CN110403993B

CN110403993B - Prescription and application of compound pulsatilla chinensis powder

Info

Publication number: CN110403993B
Application number: CN201910692944.2A
Authority: CN
Inventors: 王彦红; 高尚; 王丽扬; 许小琴; 陈素娟; 彭大新
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2019-07-30
Filing date: 2019-07-30
Publication date: 2022-01-25
Anticipated expiration: 2039-07-30
Also published as: CN110403993A

Abstract

The invention belongs to the field of traditional Chinese medicines and biotechnology, and particularly relates to a formula and application of compound pulsatilla chinensis powder, wherein the formula is mixed traditional Chinese medicine powder, and the compound pulsatilla chinensis powder is prepared by mixing and pulverizing 2-5 parts of pulsatilla chinensis, 2-3 parts of golden cypress, 2-4 parts of ash bark, 1-3 parts of scutellaria baicalensis, 4-8 parts of fructus forsythiae, 1-3 parts of radix sophorae flavescentis and 2-5 parts of liquorice in parts by weight, and is directly mixed during clinical application. The compound pulsatilla chinensis powder has the capability of inhibiting and killing salmonella typhimurium and can inhibit the formation of a biofilm; has good prevention and treatment effects on goslings artificially infected with salmonella typhimurium, and provides valuable reference for clinical medication.

Description

Prescription and application of compound pulsatilla chinensis powder

Technical Field

The invention belongs to the field of traditional Chinese medicines and biotechnology, and particularly relates to a formula of compound pulsatilla chinensis powder and application thereof.

Background

The salmonella is an important zoonosis pathogen, not only brings threat to human health, but also causes huge economic loss to the poultry industry. In poultry production, due to the use of large amounts of antibiotics and chemical antibacterial drugs to prevent and control salmonellosis, resulting in severe bacterial resistance and drug residues in poultry meat products, more and more Salmonella have become multidrug-resistant bacteria (Szych J, Cieslik A, Pacierk J, et al. antibacterial resistance in Salmonella enterica subsp. strain in polar and from 1999[ J ]. Int J. antibacterial Agents,2001,18(1): 37-42; Chiu C H, Wu T L, Su L H, the term. expression in Taiwan of fluoroquinolone resistance in Salmonella enterica strain strains [ J ]. N. Engler J. strain J., 2002-6, 2002-11. incorporated strain J. inner strain J. 9, sample J. 9. inner strain J. 1, sample J. strain, 1998, et al. 1. multiple resistance in Salmonella strain, strain J. 1, 5,12: i- ] [ J ]. Antimicrob Agents Chemother,2001,45(4):1305-8.), the generation of multiple drug-resistant strains is finally bound to harm human health, therefore, the development of novel drugs is imperative, and botanicals are hot spots, and the research shows that citrus flavonoids have the pathogenic effect of inhibiting Salmonella typhimurium (Vikram A, Jesuhasan PR, Jayaraka GK, et al. citrus flavino red repressors salmonella pathobiology island 1 and motility in S. typhimurium LT2.int J Food Microbiol,2011,145: 28-36.). Kaempferol in the traditional Chinese medicine kaempferol can inhibit the function of salmonella typhimurium to invade intestinal epithelial cells (Liliantao, Machang, Zhao Qian, and the like. Kaempferol with sub-antibacterial concentration has the inhibition effect on the salmonella typhimurium to invade the intestinal epithelial cells of chickens [ J ] China preventive veterinary science and newspaper, 2017,39(07):534-539 ]. Berberine inhibits the formation of Salmonella typhimurium biofilm under quorum sensing regulation, primarily by acting on quorum sensing signal receptors LasR and RhlR (Jamuna Bai Aswathanarayan, Ravishankar Rai vita. inhibition of bioinformation formation and quorum sensing mediated phenology by berberine in Pseudomonas aeruginosa and Salmonella typhimurium RSC adv.2018,8, 36133-36141.). Over centuries of clinical practice, it was found that the therapeutic effect of a combination formulation comprising multiple components was far superior to that of a formulation comprising a single component (Sucher NJ. the application of Chinese medicine to novel drug delivery. expert Opin. drug delivery, 2013,8, 21.).

Pulsatilla Decoction (PD) was first observed in Shang Zhong Jing Shang Han Lun, and to date, Pulsatilla powder is still a prescription recorded in pharmacopoeia of the people's republic of China (2015 edition), and has the functions of clearing away heat and toxic materials, cooling blood and stopping dysentery. It is mainly used for treating damp-heat diarrhea, dysentery with purulent blood. The pulsatilla chinensis decoction comprises pulsatilla chinensis, coptis chinensis, phellodendron amurense and ash bark, and the 4 medicines all have antibacterial active ingredients (Chenxi, WangXiongqing, Nianmin, and the like. 24 traditional Chinese medicines have in-vitro antibacterial activity on salmonella suis [ J ] Jiangsu agricultural science, 2008(05):180 + 182. + 205.). At present, in vitro And in vivo studies show that the formula has good treatment effect on infection of Escherichia coli, salmonella And the like (Jiankang Yu, Yuetian Zhang, Xu Song, et al. Effect of Modified Pulsatilla Powder on Enterotoxicosis Escherichia coli O101-Induced Diarrhea in Mice. evolution-Based comprehensive And Alternative Medicine,2017.DOI 10.1155/2017/368748; Zhuang, Hou organ, True. Pulsatilla decoction for pharmacodynamic test on chicken infected by salmonella [ J ] zootechnics And veterinarian, 2006 (35-36)). Research shows that the clinical antibacterial property of scutellaria is better than that of coptis and no drug resistance is generated (ChenShehen, WangXiongqing, substitution allergy, etc. 24 traditional Chinese medicines have in-vitro antibacterial activity on salmonella suis [ J ] Jiangsu agricultural science, 2008(05): 180-. In addition, researches prove that baicalin, the main component of scutellaria baicalensis, can regulate the virulence of bacteria and improve the immune response capability of organisms to improve the anti-salmonella typhimurium infection capability of mice (Huang Yan Hua, Zhang in the house, Niuge Wei, and the like. Meta analysis of the in-vitro antibacterial effect of rhubarb, scutellaria baicalensis, myrobalan and coptis chinensis on aeromonas hydrophila [ J ]. southwestern agriculture bulletin, 2018,31(06): 1315-1321.).

The experimental data of epidemiological investigation of bacteria for many years show that the dominant serotypes of salmonella prevalent in waterfowls are salmonella typhimurium (Huangkai, Chensu juan, Huangjun, and the like. the drug resistance analysis of salmonella of animal origin and the identification of florfenicol drug-resistant genes [ J ]. Chinese animal husbandry and veterinarian, 2015,42(2): 459) 466; Duyu. separation and identification of salmonella avicularis, biofilm determination and drug resistance analysis [ D ] Yangzhou university, 2016; Zhangwei. separation and identification of salmonella avicularis and the development of pullorum agglutination antigen [ D ]. Yangzhou university, 2017.). Therefore, the components and the proportion of the traditional prescription are determined by adding and subtracting the components and the proportion of the traditional Chinese pulsatilla root powder according to the basic composition and the proportion of the traditional Chinese pulsatilla root powder and repeated practice for many years in clinic, and the traditional Chinese pulsatilla root powder is verified in animal experiments of goose groups. Provides another good method for preventing and treating clinical salmonellosis.

Disclosure of Invention

In order to overcome the defects, the invention provides a formula and application of compound Chinese pulsatilla root powder, and the improved compound Chinese pulsatilla root powder, namely the compound Chinese pulsatilla root powder consisting of Chinese pulsatilla root, amur corktree bark, ash bark, baical skullcap root, weeping forsythia capsule, lightyellow sophora root and liquoric root, can be used for preventing and treating long-term chronic diarrhea of animals in clinical application. The compound has good prevention and treatment effect in a gosling model artificially infected by salmonella typhimurium, and provides reference for clinical prevention and treatment of salmonellosis typhimurium.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that: a formula of compound pulsatilla chinensis powder is characterized by comprising, by weight, 2-5 parts of pulsatilla chinensis, 2-3 parts of golden cypress, 2-4 parts of cortex fraxini, 1-3 parts of scutellaria baicalensis, 4-8 parts of fructus forsythiae, 1-3 parts of radix sophorae flavescentis and 2-5 parts of liquorice.

The formula is specifically as follows: 5 parts of Chinese pulsatilla root, 3 parts of golden cypress, 4 parts of ash bark, 3 parts of baical skullcap root, 8 parts of weeping forsythia, 3 parts of lightyellow sophora root and 5 parts of liquoric root, wherein the parts are in parts by weight.

3-5g of formula is required to be used for each kilogram of gosling, and 4g of formula is preferred.

A preparation method of a prescription formula of compound anemone powder comprises the following steps:

according to the weight portion, 2-5 portions of Chinese pulsatilla root, 2-3 portions of phellodendron bark, 2-4 portions of ash bark, 1-3 portions of scutellaria root, 4-8 portions of forsythia fruit, 1-3 portions of flavescent sophora root and 2-5 portions of licorice are selected to form the compound Chinese pulsatilla root powder, and the compound Chinese pulsatilla root powder is obtained by mixing and powdering. Directly mixing materials during clinical application; the compound Chinese pulsatilla root powder is a traditional Chinese medicine mixed powder.

The invention also provides application of the formula or the preparation method of the compound anemone powder in preparation of a medicine for preventing and/or treating salmonellosis of animals. The animal is poultry. Further, the animal is goose.

The invention also aims to provide application of the formula or the preparation method of the compound anemone powder in preparation of a medicine for inhibiting salmonella.

Compared with the prior art, the invention has the beneficial effects that: the compound pulsatilla chinensis powder has the capability of inhibiting and killing salmonella typhimurium and can inhibit the formation of a biofilm; has good prevention and treatment effects on goslings artificially infected with salmonella typhimurium, and provides valuable reference for clinical medication.

The compound pulsatilla chinensis powder can be added into feed, has important application value for preventing and controlling bacterial diseases, has a growth promotion effect, has no side effect or residue, is a green and environment-friendly medicine, and really realizes non-resistance feeding.

Drawings

FIG. 1 statistics of the number of different MIC/MBC strains, FIG. 1A statistics of the number of different MIC strains, and FIG. 1B statistics of the number of different MBC strains;

FIG. 24 is a schematic view of the lesion of day 9 after challenge by day-old gosling, in which

1. In blank group, pancreas is light red; 2.in the wild attack group, pancreas is red, and a plurality of white necrotic foci exist; 3. in the wild prevention group, pancreas is red; 4. in the wild defense group, pancreas is light red; 5. in the wild treatment group, pancreas is red, and a small amount of white necrotic lesions exist; 6. in the wild western medicine group, pancreas is light red; 7. in the wild Chinese and western treatment group, pancreas is red; 8. the mark attack group is marked, the pancreas is pale, and the texture is hard; 9. the mark ZhongFANG group has a pancreas part which is light red and a part which is red; 10. in the secondary and middle treatment groups, pancreas is light red; 11. in the treatment group of Bing xi, the pancreas appeared pale red.

Detailed Description

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the teachings of the present invention, and such equivalents also fall within the scope of the appended claims.

The preparation method of the compound Chinese pulsatilla root powder is a poultry feed additive which is a mixed powder of traditional Chinese medicines, and the preparation method comprises the following specific steps: selecting compound Chinese pulsatilla root powder consisting of 5 parts of Chinese pulsatilla root, 3 parts of phellodendron bark, 4 parts of ash bark, 3 parts of scutellaria baicalensis, 8 parts of forsythia, 3 parts of sophora flavescens and 5 parts of liquorice according to parts by weight, mixing, pulverizing into powder, and subpackaging for storage for later use;

the using method comprises the following steps: the dosage of each kilogram of gosling is 4 g/kg;

specifically, according to animal experiment medication, the body surface area between human and different animals is converted into an equivalent ratio (the rat is similar to a gosling), and according to the dosage: rat (200g), human (70KG) ═ 56: 1, the dosage of the Chinese pulsatilla root soup is 45g per dose, and the equivalent dose of 200g gosling is 45/56-0.8, so that the dosage per kilogram of gosling is as follows: 0.8 × 1000/200 ═ 4 g/kg.

The specific test protocol of the present invention:

1. decocting by conventional method to obtain Pulsatilla chinensis decoction with concentration of 1.5 g/ml.

Weighing 15g of the compound Chinese pulsatilla root powder, adding 100ml of 50% alcohol solution into a round-bottom flask, soaking for 30min, boiling for 30min, and decocting for 30min at 70 ℃. Filtering the liquid medicine with four layers of gauze for later use, collecting the medicine residues, adding 80ml of 50% alcohol solution, and repeatedly soaking and decocting. Filtering the liquid medicine with four layers of gauze for later use, collecting the dregs of a decoction, adding 60ml of 50% alcohol, repeatedly soaking and decocting, and filtering the liquid medicine. Mixing the three filtrates, decocting at 70 deg.C, concentrating, centrifuging, making into anemone decoction with concentration of 1.5g/ml, and storing at 4 deg.C for use.

2. Preparation of the bacterial suspension

And (4) recovering bacteria, picking the frozen salmonella typhimurium, inoculating the salmonella typhimurium to a sheep blood agar plate, and culturing for 16h at 37 ℃. Selecting the recovered salmonella typhimurium single colony, inoculating the single colony into 5ml of sterilized LB culture solution, culturing overnight at 37 ℃, absorbing 5 mu l of fresh bacterial liquid into 5ml of LB culture solution, and culturing for 5h to OD by shaking₆₀₀The value of (2) is 0.4-0.5, and then the bacterial liquid OD is added₆₀₀Adjusting the concentration of the bacterial liquid to 0.1 and the concentration of the bacterial liquid to 1.5 multiplied by 10⁸CFU/ml。

3. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

And (3) continuously diluting the decocted liquid medicine by multiple times (1500mg/ml-2.93mg/ml), repeating each concentration for three times, sequentially and respectively adding the diluted liquid medicine into a U-shaped plate with 96 holes, adding 100 mu l of the diluted liquid medicine into each hole, supplementing 100 mu l of the prepared liquid medicine into each hole, and finally adding 100 mu l of LB culture solution and the prepared liquid medicine into the two holes respectively to make negative and positive controls. Fully and evenly mixed, and incubated at 37 ℃ for 24 h. When the bacteria in the positive control holes completely grow, the experimental result is judged under the condition that the LB culture solution in the negative control holes is not polluted: the bacterial growth in the pores is completely inhibited, and the minimum drug concentration of the clarified bacterial liquid is MIC. And (3) sucking 10 mu l of culture of each well without bacterial growth, inoculating the culture on an LB agar medium, and culturing at 37 ℃ for 24h, wherein the lowest liquid medicine concentration of the colony number not more than 5 is MBC. The results show that: the compound pulsatilla chinensis bunge extract has a certain inhibiting and killing effect on 30 salmonella typhimurium strains (table 1). The Minimum Inhibitory Concentration (MIC) range is 23.45 mg/ml-375 mg/ml.

TABLE 1 Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of the compound Pulsatilla chinensis powder for Salmonella typhimurium

Note: "+" indicates bacterial growth and "-" indicates no bacterial growth

4. Determination of external bacteriostatic curve of compound pulsatilla chinensis powder on salmonella typhimurium

According to the MIC measurement result, the extract of the compound anemonin powder is diluted into three concentration gradients of 1MIC, 1/2MIC and 1/4MIC of the corresponding strains, and the concentration of the bacteria liquid is adjusted to be about 5 multiplied by 10⁶CFU/ml. 4 centrifugal tubes are arranged for each strain of bacteria, 5ml of bacteria liquid is filled in each centrifugal tube, liquid medicines with three concentration gradients are respectively added into the first three centrifugal tubes, sterile LB culture solution is added into the last centrifugal tube as blank control, the mixture is uniformly mixed and placed in a shaking incubator at 37 ℃, the centrifugal tubes are taken out at time points of 0h, 2h, 4h, 6h, 8h, 12h and 24h respectively, and each centrifugal tube is used for centrifugingMu.l of the bacterial suspension was aspirated from the tube, and the tube was placed in an EP tube containing 450. mu.l of sterile physiological saline, and serially diluted 10-fold, 10. mu.l of each dilution gradient of the bacterial suspension was counted on an LB agar plate, and each concentration was repeated 3 times, and cultured in an incubator at 37 ℃ for 24 hours. The number of colonies for each concentration was recorded to calculate the concentration of bacterial fluid in each centrifuge tube at different time points. The time is used as the abscissa, and the logarithm of the number of colonies is used as the ordinate to plot the sterilization curve.

The results show that: it was shown that the extract at the concentration of 187.5mg/ml had the highest inhibitory amount, with a very significant difference compared to the lowest inhibitory amount (FIG. 1). The extracts at three concentrations of 93.75mg/ml, 187.5mg/ml and 375mg/ml inhibited 90% of the strains. When the concentration of the extracting solution is 375mg/ml, the sterilization quantity is the largest, the sterilizing quantity reaches 16 strains of salmonella typhimurium, the percentage is 53.33 percent, and the difference is very obvious.

5. Determination of capability of traditional Chinese medicine extracting solution in inhibiting formation of salmonella typhimurium biofilm

5.1 measurement of biofilm Forming ability of Salmonella typhimurium

And (3) adopting a 96-pore plate crystal violet staining method to measure the forming capability of the salmonella typhimurium biofilm. Preparing bacterial suspension, centrifuging at 8000 Xg for 10min at 4 deg.C, removing supernatant, and resuspending to 5ml with sterile physiological saline. Each Salmonella typhimurium was replicated in 4 wells, and 180. mu.l of TSB culture solution and 20. mu.l of bacterial solution were added to each well. Only TSB culture solution is added as a negative control hole, and no TSB pollution in the negative control hole is used as an experimental condition. Culturing at 28 deg.C for 72h, discarding culture solution in the wells, washing with PBS phosphate buffer solution for 3 times, and removing floating bacteria in the wells. After natural air drying, 200 μ l of 0.4% crystal violet dye solution was added to each well, and the mixture was stained in the dark for 30min, washed 3 times with PBS phosphate buffer solution, and the excess dye solution was removed. Naturally air drying, adding 200 μ l 33% glacial acetic acid into each well, eluting the biological membrane for 30min, and measuring OD₅₅₀. With negative control OD₅₅₀The mean plus 3-fold standard deviation (ODc) values were used as a basis to classify the Salmonella biofilm-forming ability into four categories: OD₅₅₀A value of not less than ODc represents no film formation (-); ODc < OD₅₅₀A value of 2ODc or less is weak membrane formation (+); 2ODc < OD₅₅₀A value of less than or equal to 4ODc is medium film forming (++); OD₅₅₀Values > 4ODc are strong film forming (+++). The biofilm-forming ability was determined by taking the average of 4 results for each strain of bacteria.

The results show (table 2): the biofilm of 30 Salmonella typhimurium strains was quantified by crystal violet staining, and a total of 26 Salmonella typhimurium strains were able to form a biofilm (OD)₅₅₀0.165 + -0.041-0.592 + -0.117).

TABLE 2OD 550 of Salmonella typhimurium isolates_nmValue of

5.2 measurement of capability of compound anemonin powder in inhibiting formation of Salmonella typhimurium biofilm

100 mul of salmonella typhimurium strain TSB bacterial liquid capable of forming a biological membrane is inoculated into a 96-well plate, each strain of bacteria is repeatedly added for 3 times, and 100 mul of compound anemone powder (1500mg/ml-93.75mg/ml) diluted by TSB as a diluent in a continuous multiple ratio is additionally added into each well. Blank control wells containing only the culture medium and positive control wells containing only the bacterial solution were set, incubated at 28 ℃ for 72 hours, and examined by crystal violet staining (Table 3). The biofilm inhibition rate was determined using the formula: (OD positive control-OD drug group)/(OD positive control-OD blank group). times.100%.

The results show (table 4): the 16 times diluted extract (93.75mg/ml) has the best effect of inhibiting the biofilm formation capability of the salmonella typhimurium, and reaches 89.84 percent.

TABLE 3 OD550 of Salmonella typhimurium under action of compound Pulsatilla chinensis powder_nmValue of

Note: the different letter representations of the same row have statistical difference

TABLE 4 inhibition ratio (%), of Compound Pulsatilla chinensis powder, to Salmonella typhimurium biofilm

6. Comparing the in vitro bacteriostatic ability of the formula with that of other formulas

Chinese pharmacopoeia formula 1, reference (Chenxi, Wang Xiongqing, Dynasty, Guo Xiao Liang Ling, Li Yonghong. 24 Chinese medicines have in vitro antibacterial activity against Salmonella suis [ J ] Jiangsu agricultural science, 2008(05):180 + 182. +205.)

formula

2 and 3, formula 4 and 5 are derived from Chinese patent application CN104840695A (application No. 201510249215.1). A pharmaceutical composition for treating white scour of piglets and a preparation method thereof, and formula 6 (namely, the compound Baitouweng powder of the invention) studied in the subject group are prepared according to the following formula:

prescription 1 comprises radix Pulsatillae 60g, Coptidis rhizoma 30g, cortex Phellodendri 45g, and cortex Fraxini 60 g;

and prescription 2: 24g of radix sophorae flavescentis, 24g of Chinese pulsatilla root, 24g of ash bark, 18g of coptis chinensis and 24g of golden cypress;

and prescription 3: 30g of oriental wormwood, 24g of radix sophorae flavescentis, 24g of Chinese pulsatilla root, 30g of scutellaria baicalensis, 12g of akebiaquinata and 10g of ash bark;

and prescription 4: 35g of scutellaria baicalensis, 20g of cinnamon, 18g of liquorice, 32g of areca nut, 28g of coptis chinensis, 16g of angelica, 30g of Chinese pulsatilla root, 32g of golden cypress, 25g of ash bark, 22g of sargentgloryvine stem, 33g of patrinia, 24g of raw rhubarb, 18g of purslane, 20g of sophora flavescens, 14g of Chinese brake herb, 26g of groundsel, 3g of cyrtomium rhizome, 26g of indigo naturalis, 21g of suberect spatholobus stem, 30g of cockscomb and 20g of Chinese violet;

and prescription 5: 35g of scutellaria baicalensis, 20g of cinnamon, 18g of liquorice, 32g of areca nut, 28g of coptis chinensis, 16g of angelica, 30g of Chinese pulsatilla root, 32g of golden cypress, 25g of ash bark, 22g of sargentgloryvine stem, 33g of patrinia, 24g of raw rhubarb, 18g of purslane, 20g of sophora flavescens, 14g of Chinese brake herb, 26g of groundsel, 3g of cyrtomium rhizome, 26g of indigo naturalis, 21g of suberect spatholobus stem, 30g of cockscomb, 20g of Chinese violet, 17g of tsaoko amomum fruit, 22g of snakegourd fruit and 33g of honeysuckle stem;

and prescription 6: 25g of Chinese pulsatilla root, 15g of golden cypress, 20g of ash bark, 15g of baical skullcap root, 40g of weeping forsythia, 15g of lightyellow sophora root and 25g of liquoric root.

Combining the above 4 formula agents, making into anemone soup with concentration of 1.5g/ml according to the method in 1, and storing at 4 deg.C for use.

The traditional Chinese medicines are all purchased from chain company of Yangzhou Dadesheng, Daddy, China, pharmacy, China.

Remarking: prescription 4 and prescription 5, namely medicaments of viburnum sargentii, cathartic, elaeagnus pungens, chamomile dew, hosta plantaginea root, hypericum perforatum, pyrrosia lingua, coptidis duck feet and momordica cochinchinensis, are not available in the Yangzhou dade Sheng Chinese medicine shop, and are not repeated with the medicaments in the prescription in the research, so that the medicaments are not added. In addition, according to the related literature (Wu Li hong, Hu Hai Yan, Huangshi Liang, Yang De Po, fructus forsythiae and Hypericum perforatum are proved in materia Medica [ J ]. Chinese traditional medicine journal, 2002(08):55-59. Stachys, Wang Xiaoping, Baijiqing, Longkai flower and fructus forsythiae pharmacological action research of antibiosis and antivirus [ J ]. Chinese modern traditional medicine, 2013,15(11):950 and 953.), fructus forsythiae used in the prescription is a common traditional Chinese medicine, is already recorded in the pharmacopoeia China and belongs to the genus Forsythia in the family Oleaceae, and has stronger antibacterial and antiviral effects according to the modern research. Hypericum perforatum (Hypericum perforatum) is a herbaceous plant of Hypericum of Hypericaceae, has a long history of application in Europe, is the most important raw material of antidepressant botanical preparation in the international medical market, and also has antibacterial effect. Therefore, it was not added to formulations 4 and 5.

In vitro inhibition tests are respectively carried out on 3 clinically isolated goose-origin multi-drug-resistant escherichia coli by taking a wild strain JSYZ-24 (the strain with the number of 24, the multi-drug-resistant strain, the drug resistance to ceftriaxone sodium, cefotaxime, amoxicillin, mezlocillin, doxycycline and florfenicol) of the salmonella typhimurium strain and a standard strain S162. The specific method comprises the following steps: firstly, diluting a liquid medicine into 750, 500, 375, 250, 178.5, 125 and 89.25mg/ml of a LB liquid culture medium diluted by a prescription respectively, adding an equal amount of liquid medicine into each gradient, culturing overnight at 37 ℃, coating the liquid medicine of each gradient on a nutrient agar culture medium respectively by using an aseptic cotton swab, culturing for 24 hours at 37 ℃, calculating the occurrence condition of bacterial colonies, repeating the test for 3 times, taking the average value as the MIC value of each medicine, and taking the result as shown in Table 5, wherein the MIC of the prescription applied by the patent is lower than that of other prescriptions, so that the in-vitro antibacterial effect of the prescription is superior to that of other 5 prescriptions, and in addition, the traditional Chinese medicine composition used in the prescription is less than the prescriptions 4 and 5, and the composition is more optimized and simplified; compared with the Chinese patent application CN104840695A, the formula disclosed by the invention has the advantages that the components are obviously fewer, a better antibacterial effect is obtained, the formula belongs to the invention with omitted elements, and an unexpected technical effect is obtained.

In vitro bacteriostatic results for the formulations in Table 54

7. Determination of median lethal dose

The clinical isolate JSYZ-24 is used as an animal test challenge strain to establish a gosling infection model.

Determination of LD of Salmonella typhimurium strain (wild strain JSYZ-24 and standard strain S162) on 1-day-old Thai goose₅₀.3 concentrations are set, a blank group is additionally set, and 8 goslings are fed in each group. Adjusting the concentration of bacterial liquid to about 1.5 × 10⁹CFU/ml, as the highest concentration, and two concentrations were diluted down to 1.5X 10 concentrations for each of the three concentrations⁹CFU/ml，1.5×10⁸CFU/ml，1.5×10⁷CFU/ml. And (3) irrigating 1ml of bacterial liquid with corresponding concentration for each group of goslings by using an intragastric injection needle, and irrigating 1ml of normal saline for blank group of goslings. Recording the death number of each group of goslings within 7d, and calculating the LD of the two salmonella strains by an accumulative method₅₀。

Calculating the wild strain LD by Reed-muench accumulation method₅₀≈5.7×10⁷CFU, Standard Strain LD₅₀≈1.8×10⁹CFU, the virulence of the wild strain is approximately the norm30 times of the stock plant.

8. Design of animal experiments

1-day-old Taizhou geese are randomly divided into 11 groups of 10 geese, weighed and normally raised. Respectively comprises a blank control group, a toxin-counteracting infection control group, a traditional Chinese medicine prevention group, a traditional Chinese medicine treatment group, a western medicine prevention group, a western medicine treatment group and a Chinese and western medicine combined treatment group. The traditional Chinese medicine composition comprises a blank group (only water is used for gastric perfusion), a wild strain toxin-attacking group, a wild strain toxin-attacking traditional Chinese medicine prevention group (traditional Chinese medicine is attacked after 3d of gastric perfusion, namely the traditional Chinese medicine compound pulsatilla chinensis powder of the invention), a wild strain toxin-attacking traditional Chinese medicine treatment group (traditional Chinese medicine is used for gastric perfusion after attacking toxin, and 5d is continuous), a wild strain toxin-attacking western medicine prevention group (western medicine is attacked after 3d of gastric perfusion, and 5d is continuous), a wild strain toxin-attacking western medicine treatment group (western medicine is used for gastric perfusion after attacking toxin, and 5d is continuous), and a wild strain toxin-attacking traditional Chinese medicine and western medicine combination treatment group (western medicine is used for gastric perfusion after attacking toxin, and 5d is continuous); the standard strain S162 of the typhimurium (donation by Pengda Xingzhong education of veterinarian college of Yangzhou university) is used for counteracting the toxin, the standard strain is used for counteracting the toxin and is used for preventing traditional Chinese medicine (after the traditional Chinese medicine is perfused into the stomach for 3 days, the toxin is counteracted), the standard strain is used for counteracting the toxin and is used for treating traditional Chinese medicine (after the toxin is counteracted, the traditional Chinese medicine is perfused into the stomach for 5 days continuously), and the standard strain is used for counteracting the toxin and is used for treating western medicine (after the toxin is counteracted, the western medicine is perfused into the stomach for 5 days continuously).

Doxycycline is selected as a control of western medicines, the gavage starts from 1 day old in the traditional Chinese medicine prevention group and the western medicine prevention group, 1ml of doxycycline is added to each goose, the traditional Chinese medicines are 0.8g/ml, and the western medicines are 5 mg/ml. Gastrophilia irrigation and toxicity elimination at 4 days of age, according to LD₅₀As a result, the dose of the wild strain to be challenged was 5.7X 10⁷CFU, standard strain dosage 1.8 × 10⁹CFU。

The weight and blood physiological and biochemical indexes of each group of goslings are measured at 3 rd, 5 th, 7 th and 9 th days after infection, and statistical analysis is carried out. And dissecting and killing goslings at 7d and 9d after infection respectively, performing statistical analysis on organ coefficients and cecal bacteria content of each group of goslings, and comparing the prevention and treatment effects of the compound pulsatilla chinensis powder.

9. Weight change

The weights of the geese before and after challenge were statistically analyzed (table 6), and the average weight gain of the goslings in the prevention group and the treatment group was significantly higher than that in the challenge group. Wherein, the average weight gain of the Chinese/western medicine prevention group is larger than that of the Chinese/western medicine treatment group, and the average weight gain of the gosling in the Chinese/western medicine treatment group is the largest.

Body weight change before and after attacking of TABLE 64-day-old gosling

Wherein, the mark is: a standard strain; wild: a wild strain; the method comprises the following steps: traditional Chinese medicine, western: western medicines; tapping: counteracting toxic substances; prevention: prevention; treating: and (6) treating.

Note: the same column of shoulder marks contain letters with the same meaning that the difference is not significant and different meaning that the difference is significant (P <0.05)

10. Changes in leukocyte count

After the bacterial liquid is filled, 3 goslings are picked randomly in each group for jugular vein blood sampling in 3 rd, 5 th, 7 th and 9 th days, serum is separated for biochemical index analysis, and the number of leucocytes is calculated by adopting a microscope counting method.

The number of leucocytes 3, 5 and 7d in the gavage of the bacteria in the wild strain challenge group is remarkably increased compared with the prevention group and the treatment of the compound anemone powder, and the number of leucocytes 7d in the gavage of the bacteria in the standard strain challenge group is remarkably increased compared with the prevention group and the treatment of the compound anemone powder, so that the compound anemone powder can effectively prevent and treat the infection of the salmonella typhimurium (Table 7).

TABLE 74 post-challenge white blood cell count results for day-old goslings (. 10)⁹/L)

11. Variation of urea content

Urea content in blood of each group was determined at 3, 5, 7 and 9d after challenge, respectively (table 8). The content of urea after the wild strain is attacked is obviously higher than that of the blank group and the treatment group, so the damage to the kidney is serious. The traditional Chinese medicine can reduce the content of urea by preventing or treating, and the treatment result is similar to that of blank groups and western medicines. After the wild strain salmonella typhimurium is attacked, the serum urea content of each group at 9d is increased, the accumulation of urea in the body can be caused by the long duration of the kidney injury caused by bacteria, or the infection is still continuous, the treatment groups stop taking the medicine after the attacking of the wild strain salmonella typhimurium at 5d, the prevention and treatment effects are ended, the wild strain salmonella typhimurium is also increased at 9d, but the prevention group is still obviously lower than the wild strain attacking group.

Results of urea content after attacking of 84-day-old gosling

Note: the same column of shoulder marks contains lower case letters which are the same and mean no significant difference, different letters which are the same and mean significant difference (P <0.05), and upper case letters which are the same row and mean significant difference (P < 0.05).

12. Observation of pathological changes

The pathological changes were examined at 5 th, 7 th and 9 th days after infection, and the results showed that the pathological changes of the intestine and pancreas in the prevention and treatment groups of the Chinese medicine were less than those in the control group of the challenge group (fig. 2).

13. Analysis of cecal bacteria content

After pathological dissection of the gosling, 0.1g of cecum of the gosling is collected for bacterial colonization quantity analysis. The tissue was ground using an autoclaved grinding pestle and 5ml of sterile 15% glycerol PBS was added as a dilution solution. After the tissue was completely ground, 1ml of the grinding fluid was aspirated and added to 9ml of sterile 15% glycerol PBS, which was serially diluted 10-fold for 7 concentrations. And (3) sucking 10 mu l of mixed liquor with each concentration, inoculating the mixed liquor on a salmonella identification culture medium SS culture medium, culturing the mixed liquor at 37 ℃ for 24h, and counting the number of colonies with black centers. And selecting each group of black colonies for further PCR identification and serotype identification, and determining the bacteria as the challenge bacteria salmonella typhimurium.

As a result: the amount of the cecal bacteria in the wild strain and the standard strain challenge group is obviously higher than that in the blank and prevention and treatment group (P is more than 0.001 and less than 0.01), and the detail is shown in Table 9.

TABLE 9 bacterial content of cecum in each group after challenge

Note: the same column of shoulder marks containing lower case letters indicates no significant difference, the difference indicates significant difference (P <0.05),

the examples described are illustrative of the invention and are not to be construed as limiting the invention, and any variations and modifications which come within the meaning and range of equivalency of the invention are to be considered within the scope of the invention.

Claims

1. A compound Chinese pulsatilla root powder for preventing and/or treating salmonellosis of animals is characterized by comprising 2-5 parts of Chinese pulsatilla root, 2-3 parts of golden cypress, 2-4 parts of ash bark, 1-3 parts of scutellaria baicalensis, 4-8 parts of fructus forsythiae, 1-3 parts of radix sophorae flavescentis and 2-5 parts of liquorice in parts by weight;

the animal is a goose; the salmonellosis is typhimurium salmonellosis.

2. The compound pulsatilla chinensis powder for preventing and/or treating salmonellosis of animals according to claim 1, which comprises, by weight, 5 parts of pulsatilla chinensis, 3 parts of phellodendron amurense, 4 parts of cortex fraxini, 3 parts of scutellaria baicalensis, 8 parts of forsythia suspensa, 3 parts of sophora flavescens, and 5 parts of liquorice.

3. The method for preparing the compound anemonin powder as claimed in claim 1, comprising the steps of:

mixing and pulverizing 2-5 parts of Chinese pulsatilla root, 2-3 parts of golden cypress, 2-4 parts of ash bark, 1-3 parts of scutellaria baicalensis, 4-8 parts of fructus forsythiae, 1-3 parts of radix sophorae flavescentis and 2-5 parts of liquorice in parts by weight to obtain the compound Chinese pulsatilla root powder.

4. Use of the compound pulsatilla chinensis powder of claim 1 or 2 for the manufacture of a medicament for the prevention and/or treatment of salmonellosis in an animal; the animal is a goose; the salmonellosis is typhimurium salmonellosis.