CN111494312B - Oral liquid for preventing and treating chicken colibacillosis and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of preparations, and particularly relates to an oral liquid for preventing and treating chicken colibacillosis and a preparation method thereof. The application provides an oral liquid for preventing and treating chicken colibacillosis, which comprises the following raw materials: 10-20g of 3, 4-dihydroxy benzoic acid, 2-4ml of additive, 2-10g of sweetener, 2-7g of florfenicol and 100ml of solvent. 3, 4-dihydroxy benzoic acid stably exists in the oral liquid and is not easy to oxidize by adding a sweetening agent, a solvent dimethylacetamide and an additive lactic acid. Simultaneously, the preparation method of the oral liquid is also provided, and the method is simple and suitable for industrial application.

Description

Oral liquid for preventing and treating chicken colibacillosis and preparation method thereof

Technical Field

The invention belongs to the field of preparations, and particularly relates to an oral liquid for preventing and treating chicken colibacillosis and a preparation method thereof.

Disclosure of Invention

Colibacillosis in chicken is a common disease caused by Escherichia coli, and is characterized by causing pericarditis, perihepatitis, air sacculitis, peritonitis, salpingitis, synovitis, escherichia coli granuloma, omphalitis and other pathological changes.

3, 4-Dihydroxybenzoic acid [ (HO) ₂ C ₆ H ₃ COOH]Is the main component of protocatechuic acid, has the relative molecular mass of 154.12, and mainly comprises the following sources: is extracted from acanthopanax, schisandra, eucommia, holly, etc; biosynthesis is carried out by taking glucose as a substrate in a shikimic acid pathway in microorganisms. Has antioxidant, antibacterial, antiinflammatory, and antiviral effects, and has been used in medicine and health promotion fields. 3, 4-dihydroxy benzoic acid has not been approved for formal application in livestock and poultry, but related animal experiments are available. Protocatechuic acid is fed, so that stress and inflammatory reaction of weaned piglets can be relieved, intestinal flora is improved, intestinal injury is relieved, and survival rate and weight gain are improved; improving intestinal flora of yellow feather broilers and increasing weight gain; relieving heat stress of poultry and reducing oxidative damage; reducing the content of malondialdehyde in the liver of the broiler chicken, and improving the total antioxidant capacity in the liver; inhibiting virus propagation such as avian influenza, newcastle disease, infectious bursal disease, infectious bronchitis, etc., improving immunity of chicken, and reducing death rate.

The antibacterial mechanism of the 3, 4-dihydroxy benzoic acid is as follows: (1) The hydroxyl groups can be combined with lipid bilayers of cell membranes to influence the interaction of cell membrane phospholipid and protein, and change the structure, function and permeability of the cell membranes. (2) Form stable complex with iron, and the lack of iron can reduce the activity of metalloenzyme such as heme enzyme and riboside reductase, and inhibit the growth of pathogenic bacteria such as Escherichia coli. (3) The hydroxyl group is easy to be broken, and the given electron can enable O ₂ Is activated into O ^-2 Causing lipid peroxidation of bacterial cell membranes, and O ^-2 Production of H under the action of SOD ₂ O ₂ (ii) a Divalent iron ion (Fe) in pathogenic bacteria such as Escherichia coli and Staphylococcus aureus ²⁺ ) More OH is easily generated by Fenton reaction, and excessive OH grafting acts on DNA to cause breakage, thereby causing bacterial apoptosis.

3, 4-dihydroxy benzoic acid has a certain in vitro antibacterial effect, but the antibacterial effect is weaker than that of antibiotics, such as: the MIC range of the extract on escherichia coli, staphylococcus aureus and pseudomonas aeruginosa is 0.5-3.0 mg/mL, the inventor performs the bacteriostasis test on the escherichia coli (CMCC 44103), the MIC of the extract is 0.5mg/mL, and the bacteriostasis effect of the traditional Chinese medicine extract containing the extract is worse. As an antibiotic synergist, protocatechuic acid, mequindox and clinafloxacin have synergistic effect on the fish streptococci, but the synergistic effect on escherichia coli is not reported. Therefore, people lose the interest of livestock and poultry clinical antibacterial experiments, and do not search for cases for applying the antibacterial agent to livestock and poultry bacterial diseases.

3, 4-dihydroxy benzoic acid has poor solubility in cold water, and the dissolution amount in 100ml of water is less than 0.1g, so the use of the 3, 4-dihydroxy benzoic acid in the chicken raising industry is limited, because the existing large-scale chicken farm generally adopts drinking water for administration, the medicament with poor water solubility is difficult to popularize. CN201110045158.7 prepares protocatechuic acid into a liquid preparation for preventing and treating livestock and poultry viral diseases, the liquid preparation method is that protocatechuic acid, ethanol and tween are dissolved according to the weight-volume ratio of 1: 3: 4, and glycerol can be added as a solvent. However, if the preparation of the invention is mixed with chicken for drinking, four defects are caused: (1) When the medicine is diluted by 10 times of water, turbidity appears, the medicine is diluted again and still turbid, and precipitates become oil drops after the medicine is placed for 1 day, so that the medicine dosage is not uniform; (2) The drinking time of chicken comprises 4 hours of concentrated drinking or 12-24 hours of drinking, even if the preparation is diluted by 1 time to 100 times at one time (difficult to operate in an actual farm), the preparation can reach a clear state, but the color of the preparation can be changed from light yellow to yellow in 12-24 hours of drinking, the drug effect is unstable, and the preparation is not suitable for drinking in 12-24 hours; (3) Tween is used in an overdose manner, so that potential safety hazards of medication exist; (4) after being diluted by water, the taste is extremely bitter, and the chicken refuses to drink the tea.

3, 4-dihydroxy benzoic acid is a traditional Chinese medicine component with excellent antibacterial effect, and the following problems need to be solved for applying the compound to the prevention and treatment of chicken colibacillosis: (1) The in vitro bacteriostatic test of the escherichia coli standard strain cannot be limited, and the in vitro bacteriostatic action of the escherichia coli standard strain on a chicken colibacillus clinical isolate should be further researched; (2) On the premise of good in-vitro antibacterial action, the oral solution with stable quality during storage and application is prepared; (3) The problem that the water drinking amount of the chicken is greatly reduced due to bitter taste is solved, and the chicken drinking water is suitable for normal drinking of the chicken; (3) Performing in-vivo efficacy test on the target animal chicken to achieve the aim of safely and effectively preventing and treating chicken colibacillosis; (4) The synergistic effect of the antibacterial agent and the antibacterial agent is researched, and the curative effect is improved.

Disclosure of Invention

Aiming at the problems, the application provides the oral liquid for preventing and treating the chicken colibacillosis, which enables 3, 4-dihydroxy benzoic acid to exist stably and not to be oxidized easily by adding a sweetening agent, a solvent of dimethyl acetyl and an additive of lactic acid. Meanwhile, the preparation method of the oral liquid is provided, and the method is simple and suitable for industrial application.

The technical scheme of the invention is as follows: (claims)

1. An oral liquid for preventing and treating chicken colibacillosis is characterized by comprising the following raw materials: 10-20g of 3, 4-dihydroxy benzoic acid, 2-4ml of additive, 2-10g of sweetener, 2-7g of florfenicol and 100ml of solvent.

2. The oral liquid for preventing and treating chicken colibacillosis according to claim 1, wherein the additive is lactic acid.

3. The oral liquid for preventing and treating chicken colibacillosis according to claim 1, wherein the sweetener is dissolved in a solvent.

4. The oral liquid for preventing and treating chicken colibacillosis according to claim 1, wherein the solvent is an aprotic polar solvent.

5. The oral liquid for preventing and treating chicken colibacillosis according to claim 1 or 4, wherein the solvent is one or a mixture of two of dimethylacetamide and dimethylformamide.

5. The oral liquid for preventing and treating chicken colibacillosis according to claim 1 or 3, wherein the sweetening agent is: a mixture of rebaudioside and glycyrrhizin.

6. The oral liquid for preventing and treating colibacillosis in chicken as claimed in claim 1 or 3, wherein florfenicol, glycyrrhizin, in combination with 3, 4-dihydroxybenzoic acid, can exert synergistic effect (partial synergistic or additive effect) on antibacterial activity of Escherichia coli.

6. A method for preparing the oral liquid according to any one of claims 1 to 4, comprising the steps of:

(1) Adding an additive into a solvent accounting for 90 percent of the total amount, then adding 2/3 of 3, 4-dihydroxybenzoic acid, and uniformly stirring;

(2) Adding florfenicol into the solution obtained in the step (1), and uniformly stirring;

(3) Heating to 40-60 deg.C, adding the rest 3, 4-dihydroxybenzoic acid, and stirring; cooling to room temperature for later use;

(4) Adding 2 sweetening agents into the solution obtained in the step (3), stirring for dissolving, and adding the residual solvent to the whole volume; when in use, the water is added for dilution according to the instruction, and the mixture is uniformly stirred for chicken to drink.

The invention adopts lactic acid as an additive, plays a role in stabilizing 3, 4-dihydroxy benzoic acid and preventing the 3, 4-dihydroxy benzoic acid from being oxidized, and mainly acts on the easily oxidized phenolic hydroxyl of the 3, 4-dihydroxy benzoic acid. Dimethylacetamide or dimethylformamide are aprotic polar solvents, can dissolve various compounds, are completely mutually soluble with water, ether, ketone, ester and the like, have the characteristics of high thermal stability, low possibility of hydrolysis, low corrosivity, low toxicity and the like, and are excellent catalysts in organic synthesis, so that cyclization, halogenation, cyanidation, alkylation, dehydrogenation and other reactions can be accelerated, and dimethylacetamide used as a solvent in the method can also play a role in resisting oxidation and stabilizing a solution. The sweetening agent is mainly used for improving the bitter taste of the 3, 4-dihydroxy benzoic acid and has a synergistic effect, and the selected sweetening agent is dissolved in a solvent; stevioside, a novel natural sweetener extracted from stevia rebaudiana Bertoni (or stevia rebaudiana Bertoni) belonging to Compositae family herbaceous plants; glycyrrhizin, also known as glycyrrhizic acid, is a saponin component separated from traditional Chinese medicine licorice, is the main component of licorice and is also the effective component. Has corticoid-like effect, and has antiinflammatory, antiallergic, antibacterial, and toxic materials clearing away effects, and can inhibit chicken Escherichia coli proliferation and relieve inflammation and toxicity caused by Escherichia coli endotoxin. Florfenicol is an antibacterial drug special for animals, is used for treating bacterial diseases of pigs, chickens and fish caused by sensitive bacteria, and has obvious curative effect on respiratory system infection and intestinal tract infection.

The invention has the advantages of

1. Preparing an oral solution with stable quality during storage and application;

2. the problem that the water drinking amount of the chicken is greatly reduced due to bitter taste is solved, and the chicken drinking water is suitable for normal drinking of the chicken;

3. the effect is remarkable: performing in-vivo efficacy test on the target animal chicken to achieve the aim of safely and effectively preventing and treating chicken colibacillosis; the synergistic effect of the antibacterial agent and the antibacterial agent is researched, and the curative effect is improved.

Detailed Description

Example 1

A preparation method of an oral liquid for preventing and treating chicken colibacillosis comprises the following steps:

(1) Adding 4ml of lactic acid into 100ml of dimethyl acetamide with the total amount of 90 percent, then adding 13.5g of 3, 4-dihydroxybenzoic acid, and uniformly stirring;

(2) Adding 2 g of florfenicol into the solution obtained in the step (1), and uniformly stirring;

(3) Heating to 60 ℃, adding the rest 6.5 g of 3, 4-dihydroxybenzoic acid, and uniformly stirring; cooling to room temperature for later use;

(4) Adding 5g of sweetening agents of stevioside and glycyrrhizin into the solution obtained in the step (3), stirring for dissolving, and uniformly stirring; adding the residual solvent to the total volume; when in use, the water is added for dilution according to the instruction, and the mixture is uniformly stirred and is drunk by chickens.

Example 2

A preparation method of an oral liquid for preventing and treating chicken colibacillosis comprises the following steps:

(1) Adding 2 ml of lactic acid into 100ml of dimethyl acetamide with the total amount of 90 percent, then adding 6.6g of 3, 4-dihydroxybenzoic acid, and uniformly stirring;

(2) Adding 7g of florfenicol into the solution obtained in the step (1), and uniformly stirring;

(3) Heating to 40 ℃, adding 3.4 g of the rest 3, 4-dihydroxy benzoic acid, and uniformly stirring; cooling to room temperature for later use;

(4) Adding 0.5 g of sweetening agent stevioside and 1.5 g of glycyrrhizin into the solution in the step (3) before feeding, stirring for dissolving, and stirring uniformly; adding the remaining solvent to full volume; when in use, the water is added for dilution according to the instruction, and the mixture is uniformly stirred and is drunk by chickens.

Example 3

A preparation method of an oral liquid for preventing and treating chicken colibacillosis comprises the following steps:

(1) Adding 3 ml of lactic acid into 100ml of dimethylacetamide with the total amount being 90%, then adding 10g of 3, 4-dihydroxybenzoic acid, and uniformly stirring;

(2) Adding 5g of florfenicol into the solution obtained in the step (1), and uniformly stirring;

(3) Heating to 50 ℃, adding 5g of the rest 3, 4-dihydroxybenzoic acid, and uniformly stirring; cooling to room temperature for later use;

(4) Adding 2 g of sweetening agent stevioside and 3 g of glycyrrhizin into the solution obtained in the step (3), stirring for dissolving, and uniformly stirring; adding the remaining solvent to full volume; when in use, the water is added for dilution according to the instruction, and the mixture is uniformly stirred and is drunk by chickens.

Example 4

A preparation method of an oral liquid for preventing and treating chicken colibacillosis comprises the following steps:

(1) Adding 3 ml of lactic acid into 100ml of dimethylacetamide with the total amount being 90%, then adding 10g of 3, 4-dihydroxybenzoic acid, and uniformly stirring;

(2) Heating to 50 ℃, adding 5g of the rest 3, 4-dihydroxybenzoic acid, and uniformly stirring; cooling to room temperature for later use;

(3) Adding 2 g of sweetening agent stevioside and 3 g of glycyrrhizin into the solution in the step (3), stirring for dissolving, and uniformly stirring; adding the residual solvent to the total volume; when in use, the water is added for dilution according to the instruction, and the mixture is uniformly stirred and is drunk by chickens.

Comparative example 1

A preparation method of an oral liquid for preventing and treating chicken colibacillosis comprises the following steps:

(1) Adding 10g of 3, 4-dihydroxybenzoic acid into 100ml of dimethyl acetamide with the total amount of 90%, and uniformly stirring;

(2) Adding 5g of florfenicol into the solution obtained in the step (1), and uniformly stirring;

(3) Heating to 50 ℃, adding 5g of the rest 3, 4-dihydroxybenzoic acid, and uniformly stirring; cooling to room temperature for later use;

(4) Adding 2 g of sweetening agent stevioside and 3 g of glycyrrhizin into the solution obtained in the step (3), and uniformly stirring; and (5) sparsely stirring uniformly.

Examples of the effects of the invention

1. Clarity and stability of the diluted solution

Taking examples 1-4 and comparative examples 1-2 as examples, diluting 10 times, 100 times, 200 times and 400 times, and standing overnight; the results show that the oral liquid prepared in the embodiments 1 to 4 of the invention has good clarity and stability; in contrast, comparative example 1, the discoloration was severe.

2. In vitro bacteriostasis test: the oral liquid formulation of example 4 is adopted to prepare a standard drug sensitive tablet without dilution, the drug content (4 mug/tablet) is contained, and a drug sensitive test is carried out on clinically separated escherichia coli with strong drug resistance, and the results are as follows: the sensitivity rate of the invention is 100 percent, and the sensitivity rate of common antibiotics is 0-43 percent. The results are shown in Table 1.

TABLE 1 results of bacteriostasis of E.coli isolates from various sources, example 4 and other antibiotics

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3. Therapeutic effects

The oral liquids of examples 1-3 and comparative examples 1-2 were used to treat the Escherichia coli (Shouguese isolate) disease of artificially infected chickens at a dose of 40mg of 3, 4-dihydroxybenzoic acid per 1kg of body weight: the chicken mortality rate of examples 1-3 decreased from 70% to 10%; mortality of example 4 was 20%; the mortality rate of the comparative example 1 is 10% (lactic acid only has effect on the stability of the product, has no obvious effect on the direct drenching drug effect, and the curative effect is reduced because the raw materials are unstable and discolor when drinking water for chickens all day).

4. Example 4 in vitro antibacterial synergy test on E.coli in combination with antibacterial drugs

4.1 The test was carried out by the drug sensitive paper method. The drug sensitive tablet of the antibacterial drug is sold in the market; the glycyrrhizin drug sensitive tablet is self-made, and each tablet contains 10 microgram of glycyrrhizin; the drug sensitive tablet is self-made, and each tablet contains 4 mu g of 3, 4-dihydroxy benzoic acid; the combined drug sensitive tablet is prepared by dripping the invention (containing 4 mug of 3, 4-dihydroxy benzoic acid) on a commercially available drug sensitive tablet, and drying at low temperature. Coli used a standard strain (CMCC 44103). The results are shown in Table 2.

TABLE 2 evaluation of the combined bacteriostatic effect of the drug sensitive tablets and example 4

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4.2 The broth dilution checkerboard test was used. A6X 6 chessboard is adopted, the highest concentration of each drug is set as 2 XMIC according to the MIC value of each drug to an escherichia coli standard strain, then a series of double dilutions are carried out by using a culture medium, A (different antibacterial drugs) is used as a column, B (3, 4-dihydroxybenzoic acid of the invention) is used as a row, the concentration of A in each 1 row of the wells of a 96-well plate is unchanged, the concentration of B is sequentially 2, 1/2, 1/4, 1/8 and 1/16 times MIC (the MIC of B is 0.5 mg/mL), the concentration of B in each 1 row of the wells is unchanged, and the concentration of A is reduced to 1/16 times MIC. Mixing, standing at 37 deg.C, and culturing for 18-24 hr. Based on the partial antibacterial concentration FIC index as a judgment basis, FIC index = [ MIC = [) _(A) /MICA] +[MIC _(B) /MICB]Wherein MICA and MICB represent MIC of A and B drugs used alone, respectively _(A) And MIC _(B) Represent the MIC values for each of the a and B drugs when combined. FIC index is less than or equal to 0.5, and is a synergistic effect; FIC index more than 0.5 and less than or equal to 1 is partial synergy or additive effect; FIC index is more than 1 and less than or equal to 2, which is an irrelevant effect; > 2 is antagonistic. Each group was tested in 3 replicates. The results are shown in Table 3.

TABLE 3 EXAMPLE 4 in vitro bacteriostatic synergy test on E.coli in combination with antibacterial agents

。

Note: neomycin sulfate is insoluble in dimethylacetamide but is easily soluble in water, so that neomycin sulfate cannot be mixed to prepare the same solution, but can be respectively added with water and used in combination in clinic to increase the curative effect.

5. Drinking water feeding test

In the embodiment 1-4 of the invention, water is added for dilution according to the proportion of 1 to 400 times, the chicken drinks water freely, and the water drinking amount is not changed obviously; the sweetener in example 3 was removed and the chicken was diluted as well and allowed to drink water freely, reducing the water intake by 60-90%.

Claims (1)

1. A preparation method of an oral liquid for preventing and treating chicken colibacillosis is characterized by comprising the following raw materials: 10-20g of 3, 4-dihydroxy benzoic acid, 2-4ml of additive, 2-10g of sweetener, 2-7g of florfenicol and 100ml of solvent;

the additive is lactic acid;

the sweetener is dissolved in the solvent;

the solvent is one or a mixture of two of dimethylacetamide and dimethylformamide;

the sweetener is as follows: a mixture of rebaudioside, glycyrrhizin;

the preparation method comprises the following steps:

(1) Adding an additive into a solvent accounting for 90 percent of the total amount, then adding 2/3 of 3, 4-dihydroxybenzoic acid, and uniformly stirring;

(2) Adding florfenicol into the solution obtained in the step (1), and uniformly stirring;

(3) Heating to 40-60 deg.C, adding the rest 3, 4-dihydroxybenzoic acid, and stirring; cooling to room temperature for later use;

(4) Adding 2 sweetening agents into the solution obtained in the step (3), stirring for dissolving, and adding the residual solvent to the whole volume; when in use, the water is added for dilution according to the instruction, and the mixture is uniformly stirred for chicken to drink.