broiler breeder hens during the egg-laying and incubation period.

[0007] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a traditional Chinese medicine extract composition, made from the following raw materials in parts by weight: 30-40 parts of Sargentodoxa cuneata, 20-25 parts of Euphorbia humifusa, 15-20 parts of Dichroa febrifuga, 10-15 parts of Crataegus pinnatifida, and 5-12 parts of Potentilla chinensis.

[0008] Preferably, the herbal extract composition is made from the following raw materials in parts by weight: 34-36 parts of Sargentodoxa cuneata, 24-25 parts of Euphorbia humifusa, 17-19 parts of Dichroa febrifuga, 11-13 parts of Crataegus pinnatifida, and 9-11 parts of Potentilla chinensis.

[0009] Most preferably, the herbal extract composition is made from the following raw materials in parts by weight: 35 parts of Sargentodoxa cuneata, 25 parts of Euphorbia humifusa, 18 parts of Lysimachia christinae, 12 parts of Crataegus pinnatifida, and 10 parts of Potentilla chinensis.

[0010] The principle of monarch-minister-assistant-guide combination in this invention is as follows: (1) The principal drug, *Sargentodoxa cuneata*, is the core drug in the formula. It effectively inhibits pathogenic bacteria in the intestines, significantly reduces intestinal inflammatory response, clears damp-heat and blood stasis in the intestines, improves intestinal wall congestion, edema and inflammatory necrosis, blocks the development of enteritis and enterotoxic syndrome, and inhibits pathogen proliferation and intestinal tissue damage from the root.

[0011] (2) The assistant herb, Euphorbia humifusa, inherits the effects of clearing heat and detoxifying, cooling blood and stopping dysentery from the principal herb. It also has the effects of promoting diuresis and stopping diarrhea, inhibiting bacteria and anti-inflammation. It can quickly improve symptoms such as diarrhea, bloody stools and intestinal mucosal bleeding in poultry. It forms a synergistic effect of "clearing and detoxifying + stopping diarrhea" with Spatholobus suberectus, which doubles the effect of anti-enteritis and stopping dysentery, and helps to clear intestinal toxins.

[0012] (3) Adjuvant herbs: Dwarf tea and wild hawthorn: Dwarf tea clears damp heat in the intestines, reduces inflammation and swelling, alleviates intestinal wall edema and inflammatory exudation, improves intestinal permeability, relieves diarrhea and watery stools, and helps protect the intestinal mucosa from inflammatory damage; Wild hawthorn promotes digestion and regulates the spleen and stomach, improves decreased feed intake and indigestion in poultry, and also regulates intestinal flora, alleviates the adverse effects of astringent drugs on the stomach, and enhances overall digestive function. The combination of the two herbs, one clearing and the other regulating, synergistically strengthens intestinal anti-inflammatory and digestive repair, and solves the problems of indigestion and intestinal dysfunction associated with enteritis.

[0013] (4) Potentilla chinensis: harmonizes the properties of all the medicines in the formula and guides them directly to the intestinal lesions; it also has the functions of astringing the intestines and stopping diarrhea, clearing heat and detoxifying, enhancing the effect of stopping diarrhea, balancing the clearing and astringing of the whole formula, so that the efficacy of the medicine is long-lasting and does not harm the body's vital energy, achieving targeted anti-inflammatory, intestinal repair and overall conditioning.

[0014] The composition of this invention is constructed with a four-dimensional targeting mechanism that combines antibacterial, anti-inflammatory, wound-healing, and repair properties. Unlike ordinary antidiarrheal Chinese medicines, it is specifically designed to target core pathologies such as Clostridium perfringens toxin, intestinal wall necrosis, and ulceration.

[0015] Secondly, the present invention also provides a method for preparing the above-mentioned traditional Chinese medicine extract composition, comprising the following steps: (1) Wash, remove impurities, air dry, and pulverize the medicinal materials, including large-leaved vine, ground euphorbia, dwarf tea, wild hawthorn, and cinnamon bark, and preferably pass them through a 40-mesh sieve to obtain medicinal powder; (2) Weigh each medicinal powder according to the ratio, add water and heat to extract, separate the residue and collect the extract; (3) Add water to the dregs, heat and extract again, collect the extract, and combine it with the extract from step (2) and concentrate it; (4) The concentrated liquid obtained in step (3) is dried to obtain the Chinese herbal extract composition.

[0016] Preferably, in step (2), 8-12 times the total mass of the medicinal materials are added to water and heated for extraction, with an extraction time of 1-2 hours; more preferably, 10 times the total mass of the medicinal materials are added to pure water and extracted at 100°C for 1.5 hours.

[0017] Preferably, in step (3), the residue is heated and extracted again with 6-10 times the amount of water for 1-2 hours; more preferably, the residue is extracted again with 8 times the amount of pure water for 1 hour; the extracted liquid is combined with the extract from step (2) and concentrated to a relative density of 1.10-1.15.

[0018] Preferably, in step (4), the drying is spray drying, with an inlet air temperature of 180-190℃, an outlet air temperature of 85-95℃, a feed rate of 6-10mL / min, and an atomization pressure of 0.18-0.25MPa; more preferably, the inlet air temperature is 185℃, the outlet air temperature is 90℃, the feed rate is 8mL / min, and the atomization pressure is 0.22MPa.

[0019] Preferably, the obtained dry powder is passed through an 80-mesh sieve, packaged, and stored in the dark. Preferably, 1g of the herbal extract composition contains 5-10g of raw herbs; more preferably, 1g of the herbal extract composition contains about 10g of raw herbs.

[0020] Thirdly, the present invention also provides a veterinary drug composition or feed additive comprising an effective amount of the above-mentioned traditional Chinese medicine extract composition, and a pharmaceutically or feed-acceptable carrier.

[0021] Furthermore, its dosage form can be powder, premix, granules or oral liquid.

[0022] Fourthly, the present invention also provides the use of the above-mentioned traditional Chinese medicine extract composition in the preparation of pharmaceuticals or feed additives, wherein the pharmaceuticals or feed additives are used for one or more of the following purposes: (1) Inhibits Clostridium perfringens in chickens; (2) Prevention and treatment of necrotic enteritis in chickens; (3) Repairing necrotic intestinal mucosa; (4) Reduces intestinal inflammation; (5) Improve broiler production performance; (6) Ensure the egg production performance of broiler breeders; (7) Ensure the hatching performance of broiler breeders.

[0023] Preferably, the chicken includes broiler chickens, laying hens, or broiler breeders.

[0024] Fifthly, the present invention also provides a method for preventing and treating necrotizing enteritis in chickens and improving the production performance of broilers, wherein the above-mentioned Chinese herbal extract composition is added to chicken feed at a feed addition amount of 1.5-4.0 g / kg feed.

[0025] Specifically, when it is necessary to prevent necrotizing enteritis in chickens and improve broiler production performance, the dosage is 1.5-2.0 g / kg feed, for 5-7 consecutive days; when it is necessary to treat necrotizing enteritis in chickens and improve broiler production performance, the dosage is 3.0-4.0 g / kg feed, for 4-6 consecutive days. Suitable for all life cycles: safe for use in broilers, pullets, laying hens, and broiler breeder chickens during the egg-laying and incubation periods.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) Clear targeted antibacterial effect: The composition of the present invention has significant in vitro antibacterial activity against Clostridium perfringens type A. In Example 1, the diameter of the inhibition zone reached 22.6±0.3 mm, and the MIC was 6.25 mg / mL; in Example 2, the diameter of the inhibition zone was 20.4±0.4 mm, and the MIC was 12.5 mg / mL, both of which were superior to the single herbs and some comparative examples of combinations.

[0027] (2) Significant anti-infection and intestinal mucosal repair effects in vivo: Standardized challenge models confirmed that the composition of the present invention can increase daily weight gain, reduce feed conversion ratio, reduce fecal score and intestinal lesion score, reduce the number of Clostridium perfringens in the cecum, repair necrotic intestinal mucosa, and improve the intestinal barrier. Among them, Example 1 showed the best effect, and although Example 2 was not the optimal formulation, it could still achieve the technical effects of antibacterial, anti-inflammatory, intestinal mucosal repair and improved production performance.

[0028] (3) Significant clinical prevention and treatment effects: Large-scale breeding trials show that adding the composition of this invention at 3.0 g / kg feed can significantly reduce the incidence of necrotizing enteritis in chickens, mortality rate and feed conversion ratio, and improve fecal formation.

[0029] (4) Synergistic effect of compatibility: The present invention is not a simple superposition of each medicinal ingredient, but a synergistic effect produced by the combination of principal, assistant and adjuvant herbs. The basic technical effect can be achieved within the relatively wide range of proportions defined in claim 1, and the preferred proportion has the best effect.

[0030] (5) Safe and antibiotic-free, with wide applicability: The whole formula is made from the same source of medicine and food, with no antibiotics, no residues, and no drug resistance. It can be used for broilers, pullets, laying hens, and broiler breeders during the egg-laying and incubation period.

[0031] (6) The formulation and process are suitable for large-scale promotion: the extract has a small amount of additives and good palatability. It can be made into powder, premix, granules or oral liquid, which is suitable for large-scale industrial application. Detailed Implementation

[0032] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1: The herbal extract composition of the present invention This embodiment provides a traditional Chinese medicine extract composition and its preparation method, wherein the traditional Chinese medicine extract composition is made from the following raw materials in the indicated amounts: Based on a total raw medicinal material input of 100 g: 35 g of Sargentodoxa cuneata, 25 g of Euphorbia humifusa, 18 g of Dichroa febrifuga, 12 g of Crataegus pinnatifida, and 10 g of Potentilla chinensis.

[0035] The preparation method of the traditional Chinese medicine extract composition includes the following steps: (1) The medicinal materials are cleaned, impurities removed, air-dried, and pulverized through a 40-mesh sieve; (2) Using a multi-functional extraction tank, add 10 times the total mass of the medicinal materials in pure water, extract at 100℃ for 1.5 h, and collect the filtrate; (3) Add 8 times the amount of pure water to the residue and extract for 1 hour, then combine the two filtrates; (4) Centrifuge to remove impurities, and concentrate under reduced pressure to a relative density of 1.15; (5) Spray drying: inlet air 185℃, outlet air 90℃, feed rate 8 mL / min, atomization pressure 0.22 MPa, to obtain about 10g of dry powder; that is, 1g of dry powder is equivalent to about 10g of raw medicinal material; (6) The obtained dry powder is passed through an 80-mesh sieve, packaged and stored away from light.

[0036] Example 2: The herbal extract composition of the present invention This embodiment provides a traditional Chinese medicine extract composition and its preparation method, wherein the traditional Chinese medicine extract composition is made from the extracts of the following raw materials in the indicated amounts: Based on a total input of 100g of raw medicinal materials: 30 parts of *Sargentodoxa cuneata*, 23 parts of *Euphorbia humifusa*, 20 parts of *Tetracentron sinense*, 15 parts of *Crataegus pinnatifida*, and 12 parts of *Potentilla chinensis*.

[0037] The preparation method of the Chinese herbal extract composition is the same as in Example 1, yielding approximately 11g of dry powder, i.e., 1g of dry powder is equivalent to 9g of raw herb.

[0038] Comparative Example 1: Extract of single-herb sample from *Sargentodoxa cuneata* This comparative example provides the preparation of a single-herb sample extract of *Sargentodoxa cuneata*: 100g of *Sargentodoxa cuneata* was used to prepare the extract as a test verification sample. The preparation method was the same as in Example 1, yielding approximately 12g of dry powder.

[0039] Comparative Example 2: Preparation of extracts from single-herb samples of Euphorbia humifusa This comparative example provides the preparation of extracts from a single sample of Euphorbia humifusa: 100g of Euphorbia humifusa was used to prepare the extract as a test verification sample. The preparation method was the same as in Example 1, yielding approximately 10g of dry powder.

[0040] Comparative Example 3: Preparation of Extracts from Single-Flavor Tea Samples This comparative example provides the preparation of extracts from a single sample of Dwarf Tea: 100g of Dwarf Tea was used to prepare the extract as a test sample, and the preparation method was the same as in Example 1, yielding approximately 10g of dry powder.

[0041] Comparative Example 4: Preparation of Extracts from Wild Hawthorn Single Sample This comparative example provides the preparation of extract from a single wild hawthorn sample: 100g of wild hawthorn was used to prepare the extract as a test verification sample. The preparation method was the same as in Example 1, yielding approximately 13g of dry powder.

[0042] Comparative Example 5: Preparation of extract from Potentilla chinensis var. chinensis sample This comparative example provides the preparation of extracts from a sample of Potentilla chinensis: 100g of Potentilla chinensis was used to prepare the extract as a test sample. The preparation method was the same as in Example 1, yielding approximately 11g of dry powder.

[0043] Comparative Example 6: Extracts from a combination of Sargentodoxa cuneata and Euphorbia humifusa This comparative example provides the preparation of an extract from a combination of Sargentodoxa cuneata and Euphorbia humifusa: the extract was prepared from a total of 100g, of which 58.3g was Sargentodoxa cuneata and 41.7g was Euphorbia humifusa, as an experimental verification sample. The preparation method was the same as in Example 1, and about 10g of dry powder was obtained.

[0044] Comparative Example 7: Extracts from a combination of samples of *Tea gloriosa*, wild hawthorn, and *Potentilla chinensis* This comparative example provides the preparation of extracts from a combination of dwarf tea, wild hawthorn, and Potentilla chinensis: the extract was prepared from a total of 100g, including 47.4g of dwarf tea, 31.6g of wild hawthorn, and 21g of Potentilla chinensis, as an experimental verification sample. The preparation method was the same as in Example 1, yielding approximately 12g of dry powder.

[0045] Example 3: In vitro antibacterial test (Clostridium perfringens) Test strain: Clostridium perfringens type A standard strain (CVCC2030). Single colonies of Clostridium perfringens, after resuscitation and secondary activation, were picked and inoculated into vesicle culture medium for anaerobic incubation for 18 h. Counting was performed using TSC agar plates. The culture was then incubated upside down at 36±1℃ for 24 h. The bacterial concentration was corrected using sterile vesicle culture medium to achieve a viable count of 1×10⁻⁶. 6 CFU / mL was used to prepare a bacterial suspension.

[0046] The preparation process for the meat culture medium is as follows: Each liter of purified water contains 30.0 g peptone, 5.0 g beef extract, 5.0 g yeast extract, 3.0 g glucose, 2.0 g soluble starch, 5.0 g sodium dihydrogen phosphate, 0.1 g reduced iron powder, and 15.0 g agar. Adjust the pH to 7.4±0.1 (25℃). During preparation, heat and stir until completely dissolved. Beef granules can be added as needed. Autoclave at 121℃ for 20 min. After sterilization, when the temperature drops to 50-55℃, pour the plates and allow them to cool and solidify. This medium is used for plate isolation of anaerobic bacteria such as Clostridium perfringens and for Oxford cup antimicrobial tests. Incubate in an anaerobic incubator at 37℃. Agar-free meat culture medium is simply meat culture medium without agar.

[0047] (1) Oxford cup inhibition zone test Sample concentration: The herbal extract powders from Examples 1-2 and Comparative Examples 1-7 were prepared into solutions with a concentration of 200 mg / mL using sterile PBS, calculated based on the crude drug concentration (e.g., for Example 1, 0.2 g of herbal extract powder was weighed and added to 10 mL of sterile PBS to obtain a stock solution of 200 mg crude drug / mL). Negative control: Sterile PBS (phosphate buffer). Positive control: Lincomycin solution with a concentration of 2 μg / mL.

[0048] In the experiment, 15 mL of Clostridium perfringens culture medium was poured into the bottom of a sterile Petri dish and allowed to solidify horizontally. For the upper layer of bacterial agar: the Clostridium perfringens culture medium was melted and cooled to 45°C, and a suspension of Clostridium perfringens was added to bring the final bacterial concentration of the upper layer to 1×10⁻⁶. 6 Take 8 mL of CFU / mL solution, mix well, spread it on the bottom layer, and let it stand to solidify.

[0049] Sterile forceps were used to place sterile Oxford cups, pressing gently to ensure no gaps between the bottom of the cup and the agar. Four cups were placed per plate, with a spacing of ≥2.5 cm between cups to prevent overlapping inhibition zones. 200 μL of the test drug solution was added to each cup, avoiding air bubbles. Pre-diffusion was performed at 4℃ for 1.5–2 h; this step significantly improves the stability of the results due to the slow diffusion of the herbal extract. The plates were then transferred to an anaerobic incubator and anaerobically incubated at 37℃ for 20–24 h. The total diameter (mm) of the inhibition zone was measured using calipers using the cross-hatching method, with three replicates for each concentration, and the mean ± standard deviation was recorded. The test results are shown in Table 1.

[0050] Table 1. Results of inhibition zone determination of traditional Chinese medicine extracts against Clostridium perfringens type A

[0051] As shown in Table 1, no inhibition zone was observed in the negative control sterile PBS; the diameter of the inhibition zone of the positive control lincomycin was 24.5±1.3 mm; the diameter of the inhibition zone of the extract of Example 1 (200 mg / mL, based on crude drug) was 22.6±0.3 mm, and the diameter of the inhibition zone of the extract of Example 2 (200 mg / mL, based on crude drug) was 20.4±0.4 mm, which were significantly larger than those of the individual herbal comparison ratios and some combination comparison ratios. Moreover, the antibacterial effect of Example 1 was better than that of Example 2, indicating that the herbal extract composition of the present invention has a clear and stable direct in vitro antibacterial activity against Clostridium perfringens type A CVCC2030.

[0052] (2) MIC (Minimum Inhibitory Concentration) The extracts obtained from Examples 1-2 and Comparative Examples 1-7 were prepared into a stock solution of 200 mg / mL (based on crude drug) and sterilized by filtration through a 0.45 μm aqueous filter membrane. The MIC of the compound aqueous extract against Clostridium perfringens type A was determined using the 96-well micro-broth dilution method. 100 μL of sterile vesicular broth (without agar) was added to each well in columns 2-11. 100 μL of the stock solution of the extracts from the examples and comparative examples was added to column 2. After mixing the stock solution and broth, serial dilutions were performed sequentially from columns 3 to 11. After mixing in column 11, 100 μL of the diluted solution was discarded, and 100 μL of Clostridium perfringens suspension was added to each well to achieve a final bacterial concentration of 1 × 10⁻⁶. 6 The final drug concentrations in wells 2-11 were 100, 50, 25, 12.5, 6.25, 3.125, 1.562, 0.781, 0.391, and 0.195 mg / mL (based on crude drug concentration), respectively. A growth blank control was set up, and lincomycin hydrochloride was used as a positive control. The cultures were incubated in an anaerobic incubator at 37℃ for 24 h, and the minimum inhibitory concentration (MIC) was observed and determined.

[0053] The preparation process of sterile broth for vesicular meat culture medium is as follows: each liter of purified water contains 30.0 g peptone, 5.0 g beef extract, 5.0 g yeast extract, 3.0 g glucose, 2.0 g soluble starch, 5.0 g sodium dihydrogen phosphate, and 0.1 g reduced iron powder. The pH is adjusted to 7.4±0.1 (25℃). When using, test tubes are pre-filled with about 2 cm of vesicular beef granules. The broth is then dispensed until the liquid level is about 1 cm above the meat granules. 0.3-0.4 cm of liquid paraffin is added to the top to isolate oxygen. The tubes are then sterilized by autoclaving at 121℃ for 20 min. Before use, the tubes are boiled in water for 10 min to remove oxygen. After cooling, the broth can be used for antibacterial tests against anaerobic bacteria such as Clostridium perfringens.

[0054] The experimental results are shown in Table 2.

[0055] Table 2 MIC measurements for each example

[0056] As shown in Table 2, the MICs of Example 1 and Example 2 against Clostridium perfringens type A were 6.25 mg / mL and 12.5 mg / mL (based on crude drug), respectively, which were lower than those of the comparative proportions, indicating that the five-ingredient compound of the present invention has a synergistic effect in enhancing antibacterial activity.

[0057] Example 3, an in vitro antibacterial test, confirmed that the composition of Example 1 of this invention has significant antibacterial activity against Clostridium perfringens type A, the main pathogen of necrotizing enteritis in chickens, and its effect is superior to that of single herbs and some combinations, providing pharmacological basis for subsequent animal challenge experiments. The in vitro antibacterial results are only for pharmacological evidence; the actual prevention and treatment effect shall be determined by in vivo animal experiments.

[0058] Example 4: Standardized Chicken Necrotic Enteritis Challenge Model Experiment Experimental animals: 360 healthy 1-day-old yellow-feathered broiler chickens were divided into 12 groups: blank control group, model challenge group, positive control group, experimental group 1 (corresponding to Example 1), experimental group 2 (corresponding to Example 2), and control groups 1-7 (corresponding to ratios 1-7). Each group had 3 replicates, with 10 chickens per replicate. The positive group and all experimental groups were given drugs daily from day 1 until the end of the experiment, while the blank group and model group were fed normally.

[0059] Preparation of a standardized chicken necrotizing enteritis challenge model: (1) At 8 and 11 days of age, except for the control group, each chick in normally healthy reared chicks was pretreated by oral administration of 1 mL of coccidia suspension to disrupt the intestinal barrier. The coccidia suspension was obtained from commercially available live chicken coccidiosis vaccine (containing Eimeria tenella and Eimeria giantiformis, purchased from a biological products company). The vaccine was diluted 30 times the recommended dose (usually 1 dose / chick) in the vaccine instructions, i.e., 30 doses of vaccine were diluted with sterile physiological saline to 30 mL, thoroughly mixed, and the sporangia were counted under a microscope using a hemocytometer, adjusting the concentration to 3 × 10⁻⁶. 3 One sporangium / mL. Administer orally using a 1 mL disposable syringe.

[0060] (2) 15-17 days old: Except for the control group, all other groups were given oral gavage of Clostridium perfringens type A bacterial solution daily, with a challenge concentration of 2×10⁻⁶. 6 CFU / mL, 1 mL / day per bird; The preparation method of Clostridium perfringens type A bacterial suspension is as follows: The lyophilized powder of Clostridium perfringens type A standard strain CVCC2030 (purchased from the China Veterinary Microbial Culture Collection Center) was revived with vesicular broth and streaked onto TSC agar plates, then anaerobically cultured at 37°C for 24 h. Typical black single colonies were picked and inoculated into vesicular broth, then anaerobically cultured at 37°C for 18 h. The activated bacterial suspension was serially diluted 10-fold with sterile pre-reduced PBS, and the appropriate dilutions were plated onto TSC agar plates for counting. Based on the counting results, the bacterial suspension was diluted to 2 × 10⁻⁶. 6 CFU / mL. Prepare the bacterial suspension immediately before use. Store in an anaerobic chamber before gavage and use within 2 hours.

[0061] (3) Feed high-protein induced feed throughout the entire course to promote the proliferation of Clostridium perfringens in the intestine. The high-protein diet formula (by weight percentage) is as follows: corn 40.0%, soybean meal (43% CP) 20.0%, fish meal 25.0%, soybean oil 4.0%, limestone powder 1.2%, dicalcium phosphate 1.7%, salt 0.3%, lysine hydrochloride (78%) 0.2%, methionine (98%) 0.1%, 1.5% broiler chick premix, wheat bran 6.0%, total 100%. Nutritional level (calculated value): crude protein CP = 24.5%, metabolizable energy = 12.70 MJ / kg.

[0062] The study included: a blank control group consisting of 1-day-old healthy yellow-feathered broilers, fed a normal basal diet without viral challenge or medication; a model challenge group consisting of standardized chickens challenged with necrotizing enteritis, fed a normal basal diet without medication; a positive control group consisting of experimental chickens prepared according to the standardized chicken necrotizing enteritis challenge model, simultaneously fed 3 mg / kg lincomycin in their basal diet; and experimental and control groups consisting of experimental chickens prepared according to the standardized chicken necrotizing enteritis challenge model, simultaneously fed the corresponding extracts at 3 g / kg in their basal diet. The experimental period was 20 days.

[0063] The administration method is as follows: add the drug to the feed according to the above-mentioned dosage, and the chickens can eat it freely for a total of 20 days.

[0064] The treatment results for each group are shown in Table 4. The test indicators included daily feed intake, average daily weight gain, feed conversion ratio, fecal score, intestinal lesion score, and Clostridium perfringens count in the cecum. The fecal score criteria and intestinal (jejunum) score criteria are shown in Tables 5 and 6, respectively.

[0065] The necropsy and intestinal lesion scoring method was as follows: On day 20 of the experiment, three chickens were randomly selected from each replicate of each group. After euthanasia via exjugation through the jugular vein, the abdominal cavity was immediately opened, and the duodenum, jejunum, and ileum were completely separated. The intestinal tract was longitudinally cut open, and the contents were gently rinsed with sterile saline to remove them. Under sufficient light, two observers unaware of the group assignments independently scored the jejunal mucosa for gross lesions. The average of the two scores was taken as the final score for that chicken. In case of discrepancies, a third person would verify and confirm the score.

[0066] Cecal contents collection and Clostridium perfringens counting: On day 20 of the experiment, during necropsy, the cecum was aseptically cut open, and approximately 0.5g of cecal contents were weighed and placed in a pre-weighed sterile centrifuge tube. After precise weighing, 4.5mL of sterile pre-reduced PBS (phosphate buffered saline) was added, and the mixture was vortexed thoroughly to prepare 10... -1 Diluent. In an anaerobic chamber, add 100 μL of the above diluent to 900 μL of sterile PBS, and then perform a 10-fold serial dilution to a final volume of 10. -7 Take 10 respectively -5 10 -6 10 -7 Three dilutions, 100 μL each, were spread onto TSC agar plates (pancreatinine-sulfite-cyclic serine agar) containing 5% sheep blood, with three replicates for each dilution. The plates were incubated at 37°C for 24 h in an anaerobic incubator before counting. Clostridium perfringens colonies appeared black with a turbid ring around them. Typical colonies were picked and confirmed as Gram-positive, large bacilli by Gram staining and microscopic examination. Plates with colony counts between 30 and 300 were selected for counting, and the results are expressed as log10 CFU / g cecal contents.

[0067] All chicken houses maintained the same conditions, including lighting, temperature, humidity, ventilation, immunization program, disinfection procedures, and basal ration nutritional levels. Chickens had free access to feed and water, and the houses were disinfected regularly to maintain suitable temperature and humidity. The basal ration formula is shown in Table 3. Table 3. Basal Diet Formula

[0068] Note: 1% compound premix contains per kilogram of premix: VA 1,200,000 IU, VD3 300,000 IU, VE 8,000 IU, VK3 300 mg, VB1 300 mg, VB2 600 mg, VB6 400 mg, VB... 12 1.0 mg, niacin 3500 mg, calcium pantothenate 1800 mg, folic acid 80 mg, biotin 40 mg; Fe 10000 mg, Cu 1200 mg, Mn 12000 mg, Zn 10000 mg, I 150 mg, Se 40 mg, carrier zeolite powder. 2) Nutritional levels are calculated values.

[0069] Table 4. Treatment status of each group

[0070] Table 5. Stool Scoring Criteria

[0071] Table 6. Intestinal (jejunum) scoring criteria

[0072] Results of the virus challenge model experiment: (1) Production performance As shown in Table 7, the daily feed intake and daily weight gain of the model group were 21.10±0.53 g and 6.38±0.53 g, respectively, which were significantly lower than those of the control group; the feed conversion ratio was 3.32±0.33, which was significantly higher than that of the control group (P<0.05). The difference between the model group and the control group was significant (P<0.05), indicating that the model was successfully established and Clostridium perfringens could cause a decrease in feed intake in the model group.

[0073] Compared with the model group, the daily feed intake of the Example 1 group increased to 23.71±0.43 g, the daily weight gain increased to 9.94±0.43 g, and the feed conversion ratio decreased to 2.39±0.22; the daily feed intake of the Example 2 group was 23.87±0.35 g, the daily weight gain was 9.41±0.26 g, and the feed conversion ratio was 2.54±0.21. It is evident that both Example 1 and Example 2 could alleviate the decrease in feed intake caused by challenge to varying degrees. The daily weight gain of both groups was significantly higher than that of the model group (P<0.05), and there was no significant difference compared with the blank group and the positive group (P>0.05); the feed conversion ratio of both groups was significantly lower than that of the model group (P<0.05). Further comparison showed that the daily weight gain of Example 2 was at a relatively high level compared with the blank group, positive group and Example 1 group, with no significant difference (P>0.05); although its material weight ratio was slightly higher than that of the blank group, positive group and Example 1 group, it was still significantly lower than that of the model group, and lower than that of the single-herb or part-combination comparative examples such as Comparative Example 1 and Comparative Example 2.

[0074] Table 7 Production Performance

[0075] Note: Different superscript letters in each column indicate significant differences (P<0.05), while the same superscript letter or no superscript letter indicates no significant differences (P>0.05).

[0076] (2) Stool, intestinal score and cecal flora status Table 8 shows that the fecal score, jejunal lesion score, and Clostridium perfringens count in the model group were 3.67±0.58, 5.33±0.58, and 8.33±0.52 log10 CFU / g, respectively, all significantly higher than those in the control group (P<0.05), further proving the successful establishment of the model. In Example 1, the above indicators were 1.33±0.58, 1.67±0.58, and 3.67±0.48 log10 CFU / g, respectively, all significantly lower than those in the model group and each comparative group (P<0.05). However, the fecal score and intestinal score were not significantly different from the control group and positive group (P>0.05), while the Clostridium perfringens count in the cecum was significantly lower than that in the positive group (P<0.05) but not significantly different from the control group (P>0.05). This indicates that Example 1 can significantly alleviate diarrhea, repair necrotic intestinal mucosa, and inhibit the proliferation of Clostridium perfringens in the cecum. In Example 2, the fecal score was 1.66±0.58, the intestinal score was 2.00±1.00, and the cecal Clostridium perfringens count was 4.00±1.73 log10 CFU / g, all significantly lower than the model group. The intestinal score was the same as the blank group, positive group, and Example 1 group (c), indicating no significant difference compared to these groups (P>0.05). The cecal Clostridium perfringens count was also at the same low order of magnitude as the blank group, positive group, and Example 1 group. These results indicate that Example 2 also inhibits Clostridium perfringens, reduces intestinal lesion scores, alleviates diarrhea, and repairs the intestinal mucosa, achieving the basic technical effects of the present invention.

[0077] Table 8. Fecal samples, intestinal score, and cecal flora.

[0078] Note: Different superscript letters in each column indicate significant differences (P<0.05), while the same superscript letter or no superscript letter indicates no significant differences (P>0.05).

[0079] Conclusion: The composition of this invention can significantly antagonize Clostridium perfringens infection and repair intestinal damage caused by necrotizing enterocolitis. Example 1 shows the best effect. As can be seen from the comparative examples, although single herbs or partial combinations have certain antibacterial or intestinal protective effects, they are inferior to the five-herb compound in Example 1 in terms of inhibition zone, MIC, daily weight gain, feed conversion ratio, fecal score, intestinal score, and the number of Clostridium perfringens in the cecum. This indicates that the composition of this invention is not a simple superposition of individual herbs, but rather achieves a synergistic effect through the combination of principal, assistant, and adjuvant herbs.

[0080] Example 5: Large-scale clinical aquaculture application trial Under actual breeding conditions in large-scale commercial broiler farms, the clinical prevention and treatment effects of the traditional Chinese medicine extract composition described in Example 1 of this invention on necrotizing enteritis in chickens were verified, and its influence on the incidence, mortality, feed conversion ratio and fecal formation of necrotizing enteritis was evaluated, providing clinical data support for the large-scale application of this composition in antibiotic-free farming.

[0081] 5.1 Test Materials The experiment was conducted at a large-scale commercial broiler farm. 10,000 one-day-old white-feathered broilers of the same batch and breed, in good health, with no significant differences in initial weight, were selected. The experimental period was 30 days, from 1 to 30 days of age.

[0082] 10,000 chickens were randomly divided into two groups of 5,000 each, with five replicates per group and 1,000 chickens per replicate: 1) Conventional control group: fed and managed according to the conventional feeding and management plan of the chicken farm; 2) Experimental group of the Chinese medicine of this invention: on the basis of conventional feeding and management, the extract of Example 1 was added at 3.0 g / kg feed for 4 to 6 consecutive days.

[0083] The two groups of chicken houses had the same conditions, lighting, temperature, humidity, ventilation, immunization program, disinfection system, and basic nutrient level of their diet.

[0084] 5.2 Feeding Management and Drug Administration Methods The basic diet is formulated according to the broiler feeding standards (as shown in Table 9) and fed in stages: Table 9 Broiler Chicken Feeding Standards

[0085] The diet consists mainly of corn, soybean meal, wheat bran, fish meal, limestone powder, dicalcium phosphate, and premixed feed, with free access to food and water.

[0086] Vaccinations for Newcastle disease, infectious bronchitis, and infectious bursal disease were administered according to the standard immunization program for chicken farms. Regular disinfection was carried out, and the temperature and humidity inside the coop were maintained at a suitable level. The control group received preventative and necessary treatment according to the standard chicken farm protocol. This protocol involved no growth-promoting antibiotics throughout the entire treatment period; amoxicillin (13 mg / kg body weight) was administered via drinking water for 5 consecutive days only when clinical symptoms of necrotic enteritis appeared, as prescribed by a veterinarian. During the experiment, the control group did not receive prophylactic antibiotics. The only difference between the control group and the experimental group using the herbal medicine of this invention was the presence or absence of the extract of this invention; all other management measures were identical.

[0087] In the experimental group of this invention, starting at 15 days of age, the extract from Example 1 was added to the feed at a rate of 3.0 g / kg. This extract was mixed thoroughly with the basal diet using a step-by-step premixing method and fed continuously for 6 days. For flocks exhibiting suspected symptoms of necrotic enteritis, the extract could be administered at 3.0 g / kg feed for 4 consecutive days, and extended to 6 days if necessary. The conventional control group did not receive the extract from this invention.

[0088] 5.3 Observation Indicators and Judgment Criteria 5.3.1 Incidence of Necrotizing Enteritis Observe the flock's mental state, feed intake, and fecal characteristics daily. The presence of typical symptoms such as black, tarry, watery feces, bloody stools, lethargy, and decreased feed intake, along with lesions such as congestion, hemorrhage, coagulative necrosis, and ulceration of the jejunal mucosa upon necropsy of dead chickens, or a positive anaerobic culture of Clostridium perfringens from the cecal contents, indicates necrotizing enteritis.

[0089] Morbidity rate = number of sick chickens / total number of chickens in the group × 100%.

[0090] 5.3.2 Dead Rate Record the number of chickens that died and were culled during the trial.

[0091] Mortality rate = (Number of deaths + Number of culled chickens) / Total number of chickens in the group × 100%.

[0092] 5.3.3 Feed conversion ratio Record the total material consumption and total weight gain throughout the period.

[0093] Feed conversion ratio = Total feed consumption / Total weight gain over the period.

[0094] 5.3.4 Stool Formation Observe the stool appearance at a fixed time every day and score it from 0 to 4: 0 points: normal formed stool; 1 point: formed but slightly wet; 2 points: soft and flat, mild diarrhea; 3 points: watery diarrhea, severe diarrhea; 4 points: projectile watery stool, with mucus, blood streaks or necrotic debris visible.

[0095] Fecal formation rate = Number of observed chickens with a score ≤ 1 / Total number of observed chickens × 100%.

[0096] 5.4 Statistical Analysis SPSS statistical software was used for analysis. Quantitative data were expressed as mean ± standard deviation, and t-tests were used for comparisons between groups; chi-square tests were used for categorical data. A p-value < 0.05 was considered statistically significant.

[0097] 5.5 Clinical breeding results The test results are shown in Table 10.

[0098] Table 10 Clinical breeding results

[0099] Note: Each column marked with * indicates a significant difference compared to the control group, P < 0.05.

[0100] Table 10 shows that the incidence of necrotic enteritis in chickens decreased from 11.7% in the conventional control group to 2.5% in the herbal medicine experimental group of this invention, a decrease of 9.2 percentage points, or a relative decrease of approximately 78.63%; the mortality rate decreased from 3.66% in the conventional control group to 1.12% in the herbal medicine experimental group of this invention, a decrease of 2.54 percentage points, or a relative decrease of approximately 69.40%; the feed conversion ratio decreased from 1.88 in the conventional control group to 1.54 in the herbal medicine experimental group of this invention, a decrease of 0.34, or a relative decrease of approximately 18.09%; the fecal score decreased from 3.4 to 1.2, a decrease of 2.2 points, or a relative decrease of approximately 64.71%; and the fecal formation rate increased from 26.2% to 78.2%, an increase of 52 percentage points, or a relative increase of approximately 198.47%. The above indicators showed significant differences between the herbal medicine experimental group and the conventional control group (P<0.05).

[0101] This embodiment demonstrates, under actual conditions in a large-scale commercial broiler farm, that adding the herbal extract composition from Example 1 of this invention at a dose of 3.0 g / kg of feed for 4–6 consecutive days can significantly reduce the incidence of necrotic enteritis, mortality rate, and feed conversion ratio in chickens, and significantly improve fecal formation, with effects superior to conventional farm practices. This composition is convenient to use, suitable for large-scale mixed feeding, does not affect normal feeding management, and is suitable for widespread application in antibiotic-free broiler farming.

[0102] Application Examples: The extract powder obtained in Example 1 can be added to feed at a rate of 1.5-2.0 g / kg for the prevention of necrotic enteritis in broilers, for 5-7 consecutive days; or added at a rate of 3.0-4.0 g / kg for the treatment of necrotic enteritis in chickens, for 4-6 consecutive days. The obtained powder can also be mixed with a feed carrier to prepare a premix, granules, or oral liquid for use in broilers, pullets, laying hens, and broiler breeder chickens during the egg-laying and incubation period, and its application to other livestock and poultry can be explored.

[0103] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementations of the invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the invention, and these all fall within the protection scope of the invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. A composition of traditional Chinese medicine extract, characterized in that, It is made from the following ingredients in parts by weight: 30-40 parts of Sargentodoxa cuneata, 20-25 parts of Euphorbia humifusa, 15-20 parts of Dichroa febrifuga, 10-15 parts of Crataegus pinnatifida, and 5-12 parts of Potentilla chinensis.

2. The traditional Chinese medicine extract composition according to claim 1, characterized in that, It is made from the following ingredients in parts by weight: 34-36 parts of Sargentodoxa cuneata, 24-25 parts of Euphorbia humifusa, 17-19 parts of Dichroa febrifuga, 11-13 parts of Crataegus pinnatifida, and 9-11 parts of Potentilla chinensis.

3. A method for preparing the traditional Chinese medicine extract composition according to claim 1 or 2, characterized in that, Includes the following steps: (1) The medicinal materials, including *Sargentodoxa cuneata*, *Euphorbia humifusa*, *Tetracentron sinense*, *Crataegus pinnatifida*, and *Potentilla chinensis*, were processed into medicinal powders respectively; (2) Weigh each medicinal powder according to the ratio, add water and heat to extract, separate the residue and collect the extract; (3) Add water to the dregs, heat and extract again, collect the extract, and combine it with the extract from step (2) and concentrate it; (4) The concentrated liquid obtained in step (3) is dried to obtain the Chinese herbal extract composition.

4. The preparation method according to claim 3, characterized in that, In step (2), add 8-12 times the total mass of the medicinal materials to water and heat for extraction; the extraction time is 1-2 hours; and / or, In step (3), the dregs are added to 6-10 times their volume of water and heated for re-extraction; the re-extraction time is 1-2 hours; and / or, the re-extract is combined with the extract from step (2) and concentrated to a relative density of 1.10-1.15; and / or, In step (4), the drying is spray drying, with an inlet air temperature of 180-190℃, an outlet air temperature of 85-95℃, a feed rate of 6-10mL / min, and an atomization pressure of 0.18-0.25 MPa.

5. The preparation method according to claim 3 or 4, characterized in that, 1g of the herbal extract composition contains 5-10g of raw herbs.

6. A veterinary drug composition or feed additive, characterized in that, The composition comprises an effective amount of the herbal extract composition of claim 1 or 2, and a pharmaceutically or feed-grade acceptable carrier.

7. The veterinary drug composition or feed additive according to claim 6, characterized in that, Its dosage forms are powder, premix, granules or oral liquid.

8. The use of the traditional Chinese medicine extract composition according to claim 1 or 2 in the preparation of pharmaceuticals or feed additives, characterized in that, The drug or feed additive is used for one or more of the following purposes: (1) Inhibits Clostridium perfringens in chickens; (2) Prevention and treatment of necrotic enteritis in chickens; (3) Repairing necrotic intestinal mucosa; (4) Reduces intestinal inflammation; (5) Improve broiler production performance; (6) Ensure the egg production performance of broiler breeders; (7) Ensure the hatching performance of broiler breeders.

9. The application according to claim 8, characterized in that, The chickens mentioned include broilers, laying hens, or broiler breeders.

10. A method for improving broiler production performance, characterized in that, The herbal extract composition according to claim 1 or 2 is added to chicken feed at a rate of 1.5-4.0 g / kg feed.

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