Chinese herbal medicine feed additive for preventing and treating diarrhea of livestock and poultry and preparation method of Chinese herbal medicine feed additive

Abstract

The invention provides a Chinese herbal medicine feed additive for preventing and treating diarrhea of livestock and poultry and a preparation method thereof, and relates to the field of feed additives, the Chinese herbal medicine feed additive is composed of dandelion, mahonia bealei, rhizoma alismatis, gordon euryale seed, gallnut, Chinese yam, perilla leaf, pinellia ternate, red garden burnet, galangal, indigo naturalis, shiny cinquefoil root and bidens pilosa. The Chinese herbal medicine feed additive disclosed by the invention has broad-spectrum bacteriostatic activity on common pathogenic bacteria of livestock and poultry, can be used for remarkably reducing the occurrence rate of diarrhea of weaned piglets, reducing the levels of TNF-alpha and IL-6 in serum and increasing the content of IgA and IgG, and has an anti-inflammatory immune regulation effect; meanwhile, the feed additive can improve the content of TP and ALB in serum of weaned piglets, reduce the content of BUN, reduce the activity of ALT and AST, regulate protein metabolism disorder and protect liver and kidney functions; besides, the additive can optimize the intestinal microecological balance of weaned piglets, effectively prevent and treat diarrhea, and provide a safe and efficient prevention and control strategy for the diarrhea problem of livestock and poultry in large-scale breeding.

Description

Technical Field

[0001] The present invention relates to the field of feed additives, and particularly to a Chinese herbal medicine feed additive for preventing and treating diarrhea in livestock and poultry and a preparation method thereof. Background Art

[0002] In the field of livestock and poultry breeding, diarrhea is one of the common and highly prevalent diseases. Especially in the young livestock and poultry population, it shows significant morbidity characteristics and has become a key epidemic disease problem restricting the healthy development of the industry. The clinical manifestations of diseased individuals are typical symptoms such as abnormal excrement morphology, digestive function disorders (decreased appetite, vomiting, etc.) and nerve inhibition (depression). Severe cases can cause progressive dehydration due to water and electrolyte disorders, ultimately leading to death. The etiology of this disease is complex and is often caused by single or co-infection of viral and bacterial pathogens, with extremely strong group transmissibility and prone to form an epidemic outbreak situation in a short time. Therefore, building a three-level prevention and control system with prevention as the core, early monitoring as the basis, and timely intervention as the guarantee is of great practical significance for reducing breeding economic losses. The current prevention and control system mainly relies on chemically synthesized antibacterial agents and antiviral preparations. Although such drugs have advantages such as significant dose effects and rapid clinical responses, their applications have double limitations: on the one hand, drug metabolic residues may threaten human health through the food chain; on the other hand, the emergence of pathogen drug resistance accelerates the attenuation of drug efficacy. This contradictory phenomenon severely restricts the healthy development of the livestock and poultry breeding industry. In addition, with the increasing demand of consumers for food safety and eco-friendly products, traditional chemical drugs face severe tests in terms of residue control and environmental compatibility.

[0003] Chinese herbal medicine feed additives have significant advantages in the field of livestock and poultry disease prevention and control. Their characteristics such as natural safety, no drug residues, and no risk of drug resistance make them a choice for healthy breeding. These additives are prepared based on scientific formulae and modern processes, using natural medicinal plants as raw materials, and have the effects of preventing and treating diarrhea in livestock and poultry, enhancing the body's immune function, and comprehensively improving the health level of livestock and poultry. However, there are still several bottlenecks in the existing technology: firstly, the formula design mostly uses bitter and cold herbs as the main materials, and long-term feeding is likely to cause the syndrome of spleen and stomach deficiency and cold, affecting the feeding enthusiasm of livestock and poultry; secondly, the formula mode dominated by traditional experience lacks scientific design based on modern pathological mechanisms such as intestinal flora regulation and mucosal immune regulation. Therefore, there is an urgent need to develop a Chinese herbal medicine feed additive for preventing and treating diarrhea in livestock and poultry to meet the needs of the livestock and poultry breeding industry for safe, efficient, green, and drug-free feed additives. Summary of the Invention

[0004] (I) Technical Problems to be Solved

[0005] In view of the deficiencies of the prior art, the present invention provides a Chinese herbal medicine feed additive for preventing and treating diarrhea in livestock and poultry and a preparation method thereof.

[0006] (2) Technical Solution

[0007] To achieve the above object, the present invention is realized through the following technical solutions:

[0008] The present invention provides a Chinese herbal medicine feed additive for preventing and treating diarrhea in livestock and poultry. The Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 15 - 35 parts of dandelion, 18 - 38 parts of mahonia bealei, 10 - 30 parts of alisma orientale, 8 - 28 parts of euryale ferox, 12 - 32 parts of Chinese gall, 15 - 35 parts of Chinese yam, 5 - 25 parts of perilla leaf, 1 - 10 parts of pinellia ternata, 5 - 22 parts of sanguisorba officinalis, 8 - 28 parts of galangal, 15 - 35 parts of indigo naturalis, 5 - 25 parts of cynanchum paniculatum, 10 - 30 parts of bidens pilosa.

[0009] Further, the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 20 - 30 parts of dandelion, 23 - 33 parts of mahonia bealei, 15 - 25 parts of alisma orientale, 13 - 23 parts of euryale ferox, 17 - 27 parts of Chinese gall, 20 - 30 parts of Chinese yam, 10 - 20 parts of perilla leaf, 1 - 5 parts of pinellia ternata, 7 - 17 parts of sanguisorba officinalis, 13 - 23 parts of galangal, 20 - 30 parts of indigo naturalis, 10 - 20 parts of cynanchum paniculatum, 15 - 25 parts of bidens pilosa.

[0010] Further, the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 25 parts of dandelion, 28 parts of mahonia bealei, 20 parts of alisma orientale, 18 parts of euryale ferox, 22 parts of Chinese gall, 25 parts of Chinese yam, 15 parts of perilla leaf, 3 parts of pinellia ternata, 12 parts of sanguisorba officinalis, 18 parts of galangal, 25 parts of indigo naturalis, 15 parts of cynanchum paniculatum, 20 parts of bidens pilosa.

[0011] The present invention provides a preparation method of a Chinese herbal medicine feed additive for preventing and treating diarrhea in livestock and poultry, including the following steps:

[0012] (1) Weigh each raw material according to the above parts by weight, wash, dry, pulverize and pass through an 80 - mesh sieve, and then mix in a three - dimensional mixer at a rotation speed of 20 rpm for 20 - 30 min;

[0013] (2) Add absolute ethanol to the above powder at a material - liquid ratio of 1:5, adopt the ultrasonic extraction method, set the ultrasonic frequency at 40 kHz and the power at 300 W, ultrasonically extract for 30 - 50 min at 50 °C, extract twice, and combine the extraction filtrates;

[0014] (3) Transfer the filtrate to a rotary evaporator, concentrate under reduced pressure and vacuum to an extract with a relative density of 1.1 - 1.3, and then carry out spray drying and granulation to obtain the Chinese herbal medicine feed additive of the present invention.

[0015] The present invention provides the application of the Chinese herbal medicine feed additive in preventing and / or treating diarrhea in livestock and poultry.

[0016] Furthermore, the addition amount of the Chinese herbal medicine feed additive is 0.5%-2.0%.

[0017] The effects of each Chinese herbal medicine raw material in the Chinese herbal medicine feed additive of the present invention are as follows:

[0018] Dandelion: Bitter and sweet in taste, cold in nature, belonging to the liver and stomach meridians; having the effects of clearing heat and detoxifying, dissipating swelling and nodules, and promoting diuresis and relieving stranguria; used for furuncles and carbuncles, breast abscess, scrofula, red eyes, sore throat, lung abscess, intestinal abscess, damp-heat jaundice, and painful stranguria.

[0019] Mahonia bealei: Bitter in taste, cool in nature, belonging to the lung meridian; having the effects of clearing heat and tonifying deficiency, relieving cough and resolving phlegm, and drying dampness and detoxifying; used for hemoptysis due to pulmonary tuberculosis, hectic fever due to yin deficiency, dizziness and tinnitus, soreness and weakness of the waist and legs, restlessness, and red eyes.

[0020] Alisma orientale: Sweet and light in taste, cold in nature, belonging to the kidney and bladder meridians; having the effects of promoting diuresis and percolating dampness, clearing heat and discharging fire, and resolving turbidity and reducing lipid; used for oliguria, edema, diarrhea with scanty urine, phlegm retention and dizziness, painful stranguria, and hyperlipidemia.

[0021] Euryale ferox: Sweet and astringent in taste, neutral in nature, belonging to the spleen and kidney meridians; having the effects of tonifying the kidney and astringing essence, invigorating the spleen and stopping diarrhea, and removing dampness and arresting leukorrhea; used for spermatorrhea, enuresis and frequent urination, chronic diarrhea due to spleen deficiency, leukorrhea, and turbid leucorrhea.

[0022] Chinese gall: Sour and astringent in taste, cold in nature, belonging to the lung, large intestine, and kidney meridians; having the effects of astringing the lung and reducing fire, astringing the intestine and stopping diarrhea, arresting sweating, stopping bleeding, and astringing dampness and promoting granulation; used for chronic cough due to lung deficiency, cough with lung heat and phlegm, chronic diarrhea and dysentery, spontaneous sweating and night sweating, diabetes, hematochezia, hemorrhoids bleeding, traumatic bleeding, carbuncles and sores, and skin dampness and erosion.

[0023] Chinese yam: Sweet in taste, neutral in nature, belonging to the spleen, lung, and kidney meridians; having the effects of invigorating the spleen and nourishing the stomach, promoting the production of body fluid and benefiting the lung, and tonifying the kidney and astringing essence; used for poor appetite due to spleen deficiency, chronic diarrhea, cough and asthma due to lung deficiency, spermatorrhea due to kidney deficiency, leukorrhea, frequent urination, and consumptive thirst with deficiency heat. Stir-fried Chinese yam with bran invigorates the spleen and strengthens the stomach. It is used for poor appetite due to spleen deficiency, loose stools, and excessive leukorrhea.

[0024] Perilla leaf: Pungent in taste, warm in nature, belonging to the lung and spleen meridians; having the effects of inducing sweating to dispel cold, promoting qi circulation and regulating the stomach; used for wind-cold cold, cough and nausea, vomiting during pregnancy, and fish and crab poisoning.

[0025] Pinellia ternata: Pungent in taste, warm in nature; toxic. Belonging to the spleen, stomach, and lung meridians; having the effects of drying dampness and resolving phlegm, lowering adverse qi and stopping vomiting, and dissipating masses and resolving stagnation; used for damp phlegm and cold phlegm, profuse phlegm and cough, dizziness due to phlegm retention, wind-phlegm dizziness, headache due to phlegm syncope, vomiting and nausea, stuffiness and fullness in the chest and epigastrium, globus hystericus; externally used for carbuncles and phlegm nodes.

[0026] Sanguisorba officinalis: Bitter and astringent in taste, slightly cold in nature, belonging to the liver, spleen, stomach, and bladder meridians; having the effects of clearing heat and promoting diuresis, cooling blood and stopping bleeding; used for diarrhea, dysentery, indigestion, epigastric and abdominal pain, epistaxis, hematochezia, menorrhagia, postpartum hemorrhage, and traumatic injury.

[0027] Alpinia officinarum Hance: pungent in taste, hot in nature, attributing to the spleen and stomach meridians; having the effects of warming the stomach to arrest vomiting and dispelling cold to relieve pain; used for cold pain in the epigastrium and abdomen, stomach cold vomiting, belching and acid regurgitation.

[0028] Indigo Naturalis: salty in taste, cold in nature, attributing to the liver meridian; having the effects of clearing heat and detoxifying, cooling blood and eliminating macules, purging fire and calming fright; used for epidemic toxin-induced maculae, hematemesis due to blood heat, hemoptysis with chest pain, oral ulcers, mumps, sore throat, infantile convulsion.

[0029] Siphonostegia chinensis Benth: bitter and astringent in taste, cold in nature, attributing to the spleen and large intestine meridians; having the effects of clearing heat and detoxifying, astringing the intestine to stop diarrhea, cooling blood and stopping bleeding; used for dysentery with bloody and purulent stools, enteritis diarrhea, hematochezia due to intestinal wind, hemoptysis due to pulmonary tuberculosis, hematemesis, metrorrhagia and leukorrhagia, traumatic bleeding, furuncles, scalds and burns, rheumatic pain.

[0030] Bidens pilosa L.: bitter in taste, neutral in nature, attributing to the spleen, large intestine and lung meridians; having the effects of clearing heat and detoxifying, stopping diarrhea; used for enteritis diarrhea, appendicitis, sore throat due to cold, hepatitis, snake and insect bites.

[0031] (III) Beneficial effects

[0032] The present invention provides a traditional Chinese medicine feed additive for preventing and treating diarrhea in livestock and poultry and a preparation method thereof. The traditional Chinese medicine feed additive is composed of Taraxacum mongolicum Hand.-Mazz., Mahonia bealei (Fort.) Carr., Alisma orientale (Sam.) Juz., Euryale ferox Salisb., Chinese gall, Dioscorea opposita Thunb., Perilla leaf, Pinellia ternata (Thunb.) Breit., Sanguisorba officinalis L., Alpinia officinarum Hance, Indigo Naturalis, Siphonostegia chinensis Benth, Bidens pilosa L. In vitro experimental data show that the traditional Chinese medicine feed additive of the present invention exhibits significant broad-spectrum antibacterial activity in vitro, has a significant inhibitory effect on common pathogenic bacteria in livestock and poultry (including Escherichia coli, Staphylococcus aureus and Salmonella), and can also significantly reduce the secretion of key pro-inflammatory factors TNF-α, IL-1β and IL-6 by inhibiting the LPS-induced inflammatory response of macrophage RAW264.7, demonstrating its anti-inflammatory effect. In vivo experimental data show that the traditional Chinese medicine feed additive of the present invention can significantly reduce the incidence of diarrhea in weaned piglets, down-regulate the levels of serum pro-inflammatory factors TNF-α and IL-6, and simultaneously increase the contents of immunoglobulin IgA and IgG, showing a significant anti-inflammatory and immunomodulatory effect. At the same time, the traditional Chinese medicine feed additive of the present invention can increase the contents of TP and ALB in the serum of weaned piglets, reduce the content of BUN, and reduce the activities of ALT and AST, effectively regulating protein metabolism disorders and protecting liver and kidney functions. In addition, the additive can optimize the intestinal microecological balance of weaned piglets, increase the abundance of beneficial bacteria and reduce the number of pathogenic bacteria, preventing and treating diarrhea in weaned piglets from the aspect of pathogen control, and providing a safe and efficient prevention and control strategy for the problem of diarrhea in livestock and poultry in large-scale farming. Description of the drawings

[0033] Figure 1 Inhibitory effects of each test group on Escherichia coli. Note: Compared with the blank control group, *P < 0.05, ** P < 0.01.

[0034] Figure 2 These are the inhibitory effects of each experimental group on Staphylococcus aureus. Note: Compared with the blank control group, * P < 0.05, ** P < 0.01.

[0035] Figure 3 These are the inhibitory effects of each experimental group on Salmonella. Note: Compared with the blank control group, * P < 0.05, ** P < 0.01.

[0036] Figure 4 These are the comparisons of TNF-α secretion levels in each experimental group. Note: Compared with the blank control group, # P < 0.05, ## P < 0.01; compared with the model control group, * P < 0.05, ** P < 0.01.

[0037] Figure 5 These are the comparisons of IL-1β secretion levels in each experimental group. Note: Compared with the blank control group, # P < 0.05, ## P < 0.01; compared with the model control group, * P < 0.05, ** P < 0.01.

[0038] Figure 6 These are the comparisons of IL-6 secretion levels in each experimental group. Note: Compared with the blank control group, # P < 0.05, ## P < 0.01; compared with the model control group, * P < 0.05, ** P < 0.01.

[0039] Figure 7 These are the comparisons of the numbers of Escherichia coli in the intestines of weaned piglets in each experimental group. Note: Compared with the blank control group, # P < 0.05, ## P < 0.01; compared with the model control group, * P < 0.05, ** P < 0.01.

[0040] Figure 8 These are the comparisons of the numbers of Lactobacillus in the intestines of weaned piglets in each experimental group. Note: Compared with the blank control group, # P < 0.05, ## P < 0.01; compared with the model control group, *P < 0.05, ** P < 0.01. Detailed implementation manners

[0041] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0042] Embodiment 1

[0043] A traditional Chinese medicine feed additive for preventing and treating diarrhea in livestock and poultry, the traditional Chinese medicine feed additive is composed of the following raw materials in parts by weight: 25 parts of dandelion, 28 parts of mahonia bealei, 20 parts of alisma orientale, 18 parts of euryale ferox, 22 parts of Chinese gall, 25 parts of Chinese yam, 15 parts of perilla leaf, 3 parts of pinellia ternata, 12 parts of sanguisorba officinalis, 18 parts of galangal, 25 parts of indigo naturalis, 15 parts of syzygium aromaticum, 20 parts of bidens pilosa.

[0044] The preparation method of the traditional Chinese medicine feed additive includes the following steps:

[0045] (1) Weigh each raw material according to the above parts by weight, wash, dry, pulverize and pass through an 80-mesh sieve, and then mix in a three-dimensional mixer at a rotation speed of 20 rpm for 30 min;

[0046] (2) Add absolute ethanol to the above powder according to a solid-liquid ratio of 1:5, and perform ultrasonic extraction. Set the ultrasonic frequency to 40 kHz and the power to 300 W, and perform two extractions at 50 °C for 30 min each time. Combine the two extraction filtrates;

[0047] (3) Transfer the filtrate to a rotary evaporator, and concentrate it under reduced pressure and vacuum to an extract with a relative density of 1.1 - 1.3, and then perform spray drying and granulation to obtain the traditional Chinese medicine feed additive of the present invention.

[0048] Embodiment 2

[0049] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine feed additive is composed of the following raw materials in parts by weight: 32 parts of dandelion, 35 parts of mahonia bealei, 27 parts of alisma orientale, 25 parts of euryale ferox, 29 parts of Chinese gall, 32 parts of Chinese yam, 22 parts of perilla leaf, 7 parts of pinellia ternata, 20 parts of sanguisorba officinalis, 25 parts of galangal, 32 parts of indigo naturalis, 23 parts of syzygium aromaticum, 28 parts of bidens pilosa.

[0050] Embodiment 3

[0051] The difference between this embodiment and Embodiment 1 is that the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 15 parts of dandelion, 18 parts of mahonia bealei, 10 parts of alisma orientale, 8 parts of euryale ferox, 12 parts of Chinese gall, 15 parts of Chinese yam, 5 parts of perilla leaf, 1 part of pinellia ternata, 5 parts of sanguisorba officinalis, 8 parts of galangal, 15 parts of indigo naturalis, 5 parts of syzygium aromaticum, and 10 parts of bidens pilosa.

[0052] Embodiment 4

[0053] The difference between this embodiment and Embodiment 1 is that the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 20 parts of dandelion, 23 parts of mahonia bealei, 15 parts of alisma orientale, 12 parts of euryale ferox, 17 parts of Chinese gall, 20 parts of Chinese yam, 9 parts of perilla leaf, 3 parts of pinellia ternata, 9 parts of sanguisorba officinalis, 12 parts of galangal, 20 parts of indigo naturalis, 9 parts of syzygium aromaticum, and 15 parts of bidens pilosa.

[0054] Embodiment 5

[0055] The difference between this embodiment and Embodiment 1 is that the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 35 parts of dandelion, 38 parts of mahonia bealei, 30 parts of alisma orientale, 28 parts of euryale ferox, 32 parts of Chinese gall, 35 parts of Chinese yam, 25 parts of perilla leaf, 10 parts of pinellia ternata, 22 parts of sanguisorba officinalis, 28 parts of galangal, 35 parts of indigo naturalis, 25 parts of syzygium aromaticum, and 30 parts of bidens pilosa.

[0056] Embodiment 6

[0057] The difference between this embodiment and Embodiment 1 is that the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 22 parts of dandelion, 25 parts of mahonia bealei, 17 parts of alisma orientale, 15 parts of euryale ferox, 19 parts of Chinese gall, 22 parts of Chinese yam, 11 parts of perilla leaf, 4 parts of pinellia ternata, 12 parts of sanguisorba officinalis, 15 parts of galangal, 22 parts of indigo naturalis, 11 parts of syzygium aromaticum, and 17 parts of bidens pilosa.

[0058] Embodiment 7

[0059] The difference between this embodiment and Embodiment 1 is that the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 17 parts of dandelion, 20 parts of mahonia bealei, 12 parts of alisma orientale, 10 parts of euryale ferox, 14 parts of Chinese gall, 17 parts of Chinese yam, 7 parts of perilla leaf, 2 parts of pinellia ternata, 7 parts of sanguisorba officinalis, 10 parts of galangal, 17 parts of indigo naturalis, 7 parts of syzygium aromaticum, and 12 parts of bidens pilosa.

[0060] Embodiment 8

[0061] The difference between this embodiment and Embodiment 1 is that the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 30 parts of dandelion, 34 parts of mahonia bealei, 25 parts of alisma orientale, 23 parts of euryale ferox, 27 parts of Chinese gall, 30 parts of Chinese yam, 20 parts of perilla leaf, 6 parts of pinellia ternata, 17 parts of sanguisorba officinalis, 23 parts of galangal, 30 parts of indigo naturalis, 20 parts of dichotomanthes paniculata, and 25 parts of bidens pilosa.

[0062] Example 9

[0063] The difference between this embodiment and Embodiment 1 is that the Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 28 parts of dandelion, 32 parts of mahonia bealei, 22 parts of alisma orientale, 20 parts of euryale ferox, 25 parts of Chinese gall, 27 parts of Chinese yam, 18 parts of perilla leaf, 5 parts of pinellia ternata, 15 parts of sanguisorba officinalis, 20 parts of galangal, 28 parts of indigo naturalis, 18 parts of dichotomanthes paniculata, and 23 parts of bidens pilosa.

[0064] Test Example 1

[0065] 1 Materials and Methods

[0066] 1.1 Test Drugs

[0067] Test Group 1: 25 parts of dandelion, 28 parts of mahonia bealei, 20 parts of alisma orientale, 18 parts of euryale ferox, 22 parts of Chinese gall, 25 parts of Chinese yam, 15 parts of perilla leaf, 3 parts of pinellia ternata, 12 parts of sanguisorba officinalis, 18 parts of galangal, 25 parts of indigo naturalis, 15 parts of dichotomanthes paniculata, and 20 parts of bidens pilosa.

[0068] Test Group 2: 20 parts of alisma orientale, 18 parts of euryale ferox, 22 parts of Chinese gall, 25 parts of Chinese yam, 15 parts of perilla leaf, 3 parts of pinellia ternata, 12 parts of sanguisorba officinalis, 18 parts of galangal, 15 parts of dichotomanthes paniculata, and 20 parts of bidens pilosa.

[0069] Test Group 3: 25 parts of dandelion, 28 parts of mahonia bealei, 25 parts of Chinese yam, 15 parts of perilla leaf, 3 parts of pinellia ternata, 12 parts of sanguisorba officinalis, 18 parts of galangal, 25 parts of indigo naturalis, and 15 parts of dichotomanthes paniculata.

[0070] Test Group 4: 25 parts of dandelion, 28 parts of mahonia bealei, 20 parts of alisma orientale, 18 parts of euryale ferox, 22 parts of Chinese gall, 25 parts of Chinese yam, 18 parts of galangal, 25 parts of indigo naturalis, 15 parts of dichotomanthes paniculata, and 20 parts of bidens pilosa.

[0071] Test Group 5: 25 parts of dandelion, 28 parts of mahonia bealei, 20 parts of alisma orientale, 18 parts of euryale ferox, 22 parts of Chinese gall, 15 parts of perilla leaf, 3 parts of pinellia ternata, 12 parts of sanguisorba officinalis, 25 parts of indigo naturalis, and 20 parts of bidens pilosa.

[0072] Weigh each raw material according to the above parts by weight respectively, wash, dry, pulverize and pass through an 80-mesh sieve, then mix evenly. Add absolute ethanol to the medicinal powder at a material-liquid ratio of 1:5 and perform ultrasonic extraction for 30 min, filter, and concentrate the filtrate under reduced pressure and vacuum to a relative density of 1.10 - 1.15 to obtain the Chinese herbal medicine stock solutions of Test Groups 1 - 5.

[0073] 1.2 Test methods

[0074] 1.2.1 Antibacterial activity detection

[0075] Set up a blank control group and Test Groups 1 - 5. Respectively dip Escherichia coli, Staphylococcus aureus and Salmonella in a clean bench and streak them on LB nutrient agar medium. After culturing at 37°C, pick single colonies and add them to test tubes containing 5 mL of LB broth medium, and culture overnight in a constant temperature shaking incubator at 37°C to obtain bacterial suspensions. In a clean bench, dilute the bacterial suspensions with LB broth medium. Take 100 μL of the bacterial suspension with a concentration of 1×10 8 CFU / mL, evenly coat it on the surface of LB nutrient agar medium with a spreading rod. Gently place the sterilized Oxford cups on the surface of the plate, and add 100 μL of the Chinese herbal medicine stock solutions of Test Groups 1 - 5 with a concentration of 50 mg / mL to the Oxford cups respectively. Add distilled water to the blank control group, place it in a constant temperature incubator at 37°C for upright culture, observe after 24 h, and measure the diameter of the inhibition zone. Set 3 parallels for each group.

[0076] 1.2.2 Anti-inflammatory activity detection

[0077] Inoculate RAW264.7 cells in DMEM high-glucose medium containing 10% FBS and 1% double antibody (hereinafter referred to as "complete medium"), and culture them under the conditions of 37°C, 5% CO2 saturated humidity. Take RAW264.7 cells in the logarithmic growth phase and inoculate them in a 96-well plate at 1×10 5 cells / mL, 100 μL per well. After the cells adhere to the wall, divide them into a blank control group, a model control group and Test Groups 1 - 5, and set 3 replicates for each group. Add 200 μL of the complete medium containing Chinese herbal medicine stock solutions 1 - 5 (the final concentration of the Chinese herbal medicine stock solution is 200 μg / mL) to each well respectively. The blank control group is given an equal volume of complete medium. After 1 h, add LPS with a final concentration of 1 μg / mL to each well for stimulation. After culturing for 24 h, collect the culture medium, continuously centrifuge the cell culture medium of each group at 4°C and 3000 rpm for 15 min, collect the supernatant, and detect the secretion of TNF-α, IL-6 and IL-1β in the supernatant by ELISA method.

[0078] 1.3 Statistical methods

[0079] Data analysis was performed using GraphPad Prism 8.0 software. Measurement data were expressed as mean ± standard deviation and one-way ANOVA was used for comparison between groups. LSD-t test was used for pairwise comparison when the variances were homogeneous, and Tambane's T2 test was used when the variances were heterogeneous. A difference was considered statistically significant when P < 0.05.

[0080] 2 Results

[0081] 2.1 Inhibitory activity of each test drug against common pathogenic bacteria

[0082] As Figure 1-3 shown, compared with the blank control group, the test groups 1-5 showed varying degrees of antibacterial effects against Escherichia coli( Figure 1 ), Staphylococcus aureus( Figure 2 ), and Salmonella( Figure 3 ). Among them, the test group 1 showed a highly significant advantage (P < 0.01) in the inhibitory effects against the three pathogenic bacteria, and the increase in the diameter of the antibacterial circle was the most prominent. The experimental data indicate that the Chinese herbal medicine feed additive of the present invention has a significant broad-spectrum inhibitory effect on common pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Salmonella) in livestock and poultry.

[0083] 2.2 Effects of each test drug on the expression levels of inflammatory factors

[0084] As Figures 4-6 shown, compared with the blank control group, the levels of pro-inflammatory factors TNF-α( Figure 4 ), IL-1β( Figure 5 ), and IL-6( Figure 6 ) in the supernatant of RAW264.7 cells in the model control group were significantly increased (P < 0.01), indicating that LPS successfully induced a macrophage inflammation model; compared with the model control group, the test groups 1-5 all showed a regulatory effect on the cellular inflammatory response: among them, the test group 1 had the most significant inhibitory effect on the above three inflammatory factors (P < 0.01), and their secretion levels decreased by 31.69%, 31.76%, and 37.94% respectively compared with the model group; while the anti-inflammatory effects of the test groups 2-5 were weakened due to the lack of some Chinese herbal medicine raw materials. This result indicates that the Chinese herbal medicine feed additive of the present invention can effectively inhibit the LPS-induced macrophage inflammatory response through the synergistic action of multiple components, and its complete formula has a significant regulatory effect on the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6).

[0085] 3 Conclusions

[0086] The above test results indicate that the Chinese herbal medicine feed additive of the present invention exhibits significant broad-spectrum antibacterial activity in vitro and has a significant inhibitory effect on common pathogenic bacteria in livestock and poultry (including Escherichia coli, Staphylococcus aureus, and Salmonella). In addition, the Chinese herbal medicine feed additive of the present invention also shows its anti-inflammatory effect by inhibiting the inflammatory response of macrophages RAW264.7 induced by LPS and significantly reducing the secretion of pro-inflammatory factors TNF-α, IL-1β, and IL-6. Based on the synergistic effect of the above-mentioned dual antibacterial and anti-inflammatory effects, the Chinese herbal medicine feed additive of the present invention has the development potential as a feed additive for preventing and treating diarrhea in livestock and poultry.

[0087] Test Example 2

[0088] 1 Materials and Methods

[0089] 1.1 Test Drugs

[0090] The Chinese herbal medicine feed additive of the present invention was prepared by the method described in Example 1 above.

[0091] Colistin sulfate soluble powder was produced by Henan Shengshi Federal Biomedical Technology Co., Ltd.

[0092] 1.2 Test Methods

[0093] 75 healthy weaned piglets without a medical history and not immunized were randomly divided into 5 groups, namely the blank control group, the model control group, the colistin sulfate group, the low-dose group of Chinese herbal medicine feed additive, and the high-dose group, with 3 replicates in each group and 5 piglets in each replicate. The low-dose and high-dose groups of Chinese herbal medicine feed additive were fed with weaned piglet feed containing 0.5% and 1.0% of the Chinese herbal medicine feed additive of the present invention (the feed composition and nutrient components are shown in Table 1), the colistin sulfate group was fed with weaned piglet feed containing 0.02% colistin sulfate soluble powder, and the blank control group and the model control group were fed with weaned piglet feed, which was freely ingested by the piglets for 7 consecutive days. On the 8th day, after fasting for 6 hours, each group of piglets was intragastrically administered with broth culture solution containing 10 9 live Escherichia coli O139 bacteria. The blank control group was intragastrically administered with an equal amount of sterile PBS, and feeding continued for 15 days after administration. During this period, the diarrhea conditions, mental states, appetites and other clinical manifestations of each group of piglets were observed and recorded.

[0094] Table 1 Composition and Nutrient Components of Basic Feed

[0095] Raw material composition Content (%) Nutritional components Content Corn 45.07 Digestible energy (MJ / kg) 14.45 Extruded corn 15.00 Crude protein (%) 18.10 Soybean meal 14.00 Calcium (%) 0.68 Extruded soybean 10.00 Total phosphorus (%) 0.55 Glucose 2.50 Digestible lysine (%) 1.19 Fish meal 4.00 Digestible methionine (%) 0.33 Whey powder 5.00 Digestible threonine (%) 0.70 Soybean oil 0.80 Digestible tryptophan (%) 0.20 Calcium hydrogen phosphate 0.70 Limestone powder 0.50 Table salt 0.20 Zinc oxide 0.25 Compound amino acids 0.58 Choline chloride 0.10 Acidifier 0.30 Premix 1.00

[0096] Note: ① The premix provides per kilogram of diet: vitamin A 12000 IU, vitamin D2 2000 IU, vitamin E 60 IU, vitamin K3 3 mg, vitamin B 1225 μg, riboflavin 8 mg, pantothenic acid 20 mg, niacin 30 mg, folic acid 0.7 mg, vitamin B1 2.0 mg, vitamin B6 5.0 mg, biotin 0.3 mg, manganese 40 mg, iron 100 mg, zinc 250 mg, copper 100 mg, iodine 0.4 mg, selenium 0.35 mg; ② Crude protein, calcium and total phosphorus are measured values, and the rest are calculated values.

[0097] 1.3 Detection indicators

[0098] 1.3.1 Diarrhea rate

[0099] Observe and record the diarrhea situation of piglets daily, and calculate the diarrhea rate of piglets in each experimental group according to the following formula.

[0100] Diarrhea rate = (number of diarrheal pigs × number of diarrheal days) / (number of experimental days × number of experimental pigs) × 100%

[0101] 1.3.2 Serum indicators

[0102] After the experiment, randomly select 2 piglets from each replicate, draw 5 mL of blood from the anterior vena cava, let it stand at room temperature for 30 min, then centrifuge at 4 °C and 3500 rpm for 15 min to separate the serum. After aliquoting, store it at -80 °C for later use.

[0103] Use enzyme-linked immunosorbent assay to detect the levels of inflammation-related factors TNF-α, IL-6 and the contents of immunoglobulin A (IgA) and immunoglobulin G (IgG) in the serum. Use an automatic blood biochemical analyzer to measure the contents of total protein (TP), albumin (ALB), urea nitrogen (BUN) and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum.

[0104] 1.3.3 Fecal flora

[0105] On the morning before feeding on the last day of the experiment, randomly select 2 piglets from each replicate, take about 5 g of fresh rectal feces, and use the plate colony counting method to measure the number of microorganisms in the feces. Lactobacilli are anaerobically cultured using MRS medium, and Escherichia coli are aerobically cultured using EMB medium. After culturing at 37 °C for 48 h, record the number of flora.

[0106] 1.4 Statistical methods

[0107] Use GraphPad Prism 8.0 software for data analysis. Measurement data are expressed as mean ± standard deviation and one-way ANOVA is used for comparison between groups. For pairwise comparison of homogeneous variances, LSD-t test is used, and for heterogeneous variances, Tambane's T2 test is used. Statistical significance is considered when P < 0.05.

[0108] 2 Results

[0109] 2.1 Effects of Each Drug on Diarrhea Rate of Weaned Piglets

[0110] As can be seen from Table 2, compared with the blank control group, the diarrhea rate of piglets in the model control group was significantly increased (P<0.01); compared with the model control group, the diarrhea rate of piglets in each drug administration group was significantly decreased (P<0.01). Among them, the decrease in the diarrhea rate of the high-dose group was the most significant (P<0.01), and there was no significant difference compared with the positive drug colistin sulfate group (P>0.05), indicating that the Chinese herbal medicine feed additive of the present invention can effectively prevent and treat diarrhea in weaned piglets.

[0111] Table 2 Comparison of Diarrhea Rates of Piglets in Each Test Group (%)

[0112]

[0113]

[0114] Note: Compared with the blank control group, # P<0.05, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0115] 2.2 Effects of Each Drug on Serum Indexes of Weaned Piglets

[0116] As can be seen from Table 3, compared with the blank control group, the levels of serum inflammatory factors TNF-α and IL-6 in weaned piglets in the model control group were significantly increased (P<0.01); compared with the model control group, the levels of serum inflammatory factors TNF-α and IL-6 in weaned piglets in each drug administration group were significantly decreased (P<0.05, P<0.01). Among them, the decrease in the levels of TNF-α and IL-6 in the high-dose group was the most significant, indicating that the Chinese herbal medicine feed additive of the present invention has a significant anti-inflammatory effect and can effectively prevent and treat the body's inflammatory response.

[0117] Table 3 Comparison of Serum Inflammatory-related Factor Levels in Piglets in Each Test Group (pg / mL)

[0118] Grouping TNF-α IL-6 Blank control group 364.05±12.32 142.94±7.43 Model control group <![CDATA[524.80±11.28 ## > <![CDATA[364.12±13.13 ## > Colistin sulfate group <![CDATA[426.25±9.53 ** > <![CDATA[191.75±13.88 ** > Low-dose group <![CDATA[490.92±15.07 * > <![CDATA[267.68±14.72 ** > High-dose group <![CDATA[393.78±11.31 ** > <![CDATA[174.33±10.99 ** >

[0119] Note: Compared with the blank control group, # P<0.05, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0120] As can be seen from Table 4, compared with the blank control group, the contents of IgA and IgG in the serum of weaned piglets in the model control group were significantly decreased (P<0.01); compared with the model control group, the contents of IgA and IgG in the serum of weaned piglets in each drug administration group increased to some extent. Among them, the increasing trends of the contents of IgA and IgG in the serum of the high-dose group were the most significant (P<0.01), indicating that the Chinese herbal medicine feed additive of the present invention has a significant immunomodulatory effect, can effectively improve the humoral immune function of weaned piglets, and enhance their resistance.

[0121] Comparison of serum immune indexes of piglets in each experimental group (ng / mL)

[0122]

[0123]

[0124] Note: Compared with the blank control group, # P<0.05, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0125] As can be seen from Table 5, compared with the blank control group, the contents of TP and ALB in the serum of weaned piglets in the model control group were significantly decreased, the content of BUN was significantly increased (P<0.01), and the activities of ALT and AST were significantly enhanced (P<0.01); compared with the model control group, the contents of TP and ALB in the serum of weaned piglets in each drug administration group increased to some extent, and the content of BUN decreased to some extent. Among them, the increasing trends of the contents of TP and ALB and the decreasing trend of the content of BUN in the serum of the high-dose group were the most significant (P<0.01); in addition, the activities of ALT and AST in the serum of weaned piglets in each drug administration group decreased to some extent, and the decreasing trends of the activities of ALT and AST in the serum of the high-dose group were particularly significant (P<0.01). The above data indicate that the Chinese herbal medicine feed additive of the present invention has a significant liver and kidney protection function and can effectively improve protein metabolism disorders.

[0126] Comparison of serum biochemical indexes of piglets in each experimental group

[0127]

[0128] Note: Compared with the blank control group, # P<0.05, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0129] 2.3 Effects of each drug on the intestinal flora of weaned piglets

[0130] It can be seen from Figure 7 and Figure 8 that compared with the blank control group, the number of Escherichia coli in the intestines of weaned piglets in the model control group increased significantly, while the number of lactic acid bacteria decreased significantly (P<0.01); compared with the model control group, the number of Escherichia coli in the intestines of weaned piglets in each drug administration group decreased, and the number of lactic acid bacteria increased. Among them, the reduction amplitude of Escherichia coli in the intestines of weaned piglets in the high-dose group was the most significant, and the increase amplitude of lactic acid bacteria content was also the most significant (P<0.05), indicating that the Chinese herbal medicine feed additive of the present invention can improve the intestinal flora of weaned piglets, effectively prevent and treat piglet diarrhea, and show a dose-dependent effect.

[0131] 3 Conclusions

[0132] The above experimental data show that the Chinese herbal medicine feed additive of the present invention can significantly reduce the incidence of diarrhea in weaned piglets, while down-regulating the levels of serum pro-inflammatory factors TNF-α and IL-6, and simultaneously increasing the contents of immunoglobulins IgA and IgG, showing a significant anti-inflammatory and immunomodulatory effect; moreover, the Chinese herbal medicine feed additive of the present invention can increase the contents of TP and ALB in the serum of weaned piglets, reduce the content of BUN, and reduce the activities of ALT and AST, effectively regulating protein metabolism disorders and protecting liver and kidney functions; in addition, the additive can significantly optimize the intestinal microecological balance of weaned piglets, increase the abundance of beneficial bacteria, and reduce the number of pathogenic bacteria, preventing and treating diarrhea in weaned piglets from the aspect of pathogen control. The above-mentioned synergistic action mechanism shows that the Chinese herbal medicine feed additive of the present invention provides a safe and efficient prevention and control strategy for the problem of diarrhea in livestock and poultry in large-scale farming.

[0133] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A Chinese herbal medicine feed additive for preventing and treating diarrhea in livestock and poultry, characterized in that: The Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 15-35 parts of dandelion, 18-38 parts of broad-leaved mahonia, 10-30 parts of oriental rhizome, 8-28 parts of euryale, 12-32 parts of gallnut, 15-35 parts of yam, 5-25 parts of perilla leaves, 1-10 parts of pinellia, 5-22 parts of red burial uveitis, 8-28 parts of galangal, 15-35 parts of indigo, 5-25 parts of guan zhong and 10-30 parts of Bidens pilosa.

2. The Chinese herbal medicine feed additive for preventing and treating diarrhea in livestock and poultry according to claim 1, characterized in that: The Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 20-30 parts of dandelion, 23-33 parts of broad-leaved mahonia, 15-25 parts of oriental rhizome, 13-23 parts of euryale, 17-27 parts of gallnut, 20-30 parts of yam, 10-20 parts of perilla leaves, 1-5 parts of pinellia, 7-17 parts of red burial uveitis, 13-23 parts of galangal, 20-30 parts of indigo, 10-20 parts of guan zhong and 15-25 parts of bidens pilosa.

3. The Chinese herbal medicine feed additive for preventing and treating diarrhea in livestock and poultry according to claim 1, characterized in that: The Chinese herbal medicine feed additive is composed of the following raw materials in parts by weight: 25 parts of dandelion, 28 parts of broad-leaved mahonia, 20 parts of oriental rhizome, 18 parts of euryale ferox, 22 parts of gallnut, 25 parts of yam, 15 parts of perilla leaf, 3 parts of pinellia, 12 parts of red burhead elm, 18 parts of galangal, 25 parts of indigo, 15 parts of guan zhong and 20 parts of Bidens pilosa.

4. The method for preparing the Chinese herbal medicine feed additive according to any one of claims 1 to 3, characterized in that: Prepare according to the following steps: (1) Weigh the raw materials according to the above weight proportions, wash, dry, and grind them through an 80-mesh sieve, and then mix them in a three-dimensional mixer at a speed of 20 rpm for 20-30 min; (2) adding anhydrous ethanol to the above powder at a solid-liquid ratio of 1:5, using ultrasonic extraction method, setting the ultrasonic frequency to 40 kHz and the power to 300 W, ultrasonicating at 50° C. for 30-50 min, extracting twice, and combining the extraction filtrates; (3) The filtrate is transferred to a rotary evaporator and concentrated under reduced pressure to an extract with a relative density of 1.1-1.3, followed by spray drying and granulation to obtain the Chinese herbal feed additive of the present invention.

5. Use of the Chinese herbal feed additive according to any one of claims 1 to 3 in preventing and / or treating diarrhea in livestock and poultry.

6. The use of the Chinese herbal medicine feed additive according to claim 5, characterized in that: The added amount of the Chinese herbal medicine feed additive is 0.5-2.0%.

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