Use of a bis-coumarin derivative in the preparation of a drug for preventing and treating nocardiosis of fish

Abstract

The application belongs to the technical field of disease prevention and control of aquatic animals, and discloses application of a dimeric coumarin derivative in preparation of a drug for preventing and treating nocardiosis of aquatic animals. The dimeric coumarin derivative is 3,3'-(ethane-1,1-diyl)bis(4-hydroxy coumarin), which can be used as an active ingredient to prepare a drug for preventing and treating bacterial diseases of aquatic animals, and preferably in the form of feeding with the drug. Experiments prove that the derivative has an in-vitro minimum inhibitory concentration of about 3.125 μg / mL on Nocardia seriolae, and when the infected large-mouth bass is fed with the derivative at a concentration of 200 mg / kg and 2000 mg / kg, the survival rate of the large-mouth bass is 50-56% after 28 days, and the derivative has no obvious toxicity to the large-mouth bass within a feed concentration range of 100-3200 mg / kg, thereby effectively solving the problems of poor efficacy, residues and drug resistance of traditional antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal disease prevention and control technology, and relates to the application of a dicoumarin derivative in the preparation of a drug for the prevention and treatment of nocardiosis in aquatic animals. Background Technology

[0002] Dicoumarin derivatives are a class of organic compounds with well-defined structural features and a good chemical synthesis basis. Their parent nucleus is derived from natural coumarins. They are known to have potential pharmacological effects in anticoagulation, anti-inflammation, antibacterial activity and regulation of cell signaling pathways. They have been widely used in the fields of medicine and biochemistry research. However, they are still in the blank state in the prevention and control of bacterial diseases in aquatic animals, especially in the application against specific drug-resistant pathogens.

[0003] Nocardiosis in largemouth bass is caused by Nocardia amberjack (Nocardia amberjack) Nocardia seriolae This condition, characterized by nodule formation in internal organs and a high mortality rate, is a major contributing factor to the decline in the mortality rate of largemouth bass (Sinocyclocheilus var. spp.). Micropterus salmoides The main obstacle to the development of the aquaculture industry is the current reliance on traditional antibiotics for prevention and treatment. However, Nocardia amberis, as an intracellular parasite with a thick cell wall, is prone to developing resistance to traditional antibiotics, making it difficult to maintain effective drug concentrations in vivo and resulting in a high treatment failure rate. At the same time, long-term high-dose use of traditional antibiotics can easily lead to drug residues in aquaculture water and fish tissues, which not only increases the risk of drug residue exceeding standards but also puts selective pressure and environmental toxicity on the aquatic microbial environment and ecosystem. In addition, the development of highly effective and low-toxicity special drugs is lagging behind, and the rapid emergence and spread of drug-resistant bacterial strains are causing the effective use period of existing antibiotics to continue to shorten.

[0004] Therefore, the development of anti-Nocardia amurensis drugs with novel mechanisms of action, the ability to overcome drug resistance and reduce residual risks has become an urgent need in the aquaculture industry. 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin), as a biscoumarin derivative with a single active ingredient, easily elucidated pharmacological mechanism and mature industrial production, provides a feasible path to overcome the above-mentioned technical difficulties. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as poor efficacy, high risk of drug residues, and shortened effective use period due to the spread of drug-resistant strains when using traditional antibiotics to prevent and treat nocardiosis in largemouth bass, and the lack of application of dicumarol derivatives in the prevention and treatment of bacterial diseases in aquatic animals (especially against specific drug-resistant pathogens), this invention uses the dicumarol derivative 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) as the active ingredient to prevent and treat nocardiosis in largemouth bass, achieving good therapeutic effects. The specific solution provided by this invention is as follows.

[0006] Firstly, the application provides a use of a dicumarol derivative in the preparation of a drug for preventing and treating Nocardia seriolae disease of aquatic animals, wherein the dicumarol derivative is 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin).

[0007] Further, in the above use, the aquatic animals are selected from one or more of the following: large mouth bass, snakehead, spotted catfish.

[0008] Further, in the above use, the minimum inhibitory concentration of the dicumarol derivative for Nocardia seriolae of large mouth bass in vitro is 3.125 μg / mL.

[0009] Further, in the above use, the use form of the dicumarol derivative is feed mixing or medicated bath.

[0010] Further, in the above use, when the use form is feed mixing, the addition concentration of 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin) in feed is ≤3200 mg / kg.

[0011] Further, in the above use, the preparation method of 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin) comprises:

[0012] (1) adding 4-hydroxy coumarin and anhydrous ethanol into a container, and stirring to completely dissolve the 4-hydroxy coumarin;

[0013] (2) adding acetaldehyde and diethylamine into the container in sequence according to the molar ratio of 4-hydroxy coumarin, acetaldehyde and diethylamine being 2:1:2, stirring uniformly, heating the mixture to reflux state and continuously stirring, monitoring the reaction progress by thin layer chromatography during the reaction, and stopping the reaction until the raw material 4-hydroxy coumarin is completely consumed;

[0014] (3) cooling the reaction mixture to room temperature, adding hydrochloric acid dropwise to adjust the pH value of the system to 1-2, then adding cold water and continuously stirring to make the product precipitate in solid form;

[0015] (4) filtering the precipitated solid mixture by using a reduced pressure filtration device, and collecting the filter cake;

[0016] (5) washing the filter cake with cold water, removing residual impurities, collecting the filter cake by reduced pressure filtration, and vacuum drying, which is repeated for 2-3 times.

[0017] Further, in step (1), the amount of anhydrous ethanol used is 100 mL for every 16.215 g of 4-hydroxy coumarin.

[0018] Further, in step (2), the temperature of the reflux reaction is 75-85°C, and the reaction time is 3-4 h.

[0019] Further, in step (5), the vacuum drying conditions are: -90~-70℃, 3~5h.

[0020] Compared with the prior art, the application of the double coumarin derivative in the preparation of the water animal nocardiosis prevention and treatment drug has the following beneficial effects:

[0021] The double coumarin derivative 3,3'- (ethane-1,1-diyl) bis (4-hydroxycoumarin) has a clear inhibitory effect on nocardia, and in vitro tests show that the minimum inhibitory concentration (MIC) of the double coumarin derivative on the bacteria is about 3.125 μg / mL, and the growth and reproduction of the pathogenic bacteria can be effectively inhibited; in the in vivo experiment, the survival rates of the infected large-mouth bass fed with the double coumarin derivative at the concentrations of 200 mg / kg and 2000 mg / kg are 50% and 56% respectively after 28 days, which is significantly higher than that of the infection control group of 16%, and the mortality rate caused by the disease can be effectively reduced.

[0022] The double coumarin derivative has good safety for large-mouth bass, and in the concentration range of 100~3200 mg / kg of feed, the experimental fish has no discomfort or death, which meets the actual application requirements of aquaculture; and the industrial production method is mature, the active ingredient is single, the pharmacological mechanism is easy to analyze, and the preparation development and quality control are convenient. At the same time, the technology can avoid the problems of poor efficacy, drug residues and drug-resistant strain diffusion of traditional antibiotics, and provides a reliable path for the prevention and treatment of bacterial diseases of aquatic animals.

[0023] The double coumarin derivative preparation method has the following advantages: the molar ratio of raw materials (4-hydroxycoumarin, acetaldehyde and diethylamine) is clear (2:1:2), the reaction process is standardized, and the product can be obtained by refluxing, adjusting pH, precipitating, filtering, washing and drying, and the yield reaches about 94%, without the need for complex equipment and harsh reaction conditions, and the process is mature and easy to control, which is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The chemical structural formula of 3,3'- (ethane-1,1-diyl) bis (4-hydroxycoumarin) is shown.

[0025] Figure 2 The chemical structural formula of 3,3'- (ethane-1,1-diyl) bis (4-hydroxycoumarin) is shown. 1 H NMR spectrum.

[0026] Figure 3 The infrared spectrum of 3,3'- (ethane-1,1-diyl) bis (4-hydroxycoumarin) is shown.

[0027] Figure 4The in vitro test results of 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin) against Nocardia seriolae in largemouth bass.

[0028] Figure 5 The survival rate changes of largemouth bass under different dosage conditions of 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin). DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be described below in conjunction with the embodiments, apparently, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0030] The experimental supplies involved in the embodiments of the present application are as follows:

[0031] I. Test strain

[0032] Nocardia seriolae, Nocardia seriolae provided by the Aquatic Disease Laboratory of Northwest A & F University, is used for in vitro bacteriostasis and in vivo infection test. The strain (GCA_018223685.1) has been whole-genome sequenced and published on NCBI.

[0033] II. Culture medium

[0034] BHI culture medium is used for the culture of Nocardia seriolae.

[0035] III. Materials and reagents

[0036] Synthesis and purification reagents: 4-hydroxy coumarin, synthetic raw material; acetaldehyde, synthetic raw material; diethylamine, synthetic raw material; anhydrous ethanol, solvent; dilute hydrochloric acid, adjust the pH of the reaction system; cold water, used for product precipitation and filter cake washing; the molar ratio of 4-hydroxy coumarin, acetaldehyde and diethylamine is 2:1:2.

[0037] Drugs and preparation reagents: 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin), synthesized in Example 1, as the active ingredient; DMSO (dimethyl sulfoxide), used for preparing high-concentration mother liquor of the drug and dissolving the drug spray feed; resazurin solution, 0.1% concentration, sterile PBS is used for preparing the resazurin solution; sterile PBS is used for preparing the resazurin solution.

[0038] Experimental animals and feed: largemouth bass, body length 4-6 cm, body weight 3±1 g, purchased from Xianyang Aquatic Market in Shaanxi, used for in vivo safety evaluation and survival rate detection; basic feed, used for drug preparation.

[0039] IV. Instruments and equipment

[0040] Synthesis and characterization instruments: round-bottom flask, reaction vessel; thin layer chromatography (TLC) device, monitoring the progress of the synthesis reaction; vacuum filtration device, product filtration separation; vacuum drying equipment, product drying; 400 MHz nuclear magnetic resonance spectrometer, used for product 1 H NMR structure characterization, the solvent is DMSO-d6.

[0041] In vitro test instruments: 24-well plate, antibacterial experiment reaction carrier; 28°C constant temperature culture equipment, Nocardia culture and antibacterial experiment incubation; in vivo test instruments: fish tank, breeding largemouth bass, controlling water temperature at 27°C; feed drying equipment, drying the feed after mixing with drugs.

[0042] Example 1

[0043] This example describes the synthesis of 3,3'- (ethane-1, 1-diyl) bis (4-hydroxy coumarin).

[0044] I. Test method

[0045] Raw materials and reagent preparation: take 4-hydroxy coumarin, acetaldehyde, diethylamine (the molar ratio of 4-hydroxy coumarin, acetaldehyde, diethylamine is 2:1:2), anhydrous ethanol, dilute hydrochloric acid, cold water, and reserve.

[0046] Reaction process: 16.215g of 4-hydroxy coumarin was placed in a 250mL round-bottom flask, 100mL of anhydrous ethanol was added, and the 4-hydroxy coumarin was completely dissolved by stirring; then 2.81mL of acetaldehyde and 10.35mL of diethylamine were added to the round-bottom flask in the above-mentioned molar ratio, and the mixture was stirred and heated to reflux state (80°C), during which the reaction progress was monitored by thin layer chromatography (TLC), until the raw material 4-hydroxy coumarin was completely consumed (3~4h), and the reaction was stopped.

[0047] Product post-treatment: the mixture after reaction was naturally cooled to room temperature, and dilute hydrochloric acid was added dropwise to adjust the pH value of the system to 1~2 (strongly acidic) ; then 50mL of cold water was added, and the product was precipitated in solid form under the condition of continuous stirring at room temperature.

[0048] Product purification: the precipitated solid mixture was filtered by a vacuum filtration device, and the filter cake was collected; the filter cake was washed with 200mL of cold water for 4 hours to remove residual impurities, and then filtered under reduced pressure, and then dried in a vacuum drying equipment (-80°C, 4h) to obtain a yellow-brown solid crude product; the washing, filtering and drying steps were repeated to obtain 3,3'- (ethane-1, 1-diyl) bis (4-hydroxy coumarin) fine product.

[0049] II. Results

[0050] The final product, 3,3'- (ethane-1, 1-diyl) bis (4-hydroxycoumarin), was obtained as a yellow-brown solid with a yield of about 94% after the above synthesis and purification steps.

[0051] As shown in Figure 1 The target compound molecule contains two 4-hydroxycoumarin nuclei, and the two nuclei are connected by ethane-1, 1-diyl (-CH (CH3) -). Each 4-hydroxycoumarin nucleus contains a hydroxyl group (-OH) at the 4 position. The molecular formula is C 20 H 14 O6.

[0052] Example 2

[0053] This example describes the structural characterization of 3,3'- (ethane-1, 1-diyl) bis (4-hydroxycoumarin).

[0054] The obtained fine product was subjected to 1 H NMR detection using a 400 MHz nuclear magnetic resonance spectrometer. The detection solvent was DMSO-d6, and the hydrogen atom chemical shift and coupling constant were recorded. The obtained fine product was subjected to infrared (IR) detection using a Fourier transform infrared spectrometer (FT-IR). The detection method was potassium bromide (KBr) tabletting method, and the wave number and characteristic functional group absorption peak were recorded.

[0055] As shown in Figure 2 1 H NMR (400 MHz, DMSO-d6) detection data are as follows:

[0056] δ 7.84 (dd, J = 7.9, 1.7 Hz, 2H), 7.66 (ddd, J = 8.7, 7.3, 1.7 Hz, 2H), 7.46-7.22 (m, 4H, H-8 / 8'), 4.17 (s, 1H), 1.45 (d, J = 7.2 Hz).

[0057] As shown in Figure 3 −1 , the infrared (KBr, cm

[0058] Example 3

[0059] This example describes the determination of the in vitro antibacterial activity of 3,3'- (ethane-1, 1-diyl) bis (4-hydroxycoumarin) against aquatic pathogenic bacteria. ​​

[0060] I. Test method

[0061] 1. Test material

[0062] Test strain: Nocardia seriolae (ATCC 19071) Nocardia seriolae ).

[0063] Culture medium: BHI medium was used for the culture of test strain and antibacterial test.

[0064] Drugs and reagents: The purified product of 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin) synthesized in Example 1 was dissolved in DMSO to prepare a 20000 μg / mL stock solution; resazurin solution (0.1%, prepared with sterile PBS) was used to determine the growth state of bacteria.

[0065] Instrument equipment: 24-well cell culture plate, 28°C constant temperature incubator.

[0066] 2. Antibacterial test

[0067] Preparation of bacterial suspension: The Nocardia seriolae cultured at 28°C for 5 days was centrifuged at 2000 rpm / min for 1 min to gently precipitate the oversized agglomerated particles, and whether there were visible bacteria in the supernatant after centrifugation was observed. If not, the supernatant was further transferred to a new sterile centrifuge tube as the bacterial suspension.

[0068] Drug gradient setting: In the 24-well plate, drug gradient dilution holes were set in turn (the concentrations were 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 μg / mL), and bacterial liquid control holes (without drugs) and blank control holes (DMSO, without bacterial liquid) were also set.

[0069] Bacterial liquid inoculation: 50 μL of bacterial suspension was added to the drug gradient holes and bacterial liquid control holes, and the same volume of DMSO was added to the blank control holes.

[0070] Culture and result determination: The 24-well plate was placed in a 28°C constant temperature incubator for static culture for 48 h; after incubation, 100 μL of resazurin solution was added to all holes, and incubation was continued for 2 h in the dark. The color change in the holes was observed by naked eye (blue indicates that bacteria have not grown, which is negative; pink indicates that bacteria have grown, which is positive), and the minimum inhibitory concentration (MIC, i.e. the lowest drug concentration that completely inhibits bacterial growth) was recorded.

[0071] II. Test results

[0072] 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin) showed clear antibacterial activity against Nocardia seriolae: as shown in Table 1, the MIC of 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin) against Nocardia seriolae was 0.78 μg / mL. Figure 4The results of in vitro test showed that when the drug concentration was ≥3.125 μg / mL, the solution in the hole was blue (no growth of bacteria); when the drug concentration was <3.125 μg / mL (i.e. 1.56, 0.78 μg / mL), the solution in the hole was pink (normal growth of bacteria), so the minimum inhibitory concentration (MIC) of the derivative on Nocardia seriolae was about 3.125 μg / mL.

[0073] Example 4

[0074] This example describes the safety evaluation of 3,3'- (ethane-1, 1-diyl) bis (4-hydroxy coumarin) on Micropterus salmoides.

[0075] I. Test method

[0076] 1. Test material preparation

[0077] Experimental animals: healthy Micropterus salmoides with a body length of 4-6 cm and a body weight of 3±1 g were selected, and after adaptive feeding for 7 days before the test, they were confirmed to be free of disease symptoms and normal feeding for the test.

[0078] Test feed: 3,3'- (ethane-1, 1-diyl) bis (4-hydroxy coumarin) synthesized in Example 1 was dissolved with DMSO and uniformly sprayed on the surface of the basic feed, which was dried at 60°C to avoid degradation, to prepare test feed with different drug concentrations. The blank control feed was the basic feed without adding drugs, only spraying an equal amount of DMSO.

[0079] Test environment: fish tanks with consistent specifications were used, the water temperature was controlled at 27°C, continuous aeration was maintained to maintain the dissolved oxygen content ≥5 mg / L, and the water quality was kept stable.

[0080] 2. Test grouping and treatment

[0081] Grouping: the test was divided into 1 blank control group and 8 drug treatment groups, with 20 Micropterus salmoides randomly placed in each group. The drug treatment groups had feed drug concentrations of 100, 200, 400, 800, 1600, 3200, 6400, and 12800 mg / kg, respectively; the blank control group was fed with basic feed without added drugs.

[0082] Feeding and observation: all test groups were fed 3 times a day (8:00, 14:00, and 20:00), with a feeding amount of 3-5% of the fish body weight (adjusted according to feeding conditions), and continuously fed for 72 h.

[0083] The survival number of each group of large-mouth bass was recorded, and the clinical symptoms of the fish were observed, including swimming state (such as whether abnormal swimming, stationary), feeding behavior (such as whether to refuse to eat, feeding initiative), body surface and internal organs (such as whether to appear bleeding), etc., to judge the toxic effect of the drug on the large-mouth bass.

[0084] II. Test Results

[0085] Survival: In the 72h observation period, all large-mouth bass in the blank control group survived without any discomfort; the survival of the drug treatment group varied with the concentration: when the feed drug concentration was 100-3200mg / kg, no large-mouth bass died in each group, and the survival number remained 20; when the concentration increased to 6400mg / kg, 1 large-mouth bass died within 72h, and the survival rate was 95%; when the concentration reached 12800mg / kg, the number of deaths increased to 5 within 72h, and the survival rate decreased to 75%.

[0086] Clinical symptoms: In the drug treatment group with a concentration of 100-3200mg / kg, the large-mouth bass swam flexibly and actively fed, with smooth body surface and no bleeding points, showing no significant difference from the blank control group; in the 6400mg / kg and 12800mg / kg groups, the dead individuals showed symptoms of stopping feeding, and the feeding initiative of the surviving individuals decreased slightly compared with the control group, but the swimming state was basically normal.

[0087] Safe concentration range: Based on the judgment of survival and clinical symptoms, the safe feed addition concentration of 3,3'- (ethane-1,1-diyl) bis (4-hydroxy coumarin) for large-mouth bass is 3200mg / kg, and the drug concentration in subsequent efficacy tests should be controlled within this range to avoid toxicity risk.

[0088] Example 5

[0089] This example describes the efficacy test of the dicumarol derivative in the treatment of Nocardia seriolae disease in large-mouth bass.

[0090] I. Test Method

[0091] 1. Test material preparation

[0092] Test animals: same as Example 4.

[0093] Feed preparation: refer to Example 4, prepare treatment feed with drug addition concentration of 200mg / kg (low dose) and 2000mg / kg (high dose). The blank control feed is the basic feed without adding drugs, only spraying the same amount of DMSO.

[0094] Pathogenic bacteria solution preparation: Nocardia seriolae (Nocardia seriolae) was cultured in a 250ml flask containing 100ml of Gause liquid medium, and the culture was incubated at 28°C for 48h. The culture was centrifuged at 4000r / min for 10min, and the supernatant was collected and stored at 4°C for use. Nocardia seriolae) with BHI medium at 28℃ to logarithmic growth phase, centrifugal collection of bacterial cells were resuspended with sterile saline, adjusting the concentration of bacterial solution to 1.5×10 7 CFU / mL, to ensure that the infection can be stable to trigger the nocardiosis.

[0095] 2. Test grouping and treatment

[0096] Group setting: the largemouth bass were randomly divided into 4 groups, 50 tails in each group, and each group was treated as follows:

[0097] Negative control group: no infection of Nocardia seriolae, feeding blank control feed throughout the process;

[0098] Infection control group: by intraperitoneal injection of the above bacterial solution (50 μL per injection), feeding blank control feed throughout the process;

[0099] Low-dose treatment group: inoculate the bacterial solution as the infection control group, immediately after infection start feeding the feed with a drug concentration of 200 mg / kg;

[0100] High-dose treatment group: inoculate the bacterial solution as the infection control group, immediately after infection start feeding the feed with a drug concentration of 2000 mg / kg.

[0101] Rearing management: all groups were reared in fish tanks with water temperature of 27℃ and dissolved oxygen content of ≥5 mg / L, fed 3 times a day (feeding amount was 3-5% of fish body weight), observed for 28 days, and the water quality was maintained stable during the period.

[0102] 3. Observation and index calculation

[0103] The mortality of each group of largemouth bass was recorded daily, and whether the dead individuals showed typical symptoms of Nocardia seriolae disease was observed to confirm that the death was caused by the target disease; after the end of the test, the survival rate of each group was calculated according to the formula: survival rate (%) = number of survivors in the treatment group / total number in each group × 100%.

[0104] II. Test results

[0105] The survival conditions of each group are as follows: the negative control group: no any largemouth bass died in the 28-day observation period, the survival rate was 100%, and the fish always kept normal swimming and feeding state, and no any disease symptoms appeared. The infection control group: death began to appear on the 7th day after infection, and the mortality gradually increased over time, and the number of survival was only 8 at 28 days, and the survival rate was 16%; the autopsies of the dead individuals showed that the internal organs such as liver and spleen formed obvious white nodules, which was consistent with the typical pathological characteristics of Nocardiosis, and it was confirmed that the death was caused by the disease. The low-dose (200 mg / kg) treatment group: a small amount of death began to appear on the 14th day after infection, and the number of death was significantly less than that of the infection control group, and the number of survival was 25 at 28 days, and the survival rate was 50%. The high-dose (2000 mg / kg) treatment group: death appeared on the 14th day after infection, and the frequency of death was further reduced, and the number of survival was 28 at 28 days, and the survival rate was 56% (see Figure 5 ).

[0106] The above results show that 3,3'- (ethane-1, 1-diyl) bis (4-hydroxy coumarin) has a clear therapeutic effect on Nocardiosis of largemouth bass: low-dose (200 mg / kg) and high-dose (2000 mg / kg) treatment can significantly reduce the mortality after infection, and the therapeutic effect of the high-dose group is better than that of the low-dose group, which confirms that the derivative can effectively control the development of Nocardiosis of largemouth bass.

[0107] The above-described embodiments are only some of the embodiments of the present application, not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only represents selected embodiments of the application. All other embodiments obtained by related deduction and replacement made by those skilled in the art under the condition of the concept of the present application, without making creative labor, belong to the scope of protection of the present application.

Claims

1. The application of a dicumarol derivative in the preparation of a drug for the prevention and treatment of nocardiosis in aquatic animals, characterized in that, The dicoumarin derivative is: 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin).

2. The application according to claim 1, characterized in that, The aquatic animals are selected from one or more of the following: largemouth bass, snakehead, and spotted catfish.

3. The application according to claim 1, characterized in that, The minimum inhibitory concentration of the dicumarol derivative against Nocardia amberjack in vitro is 3.125 μg / mL.

4. The application according to claim 1, characterized in that, The coumarin derivative is used by mixing it into feed or by medicated bath.

5. The application according to claim 4, characterized in that, When used in feed mixing, the concentration of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) added to the feed should be ≤3200mg / kg.

6. The application according to claim 1, characterized in that, The preparation methods of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) include: (1) Add 4-hydroxycoumarin and anhydrous ethanol to the container and stir until the 4-hydroxycoumarin is completely dissolved; (2) Add acetaldehyde and diethylamine dropwise to the container in a molar ratio of 2:1:2 for 4-hydroxycoumarin, acetaldehyde and diethylamine, stir and mix well, heat the mixture to reflux and stir continuously, monitor the reaction progress by thin layer chromatography, until the raw material 4-hydroxycoumarin is completely consumed and stop the reaction. (3) Cool the mixture after the reaction to room temperature, add hydrochloric acid dropwise to adjust the pH of the system to 1~2, then add cold water and continue stirring to precipitate the product in solid form; (4) The precipitated solid mixture is filtered using a vacuum filtration device, and the filter cake is collected; (5) Wash the filter cake with cold water to remove residual impurities, then filter under reduced pressure to collect the filter cake, and vacuum dry it. Repeat this process 2 to 3 times to obtain the final product.

7. The application according to claim 6, characterized in that, In step (1), the amount of anhydrous ethanol used is: 100 mL of anhydrous ethanol for every 16.215 g of 4-hydroxycoumarin.

8. The application according to claim 6, characterized in that, In step (2), the reflux reaction temperature is 75~85℃ and the reaction time is 3~4h.

9. The application according to claim 6, characterized in that, In step (5), the vacuum drying conditions are: -90~-70℃, 3~5h.

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