Composition of pulsatilla alcohol extract and gentamicin and application of composition in inhibition of drug-resistant salmonella typhimurium

Abstract

The invention belongs to the technical field of veterinary drugs, and discloses a Chinese pulsatilla root alcohol extract and gentamicin composition and application thereof in inhibition of drug-resistant salmonella typhimurium. The pulsatilla alcohol extract composition provided by the invention comprises pulsatilla alcohol extract and gentamicin, and for salmonella typhimurium, the optimal ratio concentration of the pulsatilla alcohol extract to the gentamicin is 31.25 mg / mL: 2 [mu] g / ml; the Chinese pulsatilla root alcohol extract and the gentamicin have the synergistic bacteriostatic effect on salmonella typhimurium, the composition reduces the concentration of single bacteriostasis of the Chinese pulsatilla root alcohol extract and the gentamicin, the cost is saved, and the composition has wide application prospects and development value in the fields of food processing, medicine and breeding industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary drugs, and particularly relates to an ethanol extract of Pulsatilla chinensis and gentamicin composition and its application in inhibiting drug-resistant Salmonella typhimurium. Background Art

[0002] Salmonella typhimurium is an important zoonotic pathogen, which not only poses a threat to human health but also causes huge economic losses to the poultry industry.

[0003] Clinical trial data shows that Salmonella typhimurium isolated from different countries and regions has developed varying degrees of drug resistance. Among the Salmonella typhimurium strains isolated by Italian scholar Cucco, 61.5% of the strains were resistant to two antibiotics, and 29.5% of the strains were resistant to three antibiotics. Canadian scholar Arndt detected the drug resistance of 379 Salmonella typhimurium strains and found that most strains were resistant to tetracycline, tiamulin, and spectinomycin. Among the 1163 Salmonella typhimurium strains clinically isolated by Dutch scholar van Hout, 78.4% were resistant to tetracycline and 48.1% were resistant to clindamycin. Chinese scholars analyzed the drug resistance of 34 clinically isolated Salmonella typhimurium strains and found that 91.18% of the strains were multi-drug resistant, 82.3% - 100.0% of the strains were resistant to sulfonamides, 79.4% - 94.1% of the strains were resistant to aminoglycosides, 88.2% - 97.1% of the strains were resistant to tetracyclines, 79.4% - 85.3% of the strains were resistant to lincosamides, and 50% - 88.2% of the strains were resistant to macrolides. Therefore, finding new antibiotic alternatives, developing new antibacterial drugs, or discovering compounds that reverse antibiotic resistance are the key ways to solve the problem of antibiotic resistance.

[0004] Pulsatilla decoction (PD), first seen in Zhang Zhongjing's Treatise on Febrile Diseases, and Pulsatilla powder is still a formula included in the Chinese Veterinary Pharmacopoeia (2015 Edition), which has the functions of clearing heat and detoxifying, cooling blood and stopping dysentery. Pulsatilla powder and Pulsatilla powder are compounds, mainly containing Pulsatilla chinensis, Coptis chinensis, Phellodendron amurense, and Cortex Fraxini. These four drugs have been reported to have in vitro antibacterial effects against Salmonella in pigs. There are few reports on single Pulsatilla chinensis against Salmonella typhimurium.

[0005] The ethanol extract of Pulsatilla chinensis is mainly extracted from the stems of Pulsatilla chinensis and has biological functions such as anti-infection and anti-fungal. It is also a reducing agent that can generate free radicals, especially superoxide anions. Currently, according to literature reports, Pulsatilla powder has antibacterial effects against Salmonella typhimurium, Escherichia coli, Pseudomonas putida, Vibrio vulnificus, etc., but it is ineffective against fungi such as Fusarium oxysporum, Penicillium, and Candida albicans. In addition, the ethanol extraction process of Pulsatilla chinensis mentioned in (Gao Weiwei, Li Zhan, Lin Wenwei, Chen Dihua. Antibacterial test of Chinese herbal medicine extracts against Salmonella): Take 50 g of the crude powder of each medicinal material, add 4-6 times the amount of 90% ethanol respectively, reflux and extract for 1 h, for a total of 3 times. Combine the filtrates respectively, concentrate and recover the ethanol, and then dry in a water bath at 60 °C to obtain the respective dried extracts. By measuring the antibacterial activity of the ethanol extract of Pulsatilla chinensis against Salmonella typhimurium (C77-31) through the diameter of the inhibition zone and the minimum inhibitory concentration, it was found that the ethanol extract of Pulsatilla chinensis had no antibacterial effect on Salmonella typhimurium. Based on this speculation, the ethanol extract of Pulsatilla chinensis is not sensitive to Salmonella typhimurium, that is, the ethanol extract of Pulsatilla chinensis has no inhibitory effect on Salmonella typhimurium. And different extraction processes may lead to different main active ingredients of the drug. Studies have shown that the water extract of Pulsatilla chinensis combined with antibacterial drugs such as ceftriaxone sodium, amoxicillin, ceftazidime, cefotaxime sodium, and florfenicol has a good effect on reversing the drug resistance of drug-resistant Escherichia coli.

[0006] Gentamicin is an aminoglycoside antibiotic whose mechanism of action is to act on the 30S ribosome in bacteria, inhibit bacterial protein synthesis, and destroy the integrity of the bacterial cell membrane. Gentamicin has antibacterial effects against Escherichia coli, Klebsiella pneumoniae, Proteus, Salmonella, etc. However, in recent years, bacteria have developed serious resistance to this product, especially Escherichia coli, Salmonella, Klebsiella pneumoniae, etc. isolated clinically are highly resistant to gentamicin. Therefore, it is particularly important to find Chinese herbal medicines that can synergistically act with antibiotics. By evaluating the extraction method of single Chinese herbal medicine of Pulsatilla chinensis and its synergistic effect with antibiotics to reduce the use of antibiotics clinically, it has important guiding significance as a new type of anti-infection composition for reducing and limiting antibiotics against Salmonella typhimurium. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the problem of serious drug resistance of existing Salmonella typhimurium, and provide a composition of the ethanol extract of Pulsatilla chinensis and gentamicin.

[0008] Another object of the present invention is to provide the application of the above composition in inhibiting drug-resistant Salmonella typhimurium.

[0009] To achieve the above object, the following technical solutions are adopted:

[0010] A composition comprising an ethanol extract of Pulsatilla chinensis (Bunge) Regel and gentamicin. The preparation method of the ethanol extract of Pulsatilla chinensis (Bunge) Regel includes: pulverizing the Pulsatilla chinensis (Bunge) Regel decoction pieces, extracting with ethanol, collecting the filtrate, centrifuging the filtrate, performing rotary evaporation, and then drying to form an extract.

[0011] For the above-mentioned composition, preferably, in the preparation method of the ethanol extract of Pulsatilla chinensis (Bunge) Regel, the soaking time in the extraction step is 1 - 2 days, and the extraction is carried out 1 - 3 times;

[0012] For the above-mentioned composition, preferably, in the preparation method of the ethanol extract of Pulsatilla chinensis (Bunge) Regel, the rotary evaporation temperature is 35 - 45 °C;

[0013] For the above-mentioned composition, preferably, in the preparation method of the ethanol extract of Pulsatilla chinensis (Bunge) Regel, the alcohol concentration is 75%;

[0014] For the above-mentioned composition, preferably, in the preparation method of the ethanol extract of Pulsatilla chinensis (Bunge) Regel, the drying temperature is 50 - 60 °C.

[0015] For the above-mentioned composition, preferably, the mass ratio of the ethanol extract of Pulsatilla chinensis (Bunge) Regel to gentamicin is 15625:1.

[0016] Use of the above composition in the preparation of a drug for treating or preventing drug-resistant Salmonella typhimurium infection;

[0017] Use of the above composition in the preparation of a drug for inhibiting the growth of drug-resistant Salmonella typhimurium.

[0018] For the above-mentioned use, the drug-resistant Salmonella typhimurium has drug-resistant phenotypes such as co-trimoxazole, tetracycline, ceftriaxone, and / or chloramphenicol.

[0019] Compared with the prior art, the present invention has the following advantages and effects:

[0020] The present invention firstly provides that the ethanol extract of Pulsatilla chinensis (Bunge) Regel and gentamicin have a synergistic antibacterial effect, and this composition can be used against drug-resistant Salmonella typhimurium. The present invention has confirmed through various methods that the composition of the ethanol extract of Pulsatilla chinensis (Bunge) Regel and gentamicin has a good antibacterial effect on Salmonella typhimurium, laying a theoretical foundation for the development of new drugs for treating drug-resistant Salmonella typhimurium disease, and even providing candidate substances for the discovery of antibiotic alternatives, and having good application prospects in the prevention and treatment of drug-resistant Salmonella typhimurium disease. Detailed implementation manners

[0021] The following further illustrates the present invention in combination with specific examples of the specification, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0022] Unless otherwise specified, the reagents and materials used in the following examples are commercially available. In the examples of the present invention, drug-resistant Salmonella typhimurium Sty17 (purchased from Wuhan Fengchuang Biotechnology Co., Ltd.) and Salmonella typhimurium ATCC14028 strain are taken as examples for illustration. The compositions of the present invention also have inhibitory or killing effects on other drug-resistant Salmonella typhimurium.

[0023] Statistical analysis of the following examples of the present invention: All experiments were independently repeated at least 3 times.

[0024] Example 1:

[0025] Selection of the extraction process of Pulsatilla chinensis and its in vitro antibacterial effect against 5 kinds of bacteria in poultry:

[0026] 1. Prepare Pulsatilla chinensis extracts with a final concentration of 2 g / ml of the liquid medicine according to the following two methods

[0027] (1) Alcohol extraction method to prepare Pulsatilla chinensis liquid medicine with a concentration of 2 g / ml of the liquid medicine

[0028] 50 g of Pulsatilla chinensis cut pieces, passed through a 50-mesh sieve, added 10 times the volume of 75% ethanol of the Pulsatilla chinensis cut pieces, soaked for 1 day, collected the filtrate, repeated 2 times, combined the filtrate, centrifuged at 4000 r / min for 15 min, concentrated by rotary evaporation at 40 °C to 2 g / ml, dried in an oven at 56 °C to form an extract, and then dissolved it with sterilized distilled water to make its final concentration reach 2 g / ml, sterilized by passing through a 0.22 μm filter membrane, and stored at 4 °C. In the present invention, it is necessary to make an extract and then dissolve it to 2 g / ml, rather than directly using the alcohol extract solution obtained by rotary evaporation.

[0029] (2) Water extraction method to prepare Pulsatilla chinensis liquid medicine with a concentration of 2 g / ml of the liquid medicine

[0030] 50 g of Pulsatilla chinensis cut pieces were crushed, passed through a 50-mesh sieve, added 5 times the volume of sterilized distilled water of the Pulsatilla chinensis cut pieces, 0.5% cellulase, 0.1% protease, and enzymatically hydrolyzed and extracted at a constant temperature of 38 °C for 3 h; heated to 80 °C, added distilled water to 350 mL, continued to extract for 1 h, filtered with filter paper, and collected the filtrate; centrifuged at 4000 r / min for 15 min, centrifuged 2 times, passed through filter paper, concentrated by rotary evaporation at 65 °C to 2 g / ml, sterilized by passing through a 0.22 μm filter membrane, and stored at 4 °C.

[0031] 2. Determination of the antibacterial spectrum of Pulsatilla chinensis extract against bacteria:

[0032] 2.1 Materials

[0033] Luria-Bertani (LB) broth (purchased from Haibo Biotech Co., Ltd.), Tryptic Soy Broth (TSB) broth medium (purchased from Haibo Biotech Co., Ltd.), fetal bovine serum (purchased from Tianhang Biotech Co., Ltd.) were used for the cultivation of bacteria; Mueller-Hinton (MH) broth (purchased from BD) was used for the drug sensitivity test to detect the antibacterial efficacy of drugs; Salmonella typhimurium ATCC14028, Escherichia coli ATCC25922, and Pasteurella multocida C48-1 were sourced from the China Institute for Veterinary Drug Control, and Riemerella anatipestifer and Staphylococcus aureus were strains isolated and preserved in the laboratory, which were used to evaluate the in vitro antibacterial effect of Pulsatilla chinensis extract against 5 kinds of bacteria in poultry.

[0034] 2.2 Test methods

[0035] Salmonella typhimurium ATCC14028, Escherichia coli ATCC25922, Staphylococcus aureus, etc. preserved in the laboratory in the early stage were cultured with LB liquid medium at a volume ratio of 1:100, while Pasteurella multocida C48-1, Riemerella anatipestifer, etc. were resuscitated and cultured with TSB liquid medium containing 10% fetal bovine serum. After overnight shaking culture at 37°C and 220 rpm, the bacteria after overnight culture were transferred at a ratio of 1:1000 and shaken cultured in the above culture method until the OD 600 was approximately 0.6. Finally, the above 5 kinds of bacteria were prepared into a bacterial suspension with a concentration of 0.5 McFarland turbidity standard by the McFarland turbidity method;

[0036] The solution of Pulsatilla chinensis extract with different concentrations after serial dilution with MH liquid medium was added to a sterile 96-well polystyrene plate. The first to the tenth wells were added with the ethanol extract solution of Pulsatilla chinensis, 50 μl per well. The eleventh well was added with 50 μl of MH liquid medium as the growth control, and the twelfth well was only added with 100 μl of MH liquid medium as the negative control.

[0037] After diluting the 0.5 McFarland turbidity standard bacterial suspension 1:1000 with MH broth, 50 μl of the bacterial suspension was added to the first to the eleventh wells, and incubated at 37°C for 16 - 20 h. Observe the growth of bacteria and judge the MIC value.

[0038] 3. Results

[0039] As can be seen from Table 1, when the concentration of the water extract of Pulsatilla chinensis is 500 mg / ml, it has an inhibitory effect on the growth of Escherichia coli ATCC25922 and Salmonella typhimurium ATCC14028. When the concentration of the alcohol extract of Pulsatilla chinensis is 250 mg / ml, it has an inhibitory effect on the growth of Escherichia coli ATCC25922. However, when the concentration of the alcohol extract of Pulsatilla chinensis is 125 mg / ml, it has an inhibitory effect on the growth of Salmonella typhimurium. The results indicate that the alcohol extract of Pulsatilla chinensis has a better effect on Salmonella typhimurium and Escherichia coli than the water extract of Pulsatilla chinensis.

[0040] Table 1 Determination of the minimum inhibitory concentration of two Pulsatilla chinensis extracts against different bacteria in poultry

[0041]

[0042]

[0043] Example 2:

[0044] Determination of the minimum inhibitory concentration and minimum bactericidal concentration of the alcohol extract of Pulsatilla chinensis against drug-resistant Salmonella typhimurium Sty17 and non-drug-resistant Salmonella typhimurium ATCC14028

[0045] 1. Materials

[0046] Mueller-Hinton (MH) broth (purchased from BD Company), used for drug susceptibility testing to detect the bacteriostatic efficacy of drugs. Salmonella typhimurium ATCC14028 is a strain purchased from the National Institutes for Food and Drug Control. Salmonella typhimurium Sty17 strain (purchased from Wuhan Fengchuang Biotechnology Co., Ltd.) has drug-resistant phenotypes such as compound sulfamethoxazole, tetracycline, ceftriaxone, and chloramphenicol, and its other culture characteristics are the same as those of ATCC14028, and is used to evaluate the in vitro effect of the alcohol extract of Pulsatilla chinensis on drug-resistant Salmonella typhimurium.

[0047] 2. Test methods

[0048] According to the method in Example 1, Salmonella typhimurium ATCC14028 and drug-resistant Salmonella typhimurium Sty17 were cultured to an OD 600 of about 0.6, and a bacterial suspension with a concentration of 0.5 McFarland standard turbidity was prepared by the McFarland turbidity method;

[0049] Diluted serially with MH liquid medium, different concentrations of the alcohol extract solution of Pulsatilla chinensis were added to sterile 96-well polystyrene plates. Gentamicin solution, 50 μl per well, was added to wells 1 to 10. 50 μl of MH liquid medium was added to well 11 as a growth control, and 100 μl of only MH liquid medium was added to well 12 as a negative control.

[0050] Add 50 μl of the standard bacterial suspension to wells 1 to 11, incubate at 37 °C for 16 - 20 h, observe the growth of bacteria, and determine the MIC value. Aspirate the bacterial solution from each well and spread it on an MH plate. If no colonies grow, it is the MBC value.

[0051] 3. Results

[0052] As can be seen from Table 2, when the concentration of the ethanol extract of Pulsatilla chinensis is 125 mg / ml, it has an inhibitory effect on the growth of drug-resistant Salmonella typhimurium Sty17; when the concentration of the ethanol extract of Pulsatilla chinensis is 250 mg / ml, it has a bactericidal effect on drug-resistant Salmonella typhimurium Sty17. When the concentration of the ethanol extract of Pulsatilla chinensis is 125 mg / ml, it has an inhibitory effect on the growth of Salmonella typhimurium ATCC14028; when the concentration of the ethanol extract of Pulsatilla chinensis is 500 mg / ml, it has a bactericidal effect on Salmonella typhimurium ATCC14028.

[0053] Table 2 Minimum inhibitory concentration and minimum bactericidal concentration of the ethanol extract of Pulsatilla chinensis against Salmonella typhimurium

[0054]

[0055] Example 3:

[0056] Minimum inhibitory concentration of various antibiotics against drug-resistant Salmonella typhimurium Sty17 and Salmonella typhimurium ATCC14028 1. Materials

[0057] Mueller-Hinton (MH) broth (purchased from BD), drugs such as ampicillin, cefotaxime, tylosin, gentamicin, doxycycline, enrofloxacin (purchased from Solarbio), used for drug susceptibility testing to detect the antibacterial efficacy of drugs.

[0058] 2. Test method

[0059] Culture Salmonella typhimurium ATCC14028 and drug-resistant Salmonella typhimurium Sty17 to an OD 600 of approximately 0.6 according to the method in Example 1, and prepare a standard bacterial suspension with a concentration of 0.5 McFarland turbidity standard using the McFarland turbidity method;

[0060] Dilute ampicillin, cefotaxime, tylosin, gentamicin, doxycycline, enrofloxacin, etc. to a final concentration of 1 mg / mL with MH liquid medium by serial dilution and add them to a sterile 96-well polystyrene plate. Add 50 μl of the antibiotic solution to wells 1 to 10, add 50 μl of MH liquid medium to well 11 as a growth control, and add only 100 μl of MH liquid medium to well 12 as a negative control.

[0061] Add 50 μl of the standard bacterial suspension to wells 1 to 11, incubate at 37 °C for 16 - 20 h, observe the growth of bacteria, and determine the MIC value.

[0062] 3. Results

[0063] As can be seen from Table 3, when the concentration of ampicillin is 128 μg / ml, it has an inhibitory effect on the growth of drug-resistant Salmonella typhimurium Sty17; when the concentration of spectinomycin is 256 μg / ml, it has an inhibitory effect on the growth of drug-resistant Salmonella typhimurium Sty17; when the concentration of gentamicin is 16 μg / ml, it has an inhibitory effect on the growth of drug-resistant Salmonella typhimurium Sty17; when the concentration of cefoxitin is 8 μg / ml, it has an inhibitory effect on the growth of drug-resistant Salmonella typhimurium Sty17; when the concentrations of enrofloxacin, doxycycline, and ceftriaxone are 512 μg / ml, they have an inhibitory effect on the growth of drug-resistant Salmonella typhimurium Sty17. And Salmonella typhimurium ATCC14028 has an antibacterial effect against ampicillin, gentamicin, cefoxitin, and ceftriaxone with an MIC value of 16 μg / ml.

[0064] Table 3 Minimum inhibitory concentrations of various antibiotics against Salmonella typhimurium

[0065]

[0066]

[0067] Example 4:

[0068] The ethanol extract of Pulsatilla chinensis and gentamicin have a synergistic antibacterial effect against drug-resistant Salmonella typhimurium Sty17

[0069] 1. Materials

[0070] Mueller-Hinton (MH) broth (purchased from BD Company), used for drug sensitivity tests to detect the antibacterial efficacy of drugs.

[0071] 2. Test method

[0072] Culture drug-resistant Salmonella typhimurium Sty17 or Salmonella typhimurium ATCC14028 to an OD 600 of approximately 0.6 according to the method in Example 1, and prepare a 0.5 McFarland standard bacterial suspension by the McFarland turbidity method;

[0073] In each well from the 2nd to the 10th in each row of a sterile 96-well polystyrene plate, add 50 μl of MH liquid medium using a multichannel pipette. In the 1st and 2nd wells, add the ethanol extract solution of Pulsatilla chinensis at 4 times the MIC concentration. After pipetting and mixing well, aspirate 50 μl from the second well and add it to the third well, and so on, pipetting and mixing for serial dilution. Aspirate 50 μl from the 11th well and discard it. Add 50 μl of MH liquid medium to the 12th well as the growth control, with the concentration of the ethanol extract of Pulsatilla chinensis being the same in each column.

[0074] According to the MIC values of gentamicin for different strains, dilute gentamicin solutions at different concentrations in sterile 4-ml EP tubes using MH liquid medium, with the maximum concentration being 4 times the MIC concentration. The concentration in the second row is half of that in the first row, and so on. Calculate the concentrations of gentamicin added in 8 rows and prepare the corresponding solutions. Subsequently, add 50 μl of the solution to the 96-well polystyrene plate, with the antibiotic concentration being the same in each row.

[0075] At this time, each well in the 96-well polystyrene plate contains gentamicin and the ethanol extract solution of Pulsatilla chinensis at different concentrations. The gentamicin concentration is the same in each row of the solution, and the concentration decreases serially from the 1st to the 8th row; the concentration of the ethanol extract solution of Pulsatilla chinensis is the same in each column of the solution, and the concentration decreases serially from the 1st to the 10th column.

[0076] Add 50 μl of the standard bacterial suspension to each well from the 1st to the 10th and the 12th in each row. After adding, add 50 μl or 100 μl of MH medium to each well in the 96-well plate to make the total volume of the solution in each well 200 μl. The 11th well in each row is the negative control, containing only the ethanol extract of Pulsatilla chinensis and the gentamicin solution; the 12th well is the growth control, containing only the standard bacterial solution.

[0077] Incubate the 96-well polystyrene plate at 37 °C for 16 - 20 h, observe the growth of bacteria, and calculate the combined antibacterial index FICI according to the minimum combined inhibitory concentration of the antibiotic and the compound.

[0078] FICI calculation formula: FICI = MIC of drug A in combination / MIC of drug A alone + MIC of drug B in combination / MIC of drug B alone.

[0079] The determination method is as follows: Synergistic antibacterial effect, FICI ≤ 0.5; additive effect, 0.5 < FICI ≤ 1; no relevant effect, 1 < FICI ≤ 2; antagonistic effect, FICI > 2.

[0080] The experimental operation steps for ampicillin and cefoxitin are the same.

[0081] 3. Results

[0082] As can be seen from Table 4, when the ethanol extract of Pulsatilla chinensis is used in combination with gentamicin, the concentration of the ethanol extract of Pulsatilla chinensis is 31250 μg / ml, the concentration of gentamicin is 2 μg / ml, and the fractional inhibitory concentration index is 0.375. The two have a synergistic inhibitory effect on the growth of drug-resistant Salmonella typhimurium Sty17. However, the FICI of cefoxitin and ampicillin in combination with the ethanol extract of Pulsatilla chinensis is 1.5, and there is no synergistic effect between the two.

[0083] As can be seen from Table 5, when the ethanol extract of Pulsatilla chinensis is used in combination with gentamicin, cefoxitin and ampicillin respectively, the FICI = 2, indicating that there is no synergistic effect between the two on Salmonella typhimurium ATCC14028.

[0084] The above results show that the combination of the ethanol extract of Pulsatilla chinensis and gentamicin can reverse the resistance of drug-resistant Salmonella typhimurium, but has no synergistic antibacterial effect on non-drug-resistant Salmonella typhimurium ATCC14028.

[0085] Table 4 Minimum inhibitory concentration and fractional inhibitory concentration index of the ethanol extract of Pulsatilla chinensis in combination against drug-resistant Salmonella typhimurium Sty17

[0086]

[0087] Table 5 Minimum inhibitory concentration and fractional inhibitory concentration index of the ethanol extract of Pulsatilla chinensis in combination against Salmonella typhimurium ATCC14028

[0088]

[0089] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A composition, the composition comprising an ethanol extract of Pulsatilla chinensis and gentamicin, and the preparation method of the ethanol extract of Pulsatilla chinensis comprising: The Pulsatilla chinensis decoction pieces are crushed and extracted with ethanol, the filtrate is collected, centrifuged, and then rotary evaporated, and then dried to make an extract.

2. The composition according to claim 1, characterized in that, In the preparation method of the ethanol extract of Pulsatilla chinensis, the soaking time in the extraction step is 1-2 days, and the extraction is carried out 1-3 times.

3. The composition according to claim 1, wherein In the preparation method of the ethanol extract of Pulsatilla chinensis, the rotary evaporation temperature is 35-45 °C.

4. The composition according to claim 1, characterized in that, In the preparation method of the ethanol extract of Pulsatilla chinensis, the alcohol concentration is 75%.

5. The composition according to claim 1, wherein In the preparation method of the ethanol extract of Pulsatilla chinensis, the drying temperature is 50-60 °C.

6. The composition according to claim 1, characterized in that, The mass ratio of the ethanol extract of Pulsatilla chinensis to gentamicin is 15625:

1.

7. Use of the composition according to claim 1 in the preparation of a medicament for treating or preventing drug-resistant Salmonella typhimurium infection.

8. Use of the composition according to claim 1 in the preparation of a medicament for inhibiting the growth of drug-resistant Salmonella typhimurium.

9. According to the use of claim 7 or 8, the drug-resistant Salmonella typhimurium is resistant to co-trimoxazole, tetracycline, ceftriaxone and / or chloramphenicol.

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