CN114831937B

CN114831937B - Physcion microemulsion and application thereof

Info

Publication number: CN114831937B
Application number: CN202210411318.3A
Authority: CN
Inventors: 何诚; 刘心怡; 陈家琪; 刘佳琪; 宫启翔; 谭丽萍; 何润春
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-04-19
Filing date: 2022-04-19
Publication date: 2023-10-03
Anticipated expiration: 2042-04-19
Also published as: CN114831937A

Abstract

The invention discloses an physcion microemulsion and application thereof. The physcion microemulsion is a microemulsion system consisting of an organic solvent and a surfactant. Preferred microemulsion system formulations include one or more of N, N-dimethylformamide or N, N-dimethylacetamide, ethanol or Tween-20. In vitro experiments prove that the physcion can kill clinical drug-resistant bacillus cereus, enterococcus faecalis, chicken bacillus, bird bacillus rhinotracheitis, haemophilus paragallinarum, klebsiella, chlamydia psittaci, mycoplasma gallisepticum and mycoplasma synoviae. Meanwhile, animal experiments prove that the physcion microemulsion is suitable for preventing and treating respiratory diseases caused by clinical bird bacillus rhinotracheitis, haemophilus parasuis, klebsiella, chlamydia psittaci and mycoplasma gallisepticum, liver rupture caused by bacillus cereus, enterococcus faecalis and bacillus gallinarum, bovine mastitis and endometritis caused by klebsiella and bacillus cereus.

Description

Physcion microemulsion and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to physcion microemulsion and application thereof.

Background

Physcion (Physcion) has a structure shown in formula I, and is anthraquinone compound with CAS registration number of 521-61-9.

Chemical name: 1, 8-dihydroxy-3-methoxy-6-methylanthraquinone with molecular formula of C ₁₆ H ₁₂ O ₅ Molecular weight 284.26.

The pure product is yellow needle-shaped crystal; melting point: 203-207 ℃; vapor pressure (20-25 ℃): 4.8X10 ^-11 Pa; solubility: is dissolved in benzene, chloroform, pyridine and toluene, slightly dissolved in acetic acid and ethyl acetate, not dissolved in methanol, diethyl ether and acetone, and a small amount of physcion can be slowly dissolved in ethanol and water. Hydrolysis under strong acid conditions, slow photolysis under strong ultraviolet light.

The emodin methyl ether is a botanical pesticide extracted from root and stem of Rheum officinale of Polygonaceae, belongs to anthraquinone high activity antibacterial compound, and has the main action mechanism of oxidation and dehydrogenation of bacterial sugar and sugar metabolism intermediate products, and can inhibit ammonia nitrogen assimilation, amino acid oxidation, dehydrogenation and deamination, and inhibit synthesis of protein and nucleic acid.

The physcion has been widely studied as a high-activity plant source bactericide, and has good control effects on powdery mildew, downy mildew, gray mold, anthracnose and the like. Because the plant extract is practically nontoxic to human and livestock, the plant extract is derived from plants, is suitable for large-area planting production, and is beneficial to ecological environment protection. Physcion is also a plant protective bactericide, induces plants to produce guard reaction, inhibits pathogenic bacteria spore germination, hypha growth and aspirator formation, prevents crops from being affected by pathogenic bacteria, and achieves the effect of disease prevention. But has less report and application in preventing and treating livestock and poultry diseases.

Related reports of physcion preparations, CN112841184A discloses a physcion microemulsion sustained release agent and a preparation method thereof, wherein physcion is firstly prepared into a microemulsion, the solubility of the microemulsion is increased to obtain an adjustable release speed, and then the physcion is encapsulated in melamine formaldehyde microcapsules to obtain slow release of physcion. The microcapsule sustained release agent has the characteristics of uniform particle distribution, high drug loading rate, slow and durable drug effect, and the preparation process is easy to operate, the raw materials are cheap and easy to obtain, the microcapsule sustained release agent can be used for preventing and treating plant diseases and insect pests, especially powdery mildew of cucumber, the drug administration times can be reduced, and the microcapsule sustained release agent has important practical application value. CN102440243a discloses a process for preparing physcion soluble granule, mixing physcion with beta-cyclodextrin, then adding surfactant, filler and penetrating agent, mixing, and granulating. The obtained product can be dissolved in water, and the effective components are embedded, so that the decomposition of active substances is prevented, and the shelf life of the product is prolonged.

The application of physcion has been reported more at home and abroad. Zhang Haihui it is proved by in vitro bacteriostasis test that the physcion, one of the rhubarb extracts, has stronger bacteriostasis on Pasteurella avis, E.avium and E.coli of pigs, has strongest bacteriostasis under acidic condition, neutral time and weaker effect under alkaline condition (Zhang Haihui, etc. feed industry, 2004 (11): 24-26). Wu Zifeng and the like prove that the physcion has a certain antibacterial activity, including the escherichia coli, staphylococcus aureus, rhizopus nigricans and aspergillus niger have good antibacterial performance, and the antibacterial performance is good when 256 mug/mL is achieved, and the antibacterial rate can reach 75.78% (Wu Zifeng and the like, guangzhou chemical industry, 2017, 45 (16): 72-74). The antibacterial experiments such as Nie Lirong prove that the physcion has good inhibition effects on aeromonas miltiorrhizae, aeromonas hydrophila and proteus, and the minimum antibacterial concentrations are 61.2 mug/ml, 125 mug/ml and 31.2 mug/ml respectively, so that the physcion has certain application value on aquaculture (Nie Lirong and the like, journal of water ecology, 2010, 31 (06)). However, the clinical application of physcion is mainly limited in the field of pesticides, pathogenic bacteria and fungi are killed, and few reports are reported in the field of livestock and poultry cultivation, which directly influences the application and popularization of physcion.

With the rapid development of livestock and poultry raising industry, the development of livestock and poultry industry is seriously affected by livestock and poultry respiratory diseases caused by livestock and poultry respiratory diseases, especially chlamydia psittaci, mycoplasma gallisepticum, bird bacillus rhinotracheitis and haemophilus paragallinarum, and livestock and poultry liver rupture caused by mixed infection of bacillus cereus and bacillus gallinarum, and cow mastitis and endometritis caused by bacillus cereus, klebsiella, enterococcus faecalis and staphylococcus. The damage of the diseases to the growth and development of the livestock and poultry is particularly serious, and the healthy development of the livestock and poultry breeding industry is seriously influenced.

In order to maintain the animal source food safety and public health safety in China, feed production enterprises stop producing commercial feeds containing growth promoting drug feed additives (except traditional Chinese medicines) gradually. The traditional Chinese medicine is used for treating respiratory diseases in livestock and poultry breeding, and the problems of mastitis and genital tract diseases of milk-producing livestock, little pollution and little residue and no drug residue can be caused.

In view of the current limited use of antibiotics in livestock industry and the popularity of livestock diseases in China, the healthy development of the livestock industry is seriously endangered, and a novel preparation for replacing the antibiotics is developed to solve the technical problem of neck blocking in the livestock industry in China. The reported literature and patent technology of physcion are mainly limited in the field of pesticides, and a plurality of technical problems exist in how to solve the water solubility of physcion and to replace antibiotics when the physcion is applied to animal husbandry. Aiming at the technical problems, the invention innovates water-soluble technology and application technology.

Disclosure of Invention

The invention aims to provide an physcion microemulsion and application thereof.

In order to achieve the aim of the invention, in a first aspect, the invention provides an physcion microemulsion which is 2.5-10% physcion microemulsion prepared from N, N-dimethylformamide or N, N-dimethylacetamide, ethanol, tween-20 and water.

Further, the physcion microemulsion is prepared by mixing 2.5-10g physcion with 20-50mL of N, N-dimethylformamide or N, N-dimethylacetamide, 20-30mL of 75% -100% ethanol, 0.1-2mL of Tween-20 and the balance of water, and supplementing water until the total volume is 100mL.

Preferably, the physcion microemulsion is prepared by mixing 10g physcion with 50mL of N, N-dimethylformamide or N, N-dimethylacetamide, 20mL of absolute ethyl alcohol, 2mL of tween-20 and the balance of water, and supplementing the mixture with water until the total volume is 100mL.

The preparation method comprises the following steps: dissolving physcion in the formula amount in N, N-dimethylformamide or N, N-dimethylacetamide, adding absolute ethyl alcohol and tween-20, uniformly mixing, and slowly adding water to fix the volume to 100mL.

In a second aspect, the invention provides the use of said microemulsion in the preparation of a bacteriostatic or antibacterial agent. The medicine can be oral preparation, injection or aerosol.

The bacteria are selected from Chlamydia psittaci, mycoplasma gallisepticum, mycoplasma synoviae, bacillus cereus, enterococcus faecalis, chicken bacillus, bird bacillus rhinotracheitis, haemophilus paragallinarum, klebsiella pneumoniae and the like.

In a third aspect, the invention provides an application of the microemulsion in preparing medicines for preventing and treating livestock and poultry respiratory diseases caused by bird's-eye bacillus, haemophilus paragallinarum, klebsiella, chlamydia psittaci, mycoplasma gallisepticum and the like.

In a fourth aspect, the invention provides application of the microemulsion in preparing medicines for preventing and treating livestock and poultry digestive tract diseases caused by bacillus cereus, enterococcus faecalis, chicken bacillus and the like.

Symptoms of digestive tract diseases of livestock and poultry can be expressed as liver rupture.

In the present invention, the livestock and poultry include, but are not limited to, broiler chickens, laying hens, meat ducks, egg ducks, cows, sheep.

In a fifth aspect, the present invention provides an application of the microemulsion in preparing a medicament for preventing and treating livestock (such as dairy cows) mastitis and endometritis caused by bacillus cereus, klebsiella, enterococcus faecalis, staphylococcus and the like.

The administration mode of the physcion microemulsion is oral administration, injection, aerosol administration, breast perfusion or uterus perfusion.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the physcion microemulsion provided by the invention can prevent and treat respiratory diseases of livestock and poultry caused by clinical drug-resistant bacteria Chlamydia psittaci, mycoplasma gallisepticum, bird bacillus rhinotracheitis and haemophilus parasuis, and livestock and poultry liver rupture caused by mixed infection of bacillus cereus and chicken bacillus, and livestock mastitis and endometritis caused by klebsiella, enterococcus faecalis, bacillus cereus and staphylococcus, thereby providing an effective means for preventing and treating drug-resistant bacterial diseases caused by infections of the respiratory tract, the digestive tract and the reproductive tract of livestock and poultry, and helping the healthy development of livestock and poultry farming industry in China.

Drawings

FIG. 1 shows a 10% physcion microemulsion prepared in the preferred embodiment of the present invention.

FIG. 2 shows the color change of milk 6 days after the physcion infusion in the preferred embodiment of the invention. 1: normal fresh milk; 2 and 3: high dose of physcion; 4 and 5: the dosage of physcion; 6: low dosage of physcion.

Detailed Description

The invention aims to provide a preparation method of physcion microemulsion and application thereof in preventing and treating diseases caused by drug-resistant bacteria of livestock and poultry.

The physcion microemulsion (physcion microemulsion preparation or physcion microemulsion) is a microemulsion system composed of an organic solvent and a surfactant. Preferred microemulsion system formulations include one or more of N, N-dimethylformamide or N, N-dimethylacetamide, ethanol or Tween-20.

Preferably, the 10% physcion microemulsion is prepared from N, N-dimethylformamide or N, N-dimethylacetamide, absolute ethyl alcohol, tween-20 and water according to a volume ratio of 25:10:1:14.

The administration mode of the physcion is oral administration, injection, aerosol administration and perfusion (breast perfusion and uterus perfusion).

The invention also provides application of the physcion microemulsion in preventing and treating drug-resistant bacterial diseases caused by the infection of the respiratory tract, the digestive tract and the reproductive tract of livestock and poultry.

The livestock and poultry of the invention are short for breeding livestock and poultry animals, including broiler chickens, laying hens, meat ducks, egg ducks, dairy cows and sheep.

The physcion can kill clinical isolated drug-resistant bacteria such as Chlamydia psittaci, mycoplasma gallisepticum, avibacterium rhinotracheitis, haemophilus paragallinarum, bacillus gallinarum, enterococcus faecalis, klebsiella and bacillus cereus.

The physcion microemulsion can prevent and treat respiratory diseases of livestock and poultry caused by clinical drug-resistant bacteria Chlamydia psittaci, mycoplasma gallisepticum, bird bacillus rhinotracheitis, haemophilus parasuis and chicken bacillus, and livestock and poultry liver rupture caused by mixed infection of bacillus cereus and chicken bacillus. Livestock mastitis and endometritis caused by klebsiella, enterococcus faecalis, bacillus cereus and staphylococcus.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

The percentage "%" referred to in the present invention refers to mass percent unless otherwise specified; however, the percentage of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of solution.

The physcion used in the following examples was purchased from China food and drug identification institute, polyoxyethylene castor oil was purchased from Aba Ding Huaxue reagent Co., ltd, and ethanol and Tween-20 were purchased from national drug group chemical Co., ltd.

EXAMPLE 1 preparation of physcion microemulsion

1. The composition is as follows: physcion 10g, N-dimethylformamide or N, N-dimethylacetamide 20ml,75% ethanol 30ml, and water to 100ml.

2. The preparation method comprises the following steps:

2.1, respectively weighing the raw materials and the auxiliary materials according to the prescription amount for standby.

2.2 dissolving physcion in N, N-dimethylformamide or N, N-dimethylacetamide, adding 75% ethanol and tween-20, mixing, and slowly adding water to constant volume to 100ml.

3. Results: the prepared microemulsion appeared brown in color, 50% dissolved, and precipitation occurred after standing.

EXAMPLE 2 preparation of physcion microemulsion

1. The composition is as follows: physcion 10g, N-dimethylformamide or N, N-dimethylacetamide 20ml,95% ethanol 30ml, and water to 100ml.

2. The preparation method comprises the following steps:

2.2 dissolving physcion in N, N-dimethylformamide or N, N-dimethylacetamide, adding 95% ethanol, mixing, and slowly adding water to constant volume to 100ml.

3. Results: the prepared microemulsion appeared brown in color, 60% dissolved, and precipitation occurred after standing.

EXAMPLE 3 preparation of physcion microemulsion

1. The composition is as follows: physcion 10g, N-dimethylformamide or N, N-dimethylacetamide 40ml,95% ethanol 30ml, tween-20 2ml, and water to 100ml.

2. The preparation method comprises the following steps:

2.2 dissolving physcion in N, N-dimethylformamide, adding 95% ethanol and Tween-20, mixing, and slowly adding water to constant volume to 100ml.

3. Results: the prepared microemulsion is brown, 80% dissolved and partial precipitation.

EXAMPLE 4 preparation of physcion microemulsion

1. The composition is as follows: physcion 10g, N-dimethylformamide or N, N-dimethylformamide 50ml,95% ethanol 20ml, tween-20 2ml, and water to 100ml.

2. The preparation method comprises the following steps:

3. Results: the prepared microemulsion has brown color, and is dissolved by more than 90%, and a small amount of precipitate appears after standing.

EXAMPLE 5 preparation of physcion microemulsion

1. The composition is as follows: physcion 10g, N-dimethylformamide or N, N-dimethylformamide 50ml, absolute ethanol 20ml, tween-20 2ml, and water to 100ml.

2. The preparation method comprises the following steps:

2.2 dissolving physcion in N, N-dimethylformamide, adding absolute ethyl alcohol and tween-20, mixing, and slowly adding water to constant volume to 100ml.

3. Results: the prepared microemulsion is brown, 100% dissolved and in a dissolved state after standing.

The prepared 10% physcion microemulsion is shown in figure 1.

Experimental example 1 determination of MIC of physcion against clinical drug-resistant bacteria

1. Experimental materials:

1.1 Medium: MH broth (purchased from beijing land bridge chicken stock limited).

1.2 antibacterial drugs: the physcion was prepared by using 3% sodium carbonate to prepare a solution, and the physcion was dissolved in the solution to prepare 512. Mu.g/ml of a mother liquor.

1.3 chicken bacillus, enterococcus faecalis, klebsiella pneumoniae, bacillus subtilis and bacillus cereus, isolated and identified by the national university of agriculture animal medical institute.

1.4 working bacterial liquid: the MH broth is used for culturing chicken bacillus, enterococcus faecalis, klebsiella, bacillus and bacillus cereus, and the concentration of the bacterial liquid is regulated to be 0.5 McO.

2. Experimental method

In the first 10 cells of each row of the 96-well plate, the drug physcion was diluted to 256. Mu.g/ml, 128. Mu.g/ml, 64. Mu.g/ml, 32. Mu.g/ml, 16. Mu.g/ml, 8. Mu.g/ml, 4. Mu.g/ml, 2. Mu.g/ml, 1. Mu.g/ml, 0.5. Mu.g/ml, 0.25. Mu.g/ml, and 50. Mu.l per well. Wells 11 were used as solvent controls to add 50 μl of 3% sodium carbonate solution. Well 12 was added with 50. Mu.l of pure water as a negative control. 50 μl of working bacteria solution was added to each well, and finally 50 μl of MH broth was added, and mixed well, 3 duplicate wells were made for each concentration. And culturing for 24 hours at 37 ℃, observing with naked eyes, wherein the bottom of the last hole is clear, and the concentration of the medicine corresponding to the hole without turbidity is the minimum antibacterial concentration of the test bacteria of the medicine.

3. Experimental results

The MIC value of physcion for chicken bacillus was 64. Mu.g/ml, for enterococcus faecalis was 128. Mu.g/ml, and for Klebsiella. The MIC value of physcion for bacillus is 128 mug/ml. The MIC value of physcion for Bacillus cereus was 128. Mu.g/ml.

Experimental example 2 Physcion-Chlamydia psittaci MIC experiment

1. Experimental materials

1.1 cells: hela cells.

1.2 antibacterial drugs: physcion was prepared as a mother liquor using DMSO at 640. Mu.g/mL.

1.3 strains: chlamydia psittaci: the 6BC strain, HJ strain (GenBank accession numbers NC_017287.1, JPIH00000000.2, respectively) was isolated and identified by the university of agricultural animals medical institute in China.

2. Experimental method

Taking growth logarithmic phase Hela cells, adjusting to 1×10 with cell culture solution ⁵ Per mL, 100uL per well was seeded with 96-well plates and the cell incubator incubated for 24h. The supernatant was discarded and the cells were infected with c.psittaci at moi=5 and incubated in a 37 ℃ cell incubator for 2h. Removing supernatant, adding 100uL of Chlamydia growth solution into each well, and adding 100uL of physcion solution with different dilutions, wherein each dilution is 4Wells were repeated and 2 positive control wells (no physcion), 2 solvent control wells of corresponding concentration (sodium carbonate solution control) and 2 blank control groups were set. The cells were incubated at 37℃for 48-72 hours in an incubator, the supernatant was discarded, giemsa stained, and the lowest concentration at which inclusion bodies were not observed by microscopic examination was MIC.

3. Experimental results

The minimum inhibitory concentrations of the 6BC strain and the HJ strain are 128 mug/mL and 256 mug/mL respectively.

Experimental example 3 therapeutic Effect of physcion on Chlamydia psittaci Artificial infection of SPF chickens

1. Experimental materials

1.1 experimental animals: SPF chickens (purchased from Beijing Bo Lin Gehan vitamin Biotechnology Co., ltd.) were 25 days old, 48.

1.2 experimental drugs: the physcion microemulsion prepared in example 5.

Control drug: doxycycline (purchased from China food and drug identification institute) content is 95%.

1.3 strains:

chlamydia psittaci: HJ strain (GenBank accession number JPIH 00000000.2), 10 ⁹ IFU/ml, isolated and identified by the university of agriculture animal medical college of China.

2. Experimental grouping: the 48 SPF chickens were randomly divided into 5 groups of 8, which are respectively a physcion high-dose group, a physcion medium-dose group, a physcion low-dose group, a physcion doxycycline group and a physcion control group.

3. The experimental method comprises the following steps:

the laryngo trachea of each group is inoculated with Chlamydia psittaci, 1 multiplied by 10 ⁷ IFU/0.2 ml/chicken flock presents respiratory symptoms (after 3 days of virus attack), starts administration treatment, and carries out drenching administration according to groups, wherein the high dose group of physcion is 9 mg/day; the dosage group in physcion is 6 mg/day, the low dosage group is 4 mg/day, the doxycycline group is 3 mg/day, and the toxin-attacking control group is not dosed. The administration was continued for 6 days.

And (3) observing the indexes: record the body weight of SPF chicken before toxin attack, administration and dissection. All SPF chickens were sacrificed after drug withdrawal, and the lung and air sac pathological changes were observed by section inspection and scored according to the lesion index scoring criteria. The measurement results were performed according to the lesion integral.

The lesion index scoring criteria for the balloon and lung were as follows:

single side balloon scoring: 0-means that the air bag is normal, clean, transparent and thin; 1-slightly thickened, slightly turbid air sac, or yellowish-white dew-like spots in individual parts; 2-there is significant exudate in the balloon portion area with moderate thickening of the balloon; the 3-balloon was filled with a yellow-white cheese-like exudate and had severe thickening.

Unilateral lung score: 0-the lung is normal, no obvious pathological changes exist; 1-a hemorrhagic or necrotic change in area of about 30% of the lung; 2-a hemorrhagic or necrotic change in area around 60% of the lung; 3-all lungs exhibit ischemic or necrotic pathological changes.

Viral load determination: the spleen Chlamydia psittaci load was determined by fluorescence quantification PCR (SYBRgreen) absolute quantification.

4. Experimental results

4.1 effects of drugs on body weight of SPF chickens:

TABLE 1 Effect of drugs on SPF chicken body weight

As can be seen from the results in Table 1, after administration, the weight gain rate of each administration group was increased compared with that of the toxicity attack control group, wherein the weight gain effect of the physcion high dose group was most remarkable.

4.2 Effect of drugs on SPF chicken air bags and Lung lesions

The major lesions of Chlamydia psittaci chicken were marked by balloon turbidity, punctate bleeding of the lungs and exudation, and the lesion levels and scores are shown in Table 2.

TABLE 2 SPF chicken air bags, lung lesions and scoring after drug administration

As can be seen from the results in table 2, the severity of balloon lesions were in the order: the toxicity attack control group is more than the low-dosage group of physcion, the medium-dosage group of physcion, the high-dosage group of physcion and the doxycycline group.

The severity of lung lesions was in turn: the low-dosage group of physcion > the toxicity attack control group > the medium-dosage group of physcion > the high-dosage group of physcion > the doxycycline group.

The physcion is suggested to have a certain treatment effect on the Chlamydia psittaci infected SPF chicken, and the dose-response relationship is presented.

4.3 determination of the Chlamydia psittaci load of the lung spleen of SPF chickens by fluorescent quantitation

The SPF chicken spleen chlamydia psittaci load was measured using fluorescence quantification PCR (SYBRgreen) absolute quantification as shown in table 3.

TABLE 3 Chlamydia psittaci load

Note that: * Represents a very significant difference (P < 0.01) compared to the challenge control group.

As can be seen from table 3, the chlamydia load was reduced in the administration group compared to the challenge control group, and the differences between the high dose group, the doxycycline group and the challenge control group were very significant (P < 0.01); the difference between the low dose group and the medium dose group was not significant (P > 0.05). This suggests that the high dose of physcion has a remarkable effect of clearing Chlamydia psittaci.

5. Conclusion(s)

From the experimental results, the model modeling of the Chlamydia psittaci infection is successful, the physcion dosage reaches 9 mg/day, the anti-attack effect on the Chlamydia psittaci HJ strain is better, the weight growth rate can be increased, and the spleen Chlamydia psittaci load of the SPF chicken and the pathological change degree of the air sac are obviously reduced.

Experimental example 4 physcion prevention and treatment of liver rupture caused by clinical chicken bacillus and Bacillus cereus

1. Experimental materials

1.1 test animals: the clinical chicken bacillus and bacillus cereus are mixed to infect brown chickens, and the sick and dead chickens have lesions such as crisp liver, rupture and the like at the age of 140 days.

1.2 test drug: the physcion microemulsion prepared in example 5.

2 Experimental methods

2.1 test group: blank 19100; drug administration group 19100.

2.2 modes of administration: the feed is fully mixed with feed according to 330 g/1000 jin of feed, and the feed is continuously administered for 6 days, 1 time a day.

2.3 test index: mortality and average daily consumption.

3. Test results:

3.1 Effect of physcion on the death and panning Rate of Chicken with Mixed infection disease of Bacillus cereus

TABLE 4 mortality rate

The results in Table 4 show that physcion can significantly reduce the mortality rate of bacillus cereus infected chickens.

3.2 Effect of physcion on consumption of feed by chicken with mixed infection of Bacillus cereus

TABLE 5 consumption of materials

The results in Table 5 show that physcion can increase the average daily consumption of feed in bacillus cereus infected chickens.

4. Conclusion(s)

According to the experimental results, the physcion can be seen, the daily feed consumption of the sick chickens is increased, and the death rate of the sick chickens infected by the mixed bacillus cereus is effectively reduced.

Experimental example 5 treatment effect of physcion on Mycoplasma and clinically isolated E.coli Mixed infection

1. Experimental materials

1.1 experimental animals: SPF chicken: 21 days old, beijing Bo Lin Gehan, biotechnology Inc., 64.

2.2 experimental drugs: the physcion microemulsion prepared in example 5.

Rhein: (purchased from China food and drug identification institute).

2.3 strains:

mycoplasma gallisepticum: r strain, 10 ⁹ IFU/ml (GenBank accession number CP001872.1, supplied by the livestock veterinary institute, coastal, shandong).

3. Experimental grouping: 64 SPF chickens were randomly divided into 8 groups of 8, which are 4 physcion groups, doxycycline control group, rhein control group, blank control group and challenge control group, respectively.

4. The experimental method comprises the following steps:

the concentration of the mycoplasma bacteria liquid is adjusted to 10 ⁹ cfu/ml, 0.1ml of nasal drops and eyes per chicken in the treatment group, 3 days of continuous infection, 1 time per day. After 7 days of infection, the concentration of the coliform bacteria liquid is adjusted to 10 ⁶ cfu/ml, 0.2ml per chicken was intraperitoneally injected, 1 infection. The chicken flock presents respiratory symptoms (after 3 days of virus attack), administration treatment is started, and the physcion 1 group is administrated by drenching according to groups, wherein the physcion 1 group is 25 mg/kg/day; 13 mg/kg/day for physcion 2 group, 7 mg/day for physcion 3 group, 4 mg/kg/day for physcion 4 group, 5 mg/day for doxycycline control group and 13 mg/kg/day for rhein control group. The sections were examined 6 days after continuous administration.

And (3) observing the indexes: record the body weight of SPF chicken before toxin attack, administration and dissection. All SPF chickens were sacrificed after drug withdrawal, and the pathological changes of the air sacs were observed by section inspection and scored according to the lesion index scoring criteria. The measurement results were carried out according to the lesion integral, and the integral standard is shown in experimental example 1.

Viral load determination: the mycoplasma load was determined by fluorescence quantification PCR (SYBRgreen) absolute quantification.

5. Experimental results

5.1 effects of drugs on body weight of SPF chickens:

TABLE 6 influence of drugs on SPF chicken body weight

The results in Table 6 show that the greater the dose of physcion, the more pronounced the average weight gain. Compared with the doxycycline group, the dose group of 25mg/kg and 7mg/kg of physcion has better effect, and other lower dose groups have lower effect than the doxycycline group. Compared with rhein group, 25mg/kg of physcion has better effect, and other lower dosage groups have lower effect than rhein group.

5.2 determination of the liver E.coli load of SPF chickens by colony plate counting

The colony plate count method was used to determine the liver E.coli load of SPF chickens as shown in Table 7.

TABLE 7 E.coli load

The results in Table 7 show that physcion is effective in eliminating liver E.coli and that there is a dose-response relationship. Compared with doxycycline, the 25mg/kg dose effect of physcion is similar to that of doxycycline, and the lower dose group is less effective than that of doxycycline. Compared with rhein, the physcion has better effect.

5.3 determination of the Mycoplasma Lung-Poison-containing Capacity of SPF chickens by fluorescence quantitative method

The fluorescence quantitative PCR (SYBRgreen) absolute quantitative method is adopted to detect the mycoplasma gallisepticum load of the lung of the SPF chicken as follows:

TABLE 8 Mycoplasma chicken toxic load

The results in Table 8 show that the physcion has remarkable effect on chicken lung mycoplasma gallisepticum and has a certain dose response relationship. The effect was inferior to that of the doxycycline control group. Compared with rhein control group, except for 4mg/kg dosage group, each dosage group of physcion has better effect than rhein control group.

6. Conclusion(s)

The experimental results show that the physcion has obvious effect of removing escherichia coli and mycoplasma gallisepticum and has a dose-response relationship.

Experimental example 6 physcion prevention and treatment of cow mastitis and endometritis

1 Experimental materials

1.1 test animals: clinical cows with mastitis and clinical cows with endometritis (black white cow, 300 days old, 400 kg body weight, and clinical diagnosis of mixed infection of staphylococcus aureus, escherichia coli and streptococcus agalactiae) are provided by Ningxia northern milk industry liability company.

1.2 test drug:

1.2.1 therapeutic agents for the treatment of mastitis conditions

Physcion breast perfusion agent: the physcion microemulsion prepared in example 5.

Control drug:

fast tannin (lincomycin hydrochloride breast injectant-lactation): 8 g/piece, 20 pieces/box, hebei remote sign pharmaceutical Co., ltd., production lot: DDM200904.

Cefquinome sulfate breast injection-lactation period: 8 g/piece, 20 pieces/box, hebei remote sign pharmaceutical Co., ltd., production lot: DAV210305.

1.2.2 therapeutic agents for endometritis disorders

Physcion uterine perfusion: the physcion microemulsion prepared in example 5.

Gong Jingyou (pregnancy promoting perfusate): the main components are epimedium, motherwort and safflower, 100 ml/bottle, produced by Jiaxing Jiutianyuan Hunting biotechnology Co., ltd., production batch number: 2020062201.

2. test method

2.1 treatment group for mastitis conditions:

each case was randomly assigned to the test drug group at the following doses:

high dose group of physcion: injecting physcion into mammary gland via affected side nipple, 30ml each time, 1 time daily, and 5 days for treatment course.

Dose group of physcion: injecting into mammary gland via physcion affected side nipple, 25ml each time, 1 time daily, and 5 days as treatment course.

Low dose group of physcion: injecting into mammary gland via physcion affected side nipple, 20ml each time, 1 time daily, and 5 days for treatment course.

Control group of fast tannin injection: the mammary gland is infused into mammary gland via the nipple at affected side, 1 branch (8 g) each time, 1 time daily, and 5 days as a treatment course.

Cefprozil injection control group: the mammary gland is infused into mammary gland via the nipple at affected side, 1 branch (8 g) each time, 1 time daily, and 5 days as a treatment course.

After the medicines are injected into the nipple orifice by the dairy cow lactation needle, the medicines are gently pinched into the nipple orifice, the breasts are gently massaged, and the medicines are pushed to the upper parts of the breasts.

2.2 endometritis disorder treatment group:

high dose group of physcion: physcion 50 ml/first cow/day, 1 time daily, and 5 days as a treatment course.

Dose group of physcion: physcion 30 ml/first cow/day, 1 time daily, and 5 days as a treatment course.

Control drug: gong Jingyou the infusion is 40 ml/calf/day, 1 time a day, and the treatment course is 5 days.

3. Test results

Table 9 treatment effect of physcion on bovine mastitis

Table 10 physcion treatment effect on cow endometritis

The color change of milk 6 days after physcion infusion is shown in FIG. 2.

4. Conclusion(s)

The experimental result proves that the physcion has a certain treatment effect on cow mastitis by breast perfusion, and can improve the milk quality. The physcion can effectively reduce purulent secretion of the vulva of the dairy cows, and has obvious effect of treating endometritis.

Experimental example 7 Physcion acute toxicity experiment

1. Experimental materials

1.1 drugs: the physcion microemulsion prepared in example 5.

1.2 experimental animals: SPF grade ICR mice (purchased from Beijing university medical department) 120, body weight 18.0-22.0g, male and female halves.

2. Method of

2.1 test methods

ICR male and female mice 230 (male and female halves) were randomly divided into 23 groups according to body weight, fasted overnight without water withdrawal for 12h, and were respectively gavaged for 0.10, 0.14, 0.20, 0.28, 0.39, 0.55, 0.77, 1.08, 1.51, 2.11, 2.95, g/kg body weight doses of physcion, and a normal saline control group (see table 11 for group dosing), gavaged for one time, and continuously observed for 7 days.

Mice body weight was recorded daily and analyzed for body weight changes.

The animal toxic reaction condition and death condition, feeding condition are recorded in detail, and the trusted interval of LD50 is calculated. The symptoms of poisoning, the occurrence time of poisoning, the duration, the recovery period, etc. are recorded.

Dead animals were necropsied in time and lesions were recorded.

2.2 data analysis

Test data are presented as (X+ -SD) with EXCEL2007 and SPSS20.0 statistical software for data processing and one-dimensional analysis of variance. The difference is significant P <0.05, and the difference is extremely significant P <0.01.

Calculation of acute toxicity LD according to Bliss method using SPSS software according to observed death of mice ₅₀ Is a dose of (a).

3. Test results

3.1 Effect of physcion on ICR mouse survival

TABLE 11 acute toxicity grouping and death cases of physcion

Calculation of physcion acute toxicity LD according to Bliss method using SPSS software ₅₀ 2.525g/kg.

Conclusion 4

Compared with the acute toxicity standard of the medicine, the physcion microemulsion belongs to actual non-toxicity.

Experimental example 8 determination of the content of physcion in plasma at different time points

1. Experimental materials

1.1 experimental animals: SPF chickens (purchased from Beijing Bo Lin Gehan vitamin Biotechnology Co., ltd.) were 25 days old, 8.

1.2 experimental drugs: the physcion microemulsion prepared in example 5.

2. Experimental method

The SPF chickens were fasted for 12 hours before testing and were free to drink water. Plasma before and after administration was collected, and plasma samples were stored in a refrigerator at-80℃and administered by one-time lavage at a dose of 20mg/kg body weight.

Collecting blood of 15min, 30min, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h and 4h after administration in an anticoagulant tube, centrifuging at 8000rpm for 10min, collecting supernatant, freezing in a refrigerator at-80deg.C, collecting sample of 8-12 h, 20-24 h after administration, and storing at-80deg.C.

Adding 100uL of blood plasma into an internal standard tube, swirling for 1min, adding 400uL of acetonitrile for precipitating protein, swirling for 2min,

ultrasonic processing for 5min, centrifuging (12000 rpm,5 min), collecting 400 μl of supernatant, placing into a clean 5mL centrifuge tube, blow drying with nitrogen at 25deg.C, adding 150 μl methanol for redissolving, centrifuging (12000 rpm,5 min), and collecting supernatant. The concentration of physcion was measured at each time point by HPLC.

3. The experimental results are shown in Table 12.

TABLE 12 concentration of physcion in plasma after administration

Time	Before administration of the drug	15min	30min	1h	1.5h
						Concentration mg/ml	0	0.0296±0.0024	0.0287±0.0036	0.0352±0.0037	0.0309±0.0009
Time	2h	2.5h	3h	3.5h	4h
						Concentration mg/ml	0.0300±0.0040	0.0257±0.0013	0.0259±0.0007	0.0257±0.0008	0.0257±0.0015
Time	6h	8h	10h	12h	24h
						Concentration mg/ml	0.0250±0.0011	0.0245±0.0012	0.0190±0.0011	0.0170±0.0014	0.009±0.0011

SPF chicken is irrigated with the physcion, the concentration of physcion in the blood plasma gradually rises within 1 hour, reaches a peak value within 1 hour, and the concentration is 0.0309mg/ml, and then gradually falls.

4. Conclusion(s)

The physcion is orally taken into blood for 1 hour to reach a peak, and continuously and stably exists for 8 hours, and the half life is reached for 12 hours.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. Application of physcion microemulsion in preparing bacteriostatic agent or antibacterial drug;

the bacteria are selected from Chlamydia psittaci, mycoplasma gallisepticum, mycoplasma synoviae, chicken bacillus, bird bacillus rhinotracheitis and haemophilus paragallinarum;

the physcion microemulsion is 2.5-10% physcion microemulsion prepared from N, N-dimethylformamide or N, N-dimethylacetamide, ethanol, tween-20 and water.

2. The use according to claim 1, wherein the physcion microemulsion is prepared by mixing 2.5-10g physcion with 20-50mL of N, N-dimethylformamide or N, N-dimethylacetamide, 20-30mL of 75% -100% ethanol, 0.1-2mL tween-20 and the balance of water, and adding water to a total volume of 100mL.

3. The use according to claim 2, wherein the physcion microemulsion is prepared by mixing 10g physcion with 50mL of N, N-dimethylformamide or N, N-dimethylacetamide, 20mL of absolute ethanol, 20mL of tween-20, 2mL and the balance of water, and adding water to a total volume of 100mL.

4. Application of physcion microemulsion in preparing medicines for preventing and treating livestock and poultry respiratory diseases caused by bird's-eye bacillus, haemophilus paragallinarum, chlamydia psittaci and mycoplasma gallisepticum;

5. The use according to claim 4, wherein the physcion microemulsion is prepared by mixing 2.5-10g physcion with 20-50mL of N, N-dimethylformamide or N, N-dimethylacetamide, 20-30mL of 75% -100% ethanol, 0.1-2mL tween-20 and the balance of water, and adding water to the total volume of 100mL.

6. The use according to claim 5, wherein the physcion microemulsion is prepared by mixing 10g physcion with 50mL of N, N-dimethylformamide or N, N-dimethylacetamide, 20mL of absolute ethyl alcohol, 20mL of tween-20 and the balance of water, and adding water to a total volume of 100mL.

7. Application of physcion microemulsion in preparing medicines for preventing and treating digestive tract diseases of livestock and poultry caused by chicken bacillus;

8. The use according to claim 7, wherein the physcion microemulsion is prepared by mixing 2.5-10g physcion with 20-50mL of N, N-dimethylformamide or N, N-dimethylacetamide, 20-30mL of 75% -100% ethanol, 0.1-2mL tween-20 and the balance of water, and adding water to a total volume of 100mL.

9. The use according to claim 8, wherein the physcion microemulsion is prepared by mixing 10g physcion with 50mL of N, N-dimethylformamide or N, N-dimethylacetamide, 20mL of absolute ethanol, 20mL of tween-20, 2mL and the balance of water, and adding water to a total volume of 100mL.

10. The use according to any one of claims 7 to 9, wherein the symptoms of digestive tract diseases of livestock and poultry are manifested by liver rupture.

11. The use according to any one of claims 7 to 10, wherein the livestock and poultry comprise broiler chickens, laying hens, meat ducks, egg ducks, cows, sheep.