Application of combination of licochalcone A and antibiotics in preparation of medicine for resisting clostridium perfringens

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of combination of licochalcone A and antibiotics in preparation of drugs for resisting clostridium perfringens. According to the invention, licochalcone A (LCA) is taken as a representative drug, the bacteriostatic action of the licochalcone A on clostridium perfringens when the licochalcone A is combined with various antibiotics is explored through a drug sensitive test, a sterilization curve, a hemolysis test, a sliding test and an infected mouse treatment test, and a clostridium perfringens resisting formula taking the LCA and the antibiotics (tilmicosin or tetracycline) as main drugs is provided. According to the formula, the inhibition effect on the clostridium perfringens can be remarkably enhanced, the hemolysis of the clostridium perfringens is inhibited, the inflammatory response level caused by the clostridium perfringens is reduced, and the damage of the clostridium perfringens to muscle tissues is reduced. Therefore, when the clostridium perfringens infection is treated, the tilmicosin or tetracycline is combined with the LCA to act, so that the dosage of the medicine can be effectively reduced, and the treatment effect is improved.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to application of licorice chalcone A combined with antibiotics in the preparation of drugs against Clostridium perfringens. Background Art

[0002] Clostridium perfringens (CP) is a Gram-positive anaerobic pathogen that can cause a variety of livestock and poultry diseases, including necrotic enteritis in chickens and gas gangrene in sheep. As a foodborne zoonosis pathogen, Clostridium perfringens causes a variety of intestinal diseases, posing a serious threat to human health and public health. At present, antibiotics still play a great role in the treatment of Clostridium perfringens infection. However, due to various factors such as the abuse and irrational use of antibiotics, the drug resistance of Clostridium perfringens has increased.

[0003] As a treasure of Chinese traditional culture and a key research object in the field of new drug research, Chinese medicine has become a current research hotspot with its advantages of not easily inducing drug resistance, low cost, less residue, and less toxic and side effects. It also has a certain effect on the treatment of various diseases. The grass in the eight bowls of Chinese medicine "Shenling Shucao, Chuanxiong, Gui, Dishao" is licorice, which is a commonly used and important Chinese medicinal material with a wide range of applications. There is a saying that "nine out of ten herbs are grass". Licorice Chalcone A (LCA) is a new type of flavonoid substance isolated from licorice. The content in licorice is much higher than other ingredients. It has multiple pharmacological activities such as anticancer, antibacterial, anti-inflammatory, antioxidant, hypoglycemic, liver protection, antiparasitic, and obesity reduction. Among them, the antibacterial effect is an important pharmacological effect that is significantly underestimated. However, at present, there is no technology for the combination of licorice chalcone A and antibiotics. Therefore, based on the antibacterial effect of licorice chalcone A, it is very necessary to develop new drugs against Clostridium perfringens in combination with antibiotics. Summary of the invention

[0004] The purpose of the present invention is to provide an application of licochalcone A combined with antibiotics in the preparation of drugs against Clostridium perfringens to solve the problems of the above-mentioned prior art. The present invention produces a synergistic effect by combining licochalcone A with tetracycline or tilmicosin, and greatly reduces the amount of antibiotics used.

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

[0006] The invention provides the use of licorice chalcone A combined with antibiotics in the preparation of a drug for resisting Clostridium perfringens, wherein the antibiotic is tetracycline or tilmicosin.

[0007] Preferably, the drug comprises a veterinary drug.

[0008] Preferably, the drug achieves the effect of resisting Clostridium perfringens infection by resisting gas gangrene caused by Clostridium perfringens infection, reducing inflammatory response, tissue damage and hemolytic damage caused by Clostridium perfringens infection.

[0009] The invention provides a drug for resisting Clostridium perfringens. The effective components of the drug are licorice chalcone A and an antibiotic; the antibiotic is tetracycline or tilmicosin.

[0010] Preferably, the concentration ratio of the licorice chalcone A to the antibiotic is (1-2):1.

[0011] Preferably, the drug comprises a veterinary drug.

[0012] Preferably, the drug achieves the effect of resisting Clostridium perfringens infection by resisting gas gangrene caused by Clostridium perfringens infection, reducing inflammatory response, tissue damage and hemolytic damage caused by Clostridium perfringens infection.

[0013] The invention provides the use of licorice chalcone A combined with antibiotics in preparing a medicine for treating gas gangrene, wherein the antibiotic is tetracycline or tilmicosin.

[0014] The invention provides a medicine for treating gas gangrene. The effective ingredients of the medicine are licorice chalcone A and antibiotics; the antibiotics are tetracycline or tilmicosin.

[0015] Preferably, the concentration ratio of the licorice chalcone A to the antibiotic is (1-2):1.

[0016] The present invention discloses the following technical effects:

[0017] Due to the risk of infection by Gram-positive pathogen Clostridium perfringens in livestock and poultry production, the present invention uses licorice chalcone A (LCA) as a representative drug, and explores its antibacterial effect on Clostridium perfringens in combination with multiple antibiotics through drug sensitivity tests, bactericidal curves, hemolysis tests, sliding tests, and infected mouse treatment tests, and proposes an anti-Clostridium perfringens formula with LCA and antibiotics (tilmicosin or tetracycline) as the main drugs. The experimental results of the specific embodiments of the present invention show that the three antibiotics, clindamycin (CLDM), tetracycline (TCN), and tilmicosin (TMS), have a synergistic effect with LCA; the bactericidal curve results show that 8μg·mL -1 LCA and 16 μg mL -1 TMS can almost completely kill Clostridium perfringens; 2 μg·mL -1 LCA and 2 μg·mL -1The combined use of TMS can significantly reduce the hemolysis of bacteria, and hemolysis is almost completely inhibited (the quantitative hemolysis is 9.08%); it is worth noting that LCA has a certain promoting effect on the migration of bacteria, can improve the motility mediated by pili, and its promoting effect is significantly improved after being used in combination with TMS; the results of in vivo animal experiments show that TCN and TMS show synergistic effects when used in combination with LCA (FIC index is 0.375), and also show good synergistic effects during treatment. It can be seen that the present invention provides an anti-clostridium perfringens formula with licorice chalcone A and antibiotics (tilmicosin or tetracycline) as main drugs, which can significantly enhance the inhibitory effect on clostridium perfringens, inhibit the hemolysis of clostridium perfringens, reduce the level of inflammatory response caused by clostridium perfringens, and reduce the damage of clostridium perfringens to muscle tissue. Therefore, when treating clostridium perfringens infection, combining TMS or TCN with LCA can effectively reduce the amount of drug used and improve the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the bactericidal curve of Clostridium perfringens CVCC2030 under the action of licorice chalcone A;

[0020] Figure 2 This is the bactericidal curve of Clostridium perfringens CVCC2030 under the action of tilmicosin;

[0021] Figure 3 This is the bactericidal curve of Clostridium perfringens CVCC2030 under the combined action of Licorice Chalcone A and Tilmicosin;

[0022] Figure 4 The results of the sliding test; A is the blank control group; B is the concentration of LCA in the drug-containing medium is 1 μg·mL -1 ; C is the concentration of TMS in the drug-containing medium, 1 μg·mL -1 ; D is the concentration of LCA in the drug-containing medium, which is 0.5 μg·mL -1 , the concentration of TMS was 0.5 μg mL -1 ;

[0023] Figure 5are the results of pathological tissue observation; a is group A (negative control group); b and c are group B (positive control group); d is group C2-1; e is group C2-2; f is group C2-3; g is group C4-1; h is group C4-2; i is group C4-3; j is group C3-1; k is group C3-2; l is group C3-3; m is group C1-1; n is group C1-3;

[0024] Figure 6 These are the results of HE staining of partial processing of the right hind limb muscle of mice (200×); among them, A is group A (negative control group), which was not infected; B is group B (positive control group), which was not treated after infection; C is group C1-1; D is group C3-1; E is group C3-2; F is group C3-3; G is group C2-1; H is group C2-2; I is group C2-3; J is group C4-1; K is group C4-2; L is group C4-3. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The following examples use the Clostridium perfringens CVCC strain stored in the Veterinary Drug Residue Reference Laboratory of Huazhong Agricultural University.

[0031] Licorice chalcone A was a standard product with a purity greater than 98%, batch number A19GB145817, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0032] PCR primers: cpa.F: 5'-GTTGATAGCGCAGGACATGTTAAG-3', SEQ ID NO.1; cpa.R: 5'-CATGTAGTCATCTGTTCCAGCATC-3', SEQ ID NO.2.

[0033] Example 1

[0034] 1. Drug sensitivity test

[0035] Preparation of bacterial solution: The standard strain of Clostridium perfringens type A (CVCC2030) was provided by the National Veterinary Drug Residue Standard Laboratory of Huazhong Agricultural University. The frozen bacterial solution of glycerol was streaked on a fortified Brucella agar plate and incubated at 37°C under anaerobic conditions (80% N 2 , 10% CO 2 , 10% H 2 ) for 18 h, pick a single colony and inoculate it in FTG broth and culture it under the same conditions as above for 10 h, dip the bacterial solution and streak it on a reinforced Brucella agar plate (purchased from Qingdao Haibo, item number HB0315) and culture it for 24 h, scrape the colony and adjust it to 0.5 McFarland turbidimetric plate with sterile PBS (the number of viable bacteria is about 1×10 8 CFU / mL) to prepare bacterial suspension.

[0036] Antibiotic preparation: The broth microdilution method was used to detect the sensitivity of 20 antibiotics (erythromycin, penicillin, clindamycin, ceftiofur, oxacillin, doxycycline, florfenicol, tiamulin, tilmicosin, gentamicin, linezolid, enramycin, quinocin, enrofloxacin, kitasamycin, nosiheptide, cefoxitin, azithromycin, tetracycline and ofloxacin) and two traditional Chinese medicine monomers (berberine (BBR) and licorice chalcone A (LCA)) at different concentrations (concentrations are shown in Tables 1 and 2) to the standard strain of Clostridium perfringens CVCC2030, and the MIC values ​​of the antibiotics acting alone on Clostridium perfringens were measured.

[0037] In addition, the chessboard method was used to conduct a combined drug sensitivity test on the 20 antibiotics and the two Chinese medicine monomers. The concentrations of the antibiotics and Chinese medicine monomers are shown in Tables 1 and 2, and the FIC values ​​of their combined effects were measured.

[0038] FIC index calculation: FIC index = FIC when drug A is used in combination / MIC when drug A is used alone + FIC when drug B is used in combination / MIC when drug B is used alone. When FIC index ≤ 0.5, it indicates synergistic effect; when 0.5 < FIC index ≤ 1, it indicates additive effect; when 1 < FIC index ≤ 2, it indicates irrelevant effect; when FIC index > 2, it indicates antagonistic effect. Select the combination with synergistic effect for subsequent experiments. The results are shown in Tables 1 and 2.

[0039] Table 1 LCA drug sensitivity test and combined drug sensitivity results

[0040]

[0041]

[0042] Note: MIC LCA(-) :MIC value of Clostridium perfringens against LCA; FIC LCA(+) : MIC value of Clostridium perfringens against LCA in combined action; MIC Anti(-) :MIC value of Clostridium perfringens to antibiotics; FIC Anti(+) :MIC values ​​of Clostridium perfringens against antibiotics when acting in combination.

[0043] Table 2 BBR drug sensitivity test and combined drug sensitivity results

[0044]

[0045] Note: MIC BBR(-) :MIC value of Clostridium perfringens against BBR; FIC BBR(+) : MIC value of Clostridium perfringens against BBR in combined action; MIC Anti(-) :MIC value of Clostridium perfringens to antibiotics; FIC Anti(+) :MIC values ​​of Clostridium perfringens against antibiotics when acting in combination.

[0046] As shown in Tables 1 and 2, the synergistic effect of LCA combined with various antibiotics is generally better than that of BBR; among the antibiotics that act with LCA, clindamycin, tilmicosin and tetracycline have the lowest FIC index and the best synergistic effect with LCA.

[0047] 2. Growth curve drawing

[0048] After activation of the standard strain of Clostridium perfringens type A, a single colony was picked and inoculated into 100 mL of FTG broth medium and cultured anaerobically at 37°C overnight. The overnight cultured bacterial solution was diluted 100 times using FTG broth medium and divided into 10 groups, with 3 parallels in each group. The drug concentrations of each group are shown in Table 3. The culture was anaerobically cultured at 37°C. 100 μL of bacterial solution was sampled from each tube every 2 hours within the next 24 hours, diluted and spread on a reinforced Buchner culture dish, and counted after 24 hours of culture to draw the sterilization curve of Clostridium perfringens under the action of different drug types and concentrations. The results are shown in Figure 1-Figure 3 shown.

[0049] Table 3 Sterilization curve grouping settings

[0050] Grouping <![CDATA[Licorice chalcone A concentration (LCA) (μg·mL -1 )]]> <![CDATA[Tilmicosin (TMS) (μg·mL -1 )]]> Control 0 0 LCA8 8 0 LCA4 4 0 LCA2 2 0 TMS16 0 16 TMS8 0 8 TMS4 0 4 C4 4 4 C2 2 2 C1 1 1

[0051] Depend on Figure 1 and Figure 2 It can be seen that LCA has no obvious inhibitory effect on bacterial growth, while TMS has a stronger inhibitory effect on Clostridium perfringens, with its growth significantly inhibited and bacterial activity significantly reduced. TMS at a concentration of 2×MIC can kill bacteria to the detection line (10 CFU·mL) within 14 h. -1 ), 4×MIC concentration of TMS can kill bacteria to below the detection line within 12 hours, and there is no recovery of growth in the later period. The higher the concentration, the faster the sterilization speed and the shorter the time required, indicating that the sterilization effect of TMS on Clostridium perfringens CVCC2030 under in vitro conditions is concentration-dependent. Figure 1 and Figure 2 The results and Figure 3 The results showed that LCA and TMS at the same concentration only inhibited the growth of Clostridium perfringens and could not kill it completely, but when they acted together, the bacterial growth stopped and almost all of them died. When the drugs acted together at concentrations lower than the MIC, they had a certain inhibitory effect on the growth of bacteria, but the growth resumed after 8 hours.

[0052] 3. Hemolysis test

[0053] After washing the sheep blood with PBS, 1 mL of the precipitate was taken and diluted with PBS to a 2% volume of red blood cell suspension, and the diluted red blood cell suspension was stored at 4°C. The A-type Clostridium perfringens standard strain bacterial solution after overnight culture was diluted to 0.5 MCF with sterile PBS, and 11 groups were set up, group A was a drug-free positive control, group B was a PBS sterile negative control, and the experimental group was added with bacterial solution and different concentrations of drugs (as shown in Table 4), incubated in warm water at 37°C for 30 minutes, added with 2 mL of red blood cell suspension, incubated in warm water again for 30 minutes, and then taken out, centrifuged at 10000 rpm for 2 minutes, and the supernatant was taken to measure its OD 532 . Calculate the hemolysis quantity according to the formula: Hemolysis quantity (%) = (sample OD532 -Negative OD 532 ) / (positive OD 532 -Negative OD 532 )×100%, the drug-free group A was set as complete hemolysis, the hemolysis quantification was 100%, and the sterile group B was set as complete non-hemolysis, the hemolysis quantification was 0%; the results are shown in Table 5.

[0054] Table 4 Hemolysis test system preparation

[0055] Grouping 0.5Mc bacterial solution PBS LCA TMS Red blood cell suspension A 0.9mL 0.1mL 0 0 2mL B 0 1mL 0 0 2mL C 0.9mL 0 40μg / mL C A0.1mL 0 2mL D 0.9mL 0 20μg / mL C A0.1mL 0 2mL E 0.9mL 0 10μg / mL C A0.1mL 0 2mL F 0.9mL 0 0 40μg / mL TMS0.1mL 2mL G 0.9mL 0 0 40μg / mL TMS0.1mL 2mL H 0.9mL 0 0 40μg / mL TMS0.1mL 2mL I 0.9mL 0 40μg / mL LCA0.05mL 40μg / mL TMS0.05mL 2mL J 0.9mL 0 20μg / mL LCA0.05mL 20μg / mL TMS0.05mL 2mL K 0.9mL 0 10μg / mL LCA0.05mL 10μg / mL TMS0.05mL 2mL

[0056] Table 5 Hemolysis test results

[0057] Grouping Hemolysis quantification (%) A 100 B 0 C 14.97 D 75.03 E 91.96 F 7.65 G 42.42 H 91.90 I 9.08 J 56.80 K 92.09

[0058] As shown in Table 5, LCA and TMS both have an inhibitory effect on the hemolysis of Clostridium perfringens. The hemolysis of the standard strain of Clostridium perfringens type A is significantly reduced. The inhibitory effect is more significant after combined treatment. Compared with group D and group H, the hemolysis quantification of group I is significantly reduced, and the synergistic effect is obvious. Moreover, after different concentrations of drugs act on the standard strain of Clostridium perfringens type A, the hemolysis quantification has an obvious concentration gradient, and the hemolysis inhibition is concentration-dependent. The high concentration of drugs has a stronger inhibitory effect on bacterial hemolysis.

[0059] 4. Bacterial migration force - sliding test

[0060] Add different concentrations of LCA and TMS to BHI agar medium, mix well to prepare drug-containing culture dishes, and punch holes evenly with a puncher. Cultivate the standard strain of Clostridium perfringens type A to 1×10 8 CFU·mL -1 5 μL of bacterial liquid was dropped into the prepared drug-containing culture dish, and cultured anaerobically at 37°C for 72 hours. The size and shape of the bacterial lawn formed on the plate were observed. At the same time, the BHI agar medium without any treatment was used as the blank control group. The changes in their migration distance under the influence of drugs were compared.

[0061] The results are as follows Figure 4 As shown, both LCA and TMS have an inhibitory effect on the growth of Clostridium perfringens. However, in the research results of this example, it was found that when a low dose of LCA was used alone, the average diameter of the bacterial lawn was slightly increased compared with the blank control group, and there was no visible bacterial lawn growth at high concentrations; when TMS was used alone, the inhibitory effect on bacterial migration increased with increasing concentration, and there was no enhancing effect on the motility of Clostridium perfringens; when LCA and TMS were used in combination, the diameter of the bacterial lawn increased significantly, but the bacterial density in the bacterial lawn decreased to a certain extent.

[0062] 5. Treatment trials for Clostridium perfringens infection

[0063] Seventy 6-8 week old BALB / C male mice were divided into 14 groups, with 5 mice in each group. Each mouse was injected intramuscularly with 0.1 mL of 1×10 8 Treatment began the next day after the CFU / mLA type Clostridium perfringens standard strain was added. The dosing regimen for different groups is shown in Table 6. Each mouse was given 0.2 mL of the drug by gavage every day for six consecutive days and was killed on the seventh day. The volume percentage of the drugs used for treatment is shown in Table 6. The results are shown in Figure 5 , Figure 6 and Table 7.

[0064] Table 6 Drug administration regimen for mouse infection model

[0065] Group Medication type and dosage A Negative control, 50% volume DMSO B Positive control, 50% volume DMSO C1-1 20mg / mL LCA C1-2 60mg / mL LCA C1-3 20mg / mL LLCA+40mg / mL CLDM C2-1 20mg / mL TCN C2-2 80mg / mL TCN C2-3 20mg / mL LCA + 40mg / mL TCN C3-1 5mg / mLCLDM C3-2 10mg / mLCLDM C3-3 50mg / mLCLDM C4-1 10mg / mL TMS C4-2 50mg / mL TMS C4-3 20mg / mL LCA + 10mg / mL TMS

[0066] Table 7 Pathological changes and mortality of mice before and after infection and treatment

[0067]

[0068]

[0069] Note: -: good mental state, shiny coat, no lameness, agile movements; +: good mental state, no obvious rough coat, not obvious lameness; ++: good mental state, slightly rough coat, slight lameness, pain reaction in the right hind limb; +++: average mental state, rough coat, obvious lameness, reduced coat and slight swelling in the right hind limb; ++++: poor mental state, sparse coat, swelling, dragging on the right hind limb, pain reaction, occasional shaking; +++++: depressed mental state, withered coat, curled up and arched back, eyes closed, sparse coat, dragging on the right hind limb, obvious swelling, black and purple color, no pain reaction.

[0070] One hour after infection, mice in group A were in good spirits, with shiny fur and agile movements; mice in group B and the experimental group showed symptoms such as inconvenient right hind limb movements, lameness, mental depression, etc. All mice in the high-dose LCA group in the experimental group died, and the mortality rate of the high-concentration TCN group reached 80%, as shown in Table 7.

[0071] After drug treatment, it was found that LCA had no significant effect when used alone, and the lethality of high-concentration LCA was as high as 100%. -1 CLDM has a good antibacterial effect, but the synergistic effect of CLDM and LCA in vivo was not significantly enhanced; LCA has a more obvious synergistic effect with TMS and TCN in vivo, and the antibacterial effect of antibiotics combined with LCA is significantly better than that of the two antibiotics used alone. High-concentration TCN is highly toxic and has a high mortality rate. No deaths were observed in the low-concentration TMS group, and the high-concentration group had a low mortality rate and high safety. The overall effect is better than TCN.

[0072] During autopsy, it was found that the right hind limbs of group B and the experimental groups showed varying degrees of swelling. The hair on the right hind limbs of some mice fell off, and the skin was swollen like air-filled blisters, which felt firm to the touch. After dissection, the muscle tissue of the mice's legs was darker in color, with varying degrees of bleeding and abscesses, such as Figure 5 As shown in b and c, the leg muscles of mice in the negative control group were flesh pink with no bleeding or abscesses. Figure 5 Compared with group B, each experimental group ( Figure 5 The swelling degree of dn) in group A was alleviated to varying degrees, but there was still some improvement compared with group A.

[0073] Randomly select some mouse lesion tissues for sectioning, such as Figure 6 As shown in the figure, in group A, the nuclei of multinucleated myocytes were located at the edge of the cells, the cross-sectioned myocytes were irregular polygons, the adjacent cells were closely arranged, the boundaries between cells were clear, the gaps between muscle bundles were uniform in size, no obvious pathological changes such as necrosis, inflammation, hemorrhage, edema, etc. were observed in the tissue, and the tissue morphology and structure were normal; in group B, local myocyte necrosis was observed in the field of view of muscle tissue, the tissue morphology was destroyed, the necrotic muscle fibers were replaced by proliferating fibrous connective tissue, accompanied by a large number of inflammatory cell infiltrations. All the patients in the high-dose LCA group died, and the myocyte connective tissue hyperplasia was observed in the field of view of the low-dose group, the gaps between cells were significantly enlarged, the tissue was loosely arranged, accompanied by diffuse infiltration of inflammatory cells, but the myocyte necrosis was less than that in the positive group, and the tissue morphology was still visible.

[0074] In the low-dose CLDM group, large-area necrosis of myocytes and hyperplasia of fibrous connective tissue were still observed in the local muscle tissue field, accompanied by a large number of inflammatory cell infiltrations; in the high-dose CLDM group, mild hyperplasia of connective tissue between muscle bundles, mild enlargement of the interfascicular space, a small amount of diffuse infiltration of inflammatory cells, and significant antibacterial effect were observed. The effect of the combined use of CLDM and LCA group was not significantly enhanced compared with that of the single use.

[0075] When TCN was used alone, the effect of the low-dose group was not obvious, and muscle cell necrosis was obvious. However, after combined use with LCA, only a small amount of inflammatory cells diffusely infiltrated the tissue, but the muscle fiber morphology and structure were relatively intact, and no pathological changes such as necrosis and hemorrhage were observed in the cells. It can be seen that the combination of TCN and LCA can significantly improve the therapeutic effect; in the high-dose group, the cells were closely arranged under the microscope, and there were few inflammatory cells and connective tissue hyperplasia. The therapeutic effect was significant, but the mortality rate was high, reaching 80%.

[0076] In the microscopic field of view of the low-dose TMS group, some muscle cells in the muscle tissue were necrotic, the morphological structure of the muscle bundles was destroyed and replaced by a large amount of connective tissue hyperplasia, and a large number of inflammatory cells diffusely infiltrated in the interfascicular spaces; after the same concentration of TMS was combined with LCA, the morphological structure of the muscle cells was relatively intact, the arrangement of tissue cells was slightly loose, the infiltration of inflammatory cells was reduced, and the synergistic effect was significant. In the high-dose TMS group, the muscle cells were slightly dissolved, the muscle cell cytoplasm was lightly stained, and the connective tissue hyperplasia and inflammatory cell infiltration were not obvious.

[0077] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of licorice chalcone A combined with antibiotics in the preparation of drugs against Clostridium perfringens, characterized in that: The antibiotic is tetracycline or tilmicosin.

2. The use according to claim 1, characterized in that: The drugs include veterinary drugs.

3. The use according to claim 1, characterized in that: The drug achieves the effect of resisting the gas gangrene caused by the infection of Clostridium perfringens, reducing the inflammatory response, tissue damage and hemolytic damage caused by the infection of Clostridium perfringens, and preventing the infection of Clostridium perfringens.

4. A drug for resisting Clostridium perfringens, characterized in that: The active ingredients of the medicine are licorice chalcone A and antibiotics; the antibiotics are tetracycline or tilmicosin.

5. The drug according to claim 4, characterized in that The concentration ratio of the licorice chalcone A to the antibiotic is (1-2):

1.

6. The drug according to claim 4, characterized in that The drugs include veterinary drugs.

7. The drug according to claim 4, characterized in that The drug achieves the effect of resisting the gas gangrene caused by the infection of Clostridium perfringens, reducing the inflammatory response, tissue damage and hemolytic damage caused by the infection of Clostridium perfringens, and preventing the infection of Clostridium perfringens.

8. Use of Licorice Chalcone A combined with antibiotics in the preparation of a drug for treating gas gangrene, characterized in that: The antibiotic is tetracycline or tilmicosin.

9. A drug for treating gas gangrene, characterized in that: The active ingredients of the medicine are licorice chalcone A and antibiotics; the antibiotics are tetracycline or tilmicosin.

10. The drug according to claim 9, characterized in that The concentration ratio of the licorice chalcone A to the antibiotic is (1-2):1.

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