Traditional Chinese medicine composition for preventing and treating chicken drug-resistant colibacillosis and application thereof

Abstract

The invention relates to a traditional Chinese medicine composition for preventing and treating chicken drug-resistant colibacillosis and application thereof, and belongs to the technical field of animal medicine. The traditional Chinese medicine composition is prepared from the following components in parts by mass: fructus gardeniae, radix scutellariae, fructus forsythiae, herba taraxaci, rhizoma coptidis, radix bupleuri, liquorice root, star anise, cortex moutan, radix trichosanthis, caulis akebiae, pericarpium granati and a transfer factor, wherein the transfer factor is a chicken spleen transfer factor prepared through ultrasonic wall breaking, repeated freezing and thawing treatment, centrifugation, dialysis, ultrafiltration and the like. Through reasonable compatibility of the traditional Chinese medicine components, a multi-component and multi-target synergistic effect is exerted, and the traditional Chinese medicine composition has a remarkable inhibition effect on standard escherichia coli and mcr-1 positive drug-resistant escherichia coli. In addition, the composition has extremely high cure rate and total effective rate when being used for treating chicken colibacillosis caused by mcr-1 positive drug-resistant escherichia coli. Therefore, the composition can be used for preparing medicines and bacteriostatic agents for treating chicken colibacillosis, and has good popularization and application values.

Description

Technical Field

[0001] The invention belongs to the technical field of animal medicine, and in particular relates to a traditional Chinese medicine composition for preventing and treating drug-resistant Escherichia coli disease in chickens and an application thereof. Background Art

[0002] Colibacillosis is a common bacterial disease caused by pathogenic Escherichia coli. It is widely found in poultry farms around the world, especially in intensive farming environments. Chickens, especially those raised in high-density environments with poor ventilation and sanitary conditions, are more susceptible to infection with E. coli. The incidence of colibacillosis is usually high and the spread is fast. Once it occurs, it often causes a series of problems such as slow growth, decreased egg production, and increased mortality in chickens, causing huge economic losses to the farming industry.

[0003] Traditionally, antibiotics have been the main means of preventing and treating E. coli disease in chickens. The rational use of antibiotics can effectively inhibit the growth and spread of E. coli. However, with the long-term and irregular use of antibiotics, especially in the breeding industry, the problem of antibiotic resistance has become increasingly serious, leading to the rapid development and widespread spread of E. coli resistance. The emergence of drug-resistant strains has significantly reduced the therapeutic effects of many traditional antibiotics, or even completely failed, bringing unprecedented challenges to clinical treatment. At present, there are relatively many studies on conventional E. coli infections, but there is still a lack of research on E. coli disease caused by drug-resistant E. coli, which has gradually restricted clinical treatment methods and limited treatment effects. Therefore, finding effective drugs and treatments for the treatment of drug-resistant E. coli infections has become a key issue that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide a traditional Chinese medicine composition for preventing and treating drug-resistant Escherichia coli disease in chickens and application of the composition in preparing a medicine for treating drug-resistant Escherichia coli disease in chickens.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a traditional Chinese medicine composition for preventing and treating Escherichia coli disease in chickens, wherein the traditional Chinese medicine composition is composed of the following components in parts by weight:

[0007] Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Paeonia Suffruticosa: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Transfer Factor: 0.25-0.5 parts.

[0008] Preferably, the transfer factor is chicken spleen transfer factor;

[0009] The preparation method of the chicken spleen transfer factor comprises the following steps:

[0010] (a) Wash the chicken spleen with triple-distilled water to remove impurities, and then rinse with sterile saline;

[0011] (b) mincing the cleaned chicken spleen, adding 2 volumes of physiological saline, and preparing a homogenate using ultrasonic cell wall breaking technology;

[0012] (c) subjecting the homogenate to repeated freeze-thaw operations, and subjecting the homogenate to high-speed homogenization and pulverization after each complete thawing;

[0013] (d) After low-temperature centrifugation, the supernatant is collected and filtered under pressure, and the obtained filtrate 1 is dialyzed to obtain filtrate 2;

[0014] (e) ultrafiltration of filtrate 2 using an ultrafiltration membrane with a molecular weight of 5000, and collecting the permeate;

[0015] (f) The permeate is filtered and sterilized, and then freeze-dried to obtain chicken spleen transfer factor.

[0016] 3. The Chinese medicine composition according to claim 2, characterized in that the chicken Escherichia coli disease is chicken Escherichia coli disease caused by standard Escherichia coli or mcr-1 positive Escherichia coli.

[0017] Preferably, the chicken Escherichia coli disease is chicken Escherichia coli disease caused by standard Escherichia coli or mcr-1 positive Escherichia coli.

[0018] Preferably, the Chinese medicine composition consists of the following components in parts by mass:

[0019] Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

[0020] In a second aspect, the present invention provides a method for preparing a drug for preventing and treating chicken Escherichia coli disease, the preparation method comprising the following steps:

[0021] (1) Grind 10-20 parts of Gardenia, 10-20 parts of Scutellaria, 15-30 parts of Forsythia, 20-35 parts of Taraxacum, 5-25 parts of Coptis, 5-15 parts of Bupleurum, 6-10 parts of Licorice, 40-45 parts of Star Anise, 5-10 parts of Paeonia Suffruticosa, 5-12 parts of Radix Trichosanthis, 8-15 parts of Akebia, and 15-20 parts of Pomegranate Peel, and sieve;

[0022] (2) Add 10 times the amount of distilled water, boil at 100°C for 1.5 hours;

[0023] (3) Filter and collect the residue and filtrate 1, add 10 times the amount of distilled water of the residue, boil at 90° C. for 1.5 h, filter, and collect filtrate 2;

[0024] (4) combining filtrate 1 and filtrate 2, and concentrating the mixture to a crude drug amount of 1 g / mL to obtain a drug concentrate;

[0025] (5) After filtering and sterilizing the concentrated solution, the pH is adjusted to 7, and 0.25-0.5 parts of chicken spleen transfer factor are added. After fully dissolving, the drug is obtained.

[0026] Preferably, the chicken spleen transfer factor is prepared by the above-mentioned preparation method.

[0027] In a third aspect, the present invention provides a use of a traditional Chinese medicine composition in the preparation of a medicament for treating chicken Escherichia coli disease, wherein the traditional Chinese medicine composition is composed of the following components in parts by mass:

[0028] Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Peony Bark: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts;

[0029] The chicken spleen transfer factor is prepared by the above-mentioned method for preparing chicken spleen transfer factor.

[0030] Preferably, the chicken colibacillosis is chicken colibacillosis caused by standard Escherichia coli or mcr-1 positive Escherichia coli;

[0031] The Chinese medicine composition is composed of the following components in parts by mass:

[0032] Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

[0033] In a fourth aspect, the present invention provides an application of a traditional Chinese medicine composition in the preparation of an antibacterial agent for inhibiting mcr-1 positive Escherichia coli, characterized in that, in parts by mass, the traditional Chinese medicine composition consists of the following components:

[0034] Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Peony Bark: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts;

[0035] The chicken spleen transfer factor is prepared by the above-mentioned preparation method.

[0036] Preferably, the Chinese medicine composition consists of the following components in parts by mass:

[0037] Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

[0038] The performance of the Chinese medicine components used in the present invention is explained as follows:

[0039] Gardenia:

[0040] [Source] It is the dried mature fruit of Gardenia jasminoides Ellis, a plant of the Rubiaceae family.

[0041] [Original plant] Gardenia jasminoides Ellis.

[0042] [Harvesting and processing] Harvest the fruits when they are ripe in autumn, remove impurities, and dry them in the sun or at low temperature for later use.

[0043]

Properties

[0044] [Effects] It can relieve fire and relieve restlessness, clear away heat and dampness, cool blood and detoxify.

[0045] Scutellaria baicalensis:

[0046] [Source] It is the dried root of Scutellaria baicalensis Georgi, a plant of the Lamiaceae family.

[0047] [Original plant] Scutellaria baicalensis Georgi.

[0048] [Harvesting and processing] Dig up the roots in autumn or spring, remove the mud and sand, slice them, and dry them in the sun or at low temperature.

[0049]

Properties

[0050] [Effects] Clears away heat and dampness, purges fire and detoxifies, stops bleeding, and stabilizes the fetus.

[0051] Forsythia:

[0052] [Source] It is the dried fruit of Forsythia suspensa of the Oleaceae family.

[0053]

Original plant

[0054] [Harvesting and processing] Harvest in autumn when the fruit is just beginning to ripen and still green in color. Remove impurities, steam, and dry in the sun, commonly known as "Qingqiao"; harvest the ripe fruit, dry in the sun, remove impurities, and commonly known as "Laoqiao".

[0055]

Medicinal properties

[0056]

Effects

[0057] Dandelion:

[0058] [Source] It is the dried whole herb of dandelion, alkali dandelion or various plants of the same genus in the Asteraceae family.

[0059] [Original plant] Dandelion Taraxacum mongolicum Hand.-Mazz. Alkali soil dandelion Taraxacumsinicum Kitag.

[0060] [Harvesting and processing] Spring to autumn when the flowers first bloom

[0061]

Medicinal properties

[0062] Coptis chinensis:

[0063] [Source] It is the dried rhizome of Coptis chinensis Franch., a plant of the Ranunculaceae family.

[0064]

Original plant

[0065] [Harvesting and processing] Dig up in autumn and winter, remove the soil and fibrous roots, and dry in the sun or at low temperature.

[0066]

Properties

[0067]

Effects

[0068] Bupleurum:

[0069] [Source] It is the dried root of Bupleurum chinense DC. or Bupleurumscorzonerifolium Willd. of the Umbelliferae family.

[0070]

Original plant

[0071] [Harvesting and processing] Dig up the roots in autumn, remove the mud and fibrous roots, and dry them for later use.

[0072] [Properties] Slightly cold in nature, bitter in taste.

[0073] [Effects] Disperses and reduces fever, relieves liver qi and depression, and raises Yang Qi.

[0074] star anise:

[0075] [Source] It is the dried fruit of Illicium verum Hook.f., a plant of the Magnoliaceae family.

[0076]

Original plant

[0077] [Harvesting and processing] Pick the fruits when they are ripe, and dry them in the sun or in an oven for later use.

[0078]

Properties

[0079] [Effects] Warms the middle and regulates Qi, dispels cold and relieves pain, detoxifies and kills insects.

[0080] Trichosanthes:

[0081] [Source] It is the dried root of Trichosanthes kirilowii Maxim., a plant of the Cucurbitaceae family.

[0082]

Original plant

[0083] [Harvesting and processing] Dig up the roots in autumn and winter, remove the skin, and dry them in the sun or at low temperature.

[0084] [Properties] It is slightly cold in nature, sweet and slightly bitter in taste.

[0085]

Effects

[0086] Akebia:

[0087] [Source] It is the dried stem of Akebia quinata (Thunb.) Decne., a plant of the Akebiaceae family.

[0088]

Original plant

[0089] [Harvesting and processing] Harvest the stems in spring and autumn, remove impurities, and dry them in the sun or in an oven for later use.

[0090]

Properties

[0091]

Effects

[0092] Pomegranate peel:

[0093] [Source] It is the dried peel of pomegranate (Punica granatum L.), a plant of the Punicaceae family.

[0094]

Original plant

[0095] [Harvesting and processing] Harvest the fruits when they are ripe in autumn, take the peel, dry it in the sun or at low temperature for later use.

[0096] [Properties] Warm in nature, sour and astringent in taste.

[0097]

Effects

[0098] Peony bark:

[0099] [Source] It is the dried root bark of Paeonia suffruticosa Andr., a plant of the Ranunculaceae family.

[0100]

Original plant

[0101] [Harvesting and processing] Dig up the roots in autumn, peel off the root bark, and dry them in the sun or at low temperature.

[0102] [Properties] Slightly cold in nature, bitter and spicy in taste.

[0103]

Effects

[0104] Licorice:

[0105] [Source] It is the dried root and rhizome of the leguminous plants licorice, licorice inflated fruit or licorice glabra. The medicinal materials are commonly known as "Inner Mongolia licorice", "Xinjiang licorice" and "European licorice" respectively.

[0106]

Original plant

[0107] [Harvesting and processing] Dig up in spring and autumn, remove the fibrous roots and stem base, cut into segments of appropriate length, and dry in the sun. Some people also peel off the skin, cut into long segments and dry them in the sun, which is commonly known as "powdered licorice"

[0108]

Medicinal properties

[0109] [Effects] Tonify the spleen and replenish qi, clear away heat and detoxify, eliminate phlegm and relieve cough, relieve urgency and relieve pain, and harmonize various medicines. It should be used raw to clear away heat, and roasted to tonify the middle [Effects] Clear away heat and detoxify, reduce swelling and resolve knots, and promote diuresis and relieve stranguria.

[0110] The beneficial effects of the present invention are:

[0111] 1. The Chinese medicine composition provided by the present invention has a significant inhibitory effect on both standard Escherichia coli and mcr-1 positive resistant Escherichia coli. The in vitro antibacterial experiment verified that the Chinese medicine composition exhibited extremely sensitive and highly sensitive antibacterial effects, especially the Chinese medicine composition C was significantly better than the comparative drug and the traditional antibiotic polymyxin B in terms of the diameter of the antibacterial circle, effectively solving the problem of the difficulty in treating drug-resistant strains.

[0112] 2. The results of animal experiments show that the Chinese medicine composition of the present invention has a very high cure rate and total effective rate in treating chicken E. coli disease caused by mcr-1 positive resistant E. coli, especially the cure rate of Chinese medicine composition C is as high as 93.3%, and the total effective rate reaches 100%. Compared with traditional treatment methods, it significantly reduces the mortality rate of chickens and improves the overall treatment effect.

[0113] 3. The present invention gives full play to the synergistic effect between the components through the rational compatibility of the Chinese medicine components, and enhances the antibacterial and immunomodulatory effects. The experimental results show that the lack or replacement of components will significantly reduce the efficacy, which verifies the uniqueness and irreplaceability of the formula of the Chinese medicine composition of the present invention in the treatment of drug-resistant Escherichia coli disease.

[0114] Moreover, the Chinese medicine composition of the present invention has the characteristics of multiple components and multiple targets, and can synergistically act through multiple mechanisms such as antibacterial, anti-inflammatory, and immunity enhancement to effectively inhibit the growth of drug-resistant Escherichia coli, avoiding the drug resistance problem caused by the single target action of traditional antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 The encapsulation of the heart and liver of the chickens in the model group 3 days after the virus attack;

[0116] Figure 2 The encapsulation of the heart and liver in experimental group 4 after 5 days of treatment;

[0117] Figure 3 This is the condition of heart and liver encapsulation in the control group and experimental group 3 after 5 days of treatment. DETAILED DESCRIPTION

[0118] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0119] Example 1

[0120] This embodiment provides a Chinese medicine combination drug a for preventing and treating drug-resistant Escherichia coli disease in chickens

[0121] In this medicine, the dosage of Gardenia is 20 parts, the dosage of Scutellaria baicalensis is 20 parts, the dosage of Forsythia suspensa is 30 parts, the dosage of Taraxacum officinale is 35 parts, the dosage of Coptis chinensis is 5 parts, the dosage of Bupleurum chinense is 5 parts, the dosage of Licorice root is 6 parts, the dosage of Star Anise is 45 parts, the dosage of Paeonia suffruticosa rubra is 5 parts, the dosage of Radix Trichosanthis is 6 parts, the dosage of Atractylodes macrocephala is 8 parts, the dosage of Pomegranate peel is 15 parts, and the dosage of transfer factor is 0.25 parts.

[0122] Wherein, the transfer factor is chicken spleen transfer factor, and the preparation method of chicken spleen transfer factor is as follows:

[0123] (a) Raw material pretreatment: The raw materials were thoroughly washed with triple-distilled water, and impurities such as fascia and fat tissue on the surface were carefully removed, and then rinsed repeatedly with sterile saline;

[0124] (2) Ultrasonic wall breaking and homogenization: Cut the cleaned raw materials into small pieces of 1-2 cm, add 2 volumes of pre-cooled saline (4°C), and grind them into a uniform homogenate using ultrasonic wall breaking technology;

[0125] (3) Repeated freeze-thaw homogenization: The homogenate was quickly frozen in a -20°C refrigerator, then thawed in a 37°C water bath, and the freeze-thaw operation was repeated 6 times. After each complete thawing, a tissue homogenizer was used to perform high-speed homogenization and pulverization to further destroy the cell structure and release the cell contents.

[0126] (4) Low-temperature high-speed centrifugation: The freeze-thawed homogenate was diluted with 3 times the volume of pre-cooled physiological saline (4°C), and then centrifuged at 20,000 r / min for 30 minutes at 4°C using a low-temperature centrifuge. The supernatant was collected and pressure filtered using a filter membrane with a pore size of 0.45 μm to remove larger particles and cell debris.

[0127] (5) Dialysis: The filtrate after filtration was dialyzed using polyethersulfone hollow fiber for 4 consecutive cycles at 4°C to remove small molecular impurities.

[0128] (6) collecting the filtrate after dialysis, and then ultrafiltration using an ultrafiltration membrane with a molecular weight of 5000;

[0129] (7) The filtrate obtained by ultrafiltration is sterilized by filtration and then freeze-dried to obtain chicken spleen transfer factor.

[0130] The drug a is prepared by the following preparation method:

[0131] (1) Grind gardenia, scutellaria, forsythia, dandelion, coptis root, bupleurum, liquorice, star anise, peony bark, radix trichosanthis, akebia, and pomegranate peel, and pass through a No. 2 sieve;

[0132] (2) Add 10 times the amount of distilled water, boil at 100°C for 1.5 hours;

[0133] (3) Filter and collect the residue and filtrate 1, add 10 times the amount of distilled water of the residue, boil at 90° C. for 1.5 h, filter, and collect filtrate 2;

[0134] (4) combining filtrate 1 and filtrate 2, and concentrating the mixture to a crude drug amount of 1 g / mL to obtain a drug concentrate;

[0135] (5) The concentrated solution was filtered and sterilized using a 0.22 μm filter membrane, the pH was adjusted to 7, and chicken spleen transfer factor was added and fully dissolved to obtain the Chinese medicine combination drug a.

[0136] Example 2

[0137] This embodiment provides a Chinese medicine combination drug b for preventing and treating drug-resistant Escherichia coli disease in chickens

[0138] In this medicine, the dosage of Gardenia is 10 parts, the dosage of Scutellaria baicalensis is 10 parts, the dosage of Forsythia suspensa is 15 parts, the dosage of Dandelion is 20 parts, the dosage of Coptis chinensis is 25 parts, the dosage of Bupleurum chinense is 15 parts, the dosage of Licorice is 8 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa rubra is 10 parts, the dosage of Trichosanthes kirilowii is 12 parts, the dosage of Atractylodes lancea is 15 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0139] The preparation methods of transfer factor and drug b are the same as those in Example 1.

[0140] Example 3

[0141] This embodiment provides a Chinese medicine combination drug c for preventing and treating drug-resistant Escherichia coli disease in chickens

[0142] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum officinale is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa rubra is 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0143] The preparation methods of transfer factor and drug c are the same as those in Example 1.

[0144] Comparative Example 1

[0145] This comparative example 1 provides a comparative drug a (lacking transfer factor)

[0146] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum officinale is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa rubra is 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, and the dosage of Pomegranate peel is 20 parts.

[0147] The preparation method of the comparative drug is the same as that of Example 1.

[0148] Comparative Example 2

[0149] This comparative example 1 provides a comparative drug b (lacking Gardenia jasminoides and Scutellaria baicalensis)

[0150] In this medicine, the dosage of Forsythia suspensa is 20 parts, the dosage of dandelion is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa rubra is 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes macrocephala is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0151] The preparation method of the comparative drug is the same as that of Example 1.

[0152] Comparative Example 3

[0153] This comparative example 3 mentions a comparative drug c (lacking Forsythia suspensa and Taraxacum mongolicum)

[0154] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa rubra is 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes macrocephala is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0155] The preparation method of comparative drug c is the same as that of Example 1.

[0156] Comparative Example 4

[0157] This comparative example 4 provides a comparative drug d (lacking Rhizoma Coptidis and Radix Bupleuri)

[0158] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum mongolicum is 30 parts, the dosage of Licorice is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa is 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Ophiopogon japonicus is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of Transfer Factor is 0.5 parts.

[0159] The preparation method of comparative drug d is the same as that of Example 1.

[0160] Comparative Example 5

[0161] This comparative example 4 provides a comparative drug e (lacking liquorice and anise)

[0162] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum officinale is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Paeonia suffruticosa rubra is 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes macrocephala is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0163] The preparation method of comparative drug e is the same as that of Example 1.

[0164] Comparative Example 6

[0165] This comparative example 6 provides a comparative example medicine f (lacking peony bark and trichosanthes root)

[0166] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum officinale is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Ophiopogon japonicus is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0167] The preparation method of comparative drug f is the same as that of Example 1.

[0168] Comparative Example 7

[0169] This comparative example 7 provides a comparative example medicine g (lacking Akebia and Pomegranate peel)

[0170] In this medicine, the dosage of Gardenia is 15 parts, the dosage of Scutellaria baicalensis is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum officinale is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice is 10 parts, the dosage of Star Anise is 40 parts, the dosage of Paeonia suffruticosa is 8 parts, the dosage of Radix Trichosanthis is 5 parts, and the dosage of transfer factor is 0.5 parts.

[0171] The preparation method of comparative drug g is the same as that of Example 1.

[0172] Comparative Example 8

[0173] This comparative example 8 provides a comparative drug h (Gardenia jasminoides is replaced by Fructus Aurantii Immaturus, and Scutellaria baicalensis is replaced by Atractylodes macrocephala)

[0174] In this medicine, the dosage of Citrus aurantium is 15 parts, the dosage of Atractylodes macrocephala is 15 parts, the dosage of Forsythia suspensa is 20 parts, the dosage of Taraxacum mongolicum is 30 parts, the dosage of Coptis chinensis is 15 parts, the dosage of Bupleurum chinense is 10 parts, the dosage of Licorice root is 10 parts, the dosage of Star anise is 40 parts, the dosage of Paeonia suffruticosa rubra is 8 parts, the dosage of Radix Trichosanthis is 5 parts, the dosage of Atractylodes lancea is 12 parts, the dosage of Pomegranate peel is 20 parts, and the dosage of transfer factor is 0.5 parts.

[0175] The preparation method of comparative drug h is the same as that of Example 1.

[0176] Example 4

[0177] In vitro antibacterial experiments to examine the antibacterial effects of different drugs

[0178] (1) Strain extraction and separation:

[0179] The diseased samples were collected from the market, isolated and PCR-identified for drug-resistant genes, and mcr-1-positive drug-resistant Escherichia coli and the standard strain of Escherichia coli CMCC.B.44103 were screened out.

[0180] (2) Strain culture:

[0181] The standard strain of E. coli and the positive resistant bacteria were streaked onto LB nutrient agar for activation and cultured at 37°C for 24 hours. A single colony was picked and inoculated into 5 mL of LB liquid medium and cultured in a constant temperature shaker at 180 r / min and 37°C for 24 hours. The plate count method was then used to count the bacteria and the bacterial solution was diluted with sterile saline to adjust the concentration to 1.2×10 7 CFU / mL, for future use.

[0182] (3) Experimental testing:

[0183] Absorb 1.2×10 7 100 μL of the bacterial suspension of CFU / mL Escherichia coli standard strain and positive drug-resistant strains was evenly spread on the LB nutrient agar plate. When the surface was slightly dry, the autoclaved Oxford cup was placed on the plate after the bacteria were spread.

[0184] Drugs were added according to the following experimental groups:

[0185] The control group was added with 200 μL sterile water; the experimental group 1 was added with 5 μg / mL polymyxin B; the experimental group 2 was added with Chinese medicine combination drug a; the experimental group 3 was added with Chinese medicine combination drug b; the experimental group 4 was added with Chinese medicine combination drug c;

[0186] Comparative experimental groups 1-8 were added with comparative drugs ah prepared in comparative examples 1-8 respectively;

[0187] Three replicate Oxford cups were set up for each bacterial treatment.

[0188] After adding the drug, the culture dish was placed in a 37°C constant temperature incubator for 24 hours, and the diameter of the inhibition zone was measured. The diameter of the inhibition zone was expressed as "mean ± standard deviation".

[0189] Table 1 Differences in the diameters of the inhibition zones in different experimental groups

[0190]

[0191]

[0192] The criteria for judging the diameter of the inhibition zone are: diameter ≥ 20mm, extremely sensitive "++++"; 15mm≤diameter<20mm, highly sensitive "+++"; 10mm≤diameter<15mm, moderately sensitive "++"; diameter<10mm is low-sensitivity "+"; no inhibition zone means insensitive "-".

[0193] From the analysis of the results in Table 1, it can be seen that polymyxin B in experimental group 1 has a good inhibitory effect on standard Escherichia coli, but has a poor inhibitory effect on mcr-1-positive resistant Escherichia coli, which is consistent with expectations.

[0194] The Chinese medicine composition a, Chinese medicine composition b and Chinese medicine composition c of experimental groups 2, 3 and 4 all had excellent inhibitory effects on standard Escherichia coli and mcr-1-positive drug-resistant Escherichia coli. Although the inhibitory effect on mcr-1-positive drug-resistant Escherichia coli was slightly reduced, it was still at the extremely sensitive and highly sensitive levels.

[0195] Among them, the effect of Chinese medicine composition c is significantly better than that of Chinese medicine composition a and Chinese medicine composition b. For standard Escherichia coli, the antibacterial effect of Chinese medicine composition c is 45.21% higher than that of Chinese medicine composition a and 40.03% higher than that of Chinese medicine composition b. For mcr-1 positive drug-resistant Escherichia coli, the antibacterial effect of Chinese medicine composition c is 31.49% higher than that of Chinese medicine composition a and 32.04% higher than that of Chinese medicine composition b. This result shows that the medicine prepared according to the formula of Chinese medicine composition c can more effectively synergize the effects of various Chinese medicine components and transfer factors, thereby achieving the best antibacterial effect.

[0196] The results of the comparative experiment group 1 show that the transfer factor plays an important role in enhancing the inhibitory effect of the drug of the present invention on Escherichia coli. In particular, for mcr-1 positive drug-resistant Escherichia coli, the absence of the transfer factor will lead to a significant reduction in the antibacterial effect.

[0197] The results of experimental groups 2 to 7 were compared and showed that the lack of specific ingredients significantly reduced the antibacterial effect of the control group drug, and its antibacterial effect was significantly weakened compared to the complete Chinese medicine composition C. Especially when targeting mcr-1 positive resistant Escherichia coli, the decline in antibacterial effect is more significant. This result shows that the integrity of the Chinese medicine ingredients of the drug of the present invention plays a key role in the effect of the drug in inhibiting Escherichia coli, especially in inhibiting mcr-1 positive resistant Escherichia coli. The lack of certain ingredients may lead to a substantial decrease in drug efficacy. Therefore, ensuring the integrity of Chinese medicine ingredients is an important guarantee for the drug to achieve the best antibacterial effect, especially for drug-resistant strains.

[0198] The results of comparative experimental group 8 showed that after replacing Gardenia with Fructus Aurantii Immaturus and Scutellaria Baicalensis with Atractylodes Macrocephala, the diameter of the inhibition zone was smaller than that of the experimental group. This result further verified that after replacing the components in the Chinese medicine composition of the present invention, the effect of the composition of the present invention could not be exerted, further proving the irreplaceability and uniqueness of the components in the Chinese medicine composition of the present invention.

[0199] Example 5

[0200] Animal experiments to test the therapeutic effects of different drugs on Escherichia coli disease caused by mcr-1 positive Escherichia coli

[0201] (1) The test animals of the present invention were 9-day-old SPF chickens, 250 in total, purchased from Beijing Boehringer Ingelheim Weitong Biotechnology Co., Ltd.

[0202] (2) The mcr-1 positive Escherichia coli obtained in Example 4 was inoculated into a plate and cultured at 37°C for rejuvenation. The concentration of the bacterial solution was determined by the plate surface live bacteria counting method, so that the concentration of the bacterial stock solution reached 1×10 8 cfu / mL, for future use.

[0203] (3) All chickens were weighed at 10 days of age and randomly divided into a blank control group (30 chickens) and a model group (220 chickens);

[0204] The chickens in the blank control group continued to be raised in a negative pressure isolator, while the chickens in the model group were moved to a temperature and humidity controlled, completely disinfected, relatively closed room for 3 days to adapt to the feeding. At 12 days of age, the chickens in the model group were gavaged with 0.5 ml of bacterial stock solution, and the blank group was given an equal amount of sterile PBS in the same way. Three days after the infection, 10 chickens were randomly dissected to observe whether the chickens showed typical symptoms such as pericardial perihepatitis (such as Figure 1 As shown, 3 days after the infection, obvious encapsulation of the heart and liver occurred).

[0205] (4) After successful modeling, the sick chickens were randomly divided into 7 groups, 30 in each group, namely experimental groups 1-4 and comparative experimental groups 1-3;

[0206] Experimental groups 1-4 were fed with 0.5 ml of water containing polymyxin B, Chinese medicine combination drug a, Chinese medicine combination drug b and Chinese medicine combination drug c respectively;

[0207] Control groups 1-3 were fed with water containing comparative drug a, comparative drug g and comparative drug h, respectively (since the above experiments have proved that the absence of different drugs will lead to a decrease in the antibacterial effect, the present invention only selects comparative drug a, comparative drug g and comparative drug h with better effects among the comparative drugs for animal experiments);

[0208] Cut off water supply at 3:00 pm every day, and administer medication in drinking water at 5:00 pm for 4 hours, for 5 consecutive days.

[0209] (5) During the experiment, the disease progression of each group of experimental chickens was recorded twice a day at 9:00 am and 4:00 pm. During the experiment, clinical symptoms such as mental state, appetite, and feces were observed and recorded. The cure rate, effective rate, and mortality rate were calculated.

[0210] Efficacy criteria

[0211] Cured: Mental state, diet and defecation return to normal, and symptoms completely disappear;

[0212] Effective: Mental state, diet, and defecation have improved, and symptoms have been significantly alleviated;

[0213] Ineffective: No improvement in symptoms or even death; autopsy reveals varying degrees of pericarditis, perihepatitis and other lesions.

[0214] Table 2 The therapeutic effect of different experimental groups on mcr-positive drug-resistant Escherichia coli disease

[0215]

[0216]

[0217] First, if Figure 2 This is the result of experimental group 4 after treatment. Combined with actual observations, it can be seen that after treatment, the symptoms of pericarditis and perihepatitis disappeared and returned to normal, and the mental state and stool were normal.

[0218] Figure 3 This is the result after treatment of control experimental group 3. It can be seen that there are still mild pericarditis and perihepatitis symptoms after treatment.

[0219] Combining the results in Table 2, we can see that

[0220] Experimental group 1: The cure rate of this group was only 13.3%, the effective rate was 16.7%, and the total effective rate was only 30%. This shows that polymyxin B is not ideal for the treatment of chickens infected with mcr-1 positive drug-resistant Escherichia coli.

[0221] Experimental Group 2, Experimental Group 3 and Experimental Group 4 (Chinese medicine compositions a, b, c): The therapeutic effects of these groups are significantly better than those of Experimental Group 1, especially Experimental Group 4 (Chinese medicine composition c). The cure rates of Experimental Group 2 and Experimental Group 3 were 73.3% and 76.7%, respectively, the effective rates were 1.00% and 1.33%, respectively, and the total effective rates were 83.3% and 90%, respectively. The cure rate of Experimental Group 4 was as high as 93.3%, the effective rate was 0.67%, and the total effective rate reached 100%, showing a significant therapeutic effect. Chinese medicine composition c showed the best efficacy in the treatment of mcr-1 positive Escherichia coli infection, which is consistent with its strong antibacterial effect in Table 1.

[0222] Control groups 1-3: These groups used different comparative drugs a, g, and h, respectively, and their cure rates and total effective rates were generally lower than those of the experimental groups. The cure rate of control group 1 was 26.7%, and the total effective rate was 56.7%; the cure rate of control group 2 was 23.3%, and the total effective rate was 53.3%; the cure rate of control group 3 was 30%, and the total effective rate was 63.3%. This result further proves the uniqueness and efficacy of the Chinese medicine composition of the present invention, and the integrity and synergy of the ingredients are crucial in improving the therapeutic effect. Whether it is the absence or replacement of some ingredients, it will lead to a significant reduction in the efficacy.

[0223] In summary, this study showed that the Chinese medicine composition C had a significant therapeutic effect in the treatment of mcr-1-positive Escherichia coli infection, and its efficacy was significantly better than polymyxin B and other comparative drugs.

Claims

1. A Chinese medicinal composition for preventing and treating Escherichia coli disease in chickens, characterized in that: The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Paeonia Suffruticosa: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Transfer Factor: 0.25-0.5 parts.

2. The Chinese medicine composition according to claim 1, characterized in that: The transfer factor is chicken spleen transfer factor; The preparation method of the chicken spleen transfer factor comprises the following steps: (a) Wash the chicken spleen with triple-distilled water to remove impurities, and then rinse with sterile saline; (b) mincing the cleaned chicken spleen, adding 2 volumes of physiological saline, and preparing a homogenate using ultrasonic cell wall breaking technology; (c) subjecting the homogenate to repeated freeze-thaw operations, and subjecting the homogenate to high-speed homogenization and pulverization after each complete thawing; (d) After low-temperature centrifugation, the supernatant is collected and filtered under pressure, and the obtained filtrate 1 is dialyzed to obtain filtrate 2; (e) ultrafiltration of filtrate 2 using an ultrafiltration membrane with a molecular weight of 5000, and collecting the permeate; (f) The permeate is filtered and sterilized, and then freeze-dried to obtain chicken spleen transfer factor.

3. The Chinese medicine composition according to claim 2, characterized in that: The chicken Escherichia coli disease is chicken Escherichia coli disease caused by standard Escherichia coli or mcr-1 positive Escherichia coli.

4. The Chinese medicine composition according to claim 3, characterized in that: The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

5. A method for preparing a drug for preventing and treating chicken Escherichia coli disease, characterized in that: The preparation method comprises the following steps: (1) Grind 10-20 parts of Gardenia, 10-20 parts of Scutellaria, 15-30 parts of Forsythia, 20-35 parts of Taraxacum, 5-25 parts of Coptis, 5-15 parts of Bupleurum, 6-10 parts of Licorice, 40-45 parts of Star Anise, 5-10 parts of Paeonia Suffruticosa, 5-12 parts of Radix Trichosanthis, 8-15 parts of Akebia, and 15-20 parts of Pomegranate Peel, and sieve; (2) Add 10 times the amount of distilled water, boil at 100°C for 1.5 hours; (3) Filter and collect the residue and filtrate 1, add 10 times the amount of distilled water of the residue, boil at 90°C for 1.5 h, filter, and collect filtrate 2; (4) combining filtrate 1 and filtrate 2, and concentrating the mixture to a crude drug amount of 1 g / mL to obtain a drug concentrate; (5) After filtering and sterilizing the concentrated solution, the pH is adjusted to 7, and 0.25-0.5 parts of chicken spleen transfer factor are added. After fully dissolving, the drug is obtained.

6. The preparation method according to claim 5, characterized in that: The chicken spleen transfer factor is prepared by the preparation method described in claim 2.

7. Use of a Chinese medicine composition in the preparation of a drug for treating chicken Escherichia coli disease, characterized in that: The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Peony Bark: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts; The chicken spleen transfer factor is prepared by the preparation method described in claim 2.

8. The use according to claim 7, characterized in that: The chicken colibacillosis is chicken colibacillosis caused by standard Escherichia coli or mcr-1 positive Escherichia coli; The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

9. Use of a Chinese medicine composition in the preparation of an antibacterial agent for inhibiting mcr-1 positive Escherichia coli, characterized in that: The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 10-20 parts, Scutellaria: 10-20 parts, Forsythia: 15-30 parts, Taraxacum: 20-35 parts, Coptis: 5-25 parts, Bupleurum: 5-15 parts, Licorice: 6-10 parts, Star Anise: 40-45 parts, Peony Bark: 5-10 parts, Radix Trichosanthis: 5-12 parts, Akebia: 8-15 parts, Pomegranate Peel: 15-20 parts, Chicken Spleen Transfer Factor: 0.25-0.5 parts; The chicken spleen transfer factor is prepared by the preparation method described in claim 2.

10. The use according to claim 9, characterized in that: The Chinese medicine composition is composed of the following components in parts by mass: Gardenia: 15 parts, Scutellaria: 15 parts, Forsythia: 20 parts, Taraxacum: 30 parts, Coptis: 15 parts, Bupleurum: 10 parts, Licorice: 10 parts, Star Anise: 40 parts, Paeonia Suffruticosa: 8 parts, Radix Trichosanthis: 5 parts, Akebia: 12 parts, Pomegranate Peel: 20 parts, Transfer Factor: 0.5 parts.

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