Application of biscoumarin derivative in preparation of medicine for preventing and treating nocardia seriola of aquatic animals

Abstract

The invention belongs to the technical field of prevention and control of diseases of aquatic animals, and discloses application of a biscoumarin derivative in preparation of a medicine for preventing and treating nocardia seriolae diseases of the aquatic animals. The bishydroxycoumarin derivative is 3, 3 '-(ethane-1, 1-diyl) bis (4-hydroxycoumarin), and can be used as an active ingredient for preparing a medicine for preventing and treating bacterial diseases of aquatic animals, and preferably, a mixed feeding use form is adopted. Experiments prove that the minimum in-vitro inhibitory concentration of the derivative to nocardia seriolae is about 3.125 mu g / mL, the survival rate of the derivative in 28 days is 50-56% when the derivative is mixed with largemouth bass infected with the nocardia seriolae at the concentrations of 200 mg / kg and 2000 mg / kg, and the derivative has no obvious toxicity to the largemouth bass within the feed concentration range of 100-3200 mg / kg, and can effectively solve the problems of poor curative effect, residue, drug resistance and the like 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, an 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] First, the present invention provides the application of a dicumarol derivative in the preparation of a drug for the prevention and treatment of Nocardia purpureus infection in aquatic animals, wherein the dicumarol derivative is: 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin).

[0007] Furthermore, in the above applications, the aquatic animals are selected from one or more of the following: largemouth bass, snakehead, and spotted catfish.

[0008] Furthermore, in the above applications, the minimum inhibitory concentration of the dicoumarin derivative against Nocardia amberjack in vitro is 3.125 μg / mL.

[0009] Furthermore, in the above applications, the coumarin derivative is used in the form of feed mixing or medicated bath.

[0010] Furthermore, in the above applications, when used in the form of feed mixing, the concentration of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) added to the feed is ≤3200mg / kg.

[0011] Furthermore, in the above applications, the preparation method of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) includes: (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.

[0012] Furthermore, in step (1), the amount of anhydrous ethanol used is: 100 mL of anhydrous ethanol is used in combination with every 16.215 g of 4-hydroxycoumarin.

[0013] Furthermore, in step (2), the reflux reaction temperature is 75~85℃ and the reaction time is 3~4h.

[0014] Furthermore, in step (5), the vacuum drying conditions are: -90~-70℃, 3~5h.

[0015] Compared with the prior art, the present invention, "Application of a coumarin derivative in the preparation of a drug for the prevention and treatment of nocardiosis in aquatic animals," has the following beneficial effects: The 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) dicoumarin derivative described in this invention has a clear inhibitory effect on Nocardia amberis. In vitro tests show that its minimum inhibitory concentration (MIC) against this bacterium is approximately 3.125 μg / mL, which can effectively inhibit the growth and reproduction of the pathogen. In in vivo experiments, when largemouth bass infected with Nocardia amberis were fed with feed at concentrations of 200 mg / kg and 2000 mg / kg, the 28-day survival rates were 50% and 56%, respectively, which were significantly higher than the 16% of the infected control group, effectively reducing the mortality rate caused by the disease.

[0016] The dicumarol derivative described in this invention exhibits good safety in largemouth bass. Within a feed concentration range of 100–3200 mg / kg, experimental fish showed no adverse symptoms or mortality, meeting the practical application requirements of aquaculture. Furthermore, its industrial production method is mature, its active ingredient is singular, and its pharmacological mechanism is easily elucidated, facilitating formulation development and quality control. Simultaneously, this technology avoids the problems of poor efficacy, drug residues, and the spread of drug-resistant strains associated with traditional antibiotics, providing a reliable pathway for the prevention and control of bacterial diseases in aquatic animals.

[0017] The method for preparing the dicoumarin derivative has significant advantages. The molar ratio of raw materials is well-defined (4-hydroxycoumarin, acetaldehyde and diethylamine are 2:1:2), the reaction process is standardized, and the product can be obtained by reflux, pH adjustment, precipitation, filtration, washing and drying, with a yield of about 94%. It does not require complex equipment or harsh reaction conditions, the process is mature and easy to control, and it is suitable for industrial production. Attached Figure Description

[0018] Figure 1 The chemical structural formula is 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin).

[0019] Figure 2 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) 1 H NMR spectrum.

[0020] Figure 3 The infrared spectrum of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin).

[0021] Figure 4 The results of in vitro tests on 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) against Nocardia amberjack.

[0022] Figure 5The survival rate of largemouth bass was observed to vary under different dosage conditions for 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin). Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] The experimental supplies involved in this embodiment of the invention are as follows: I. Test strains Nocardia amber ( Nocardia seriolae The strain (GCA_018223685.1), provided by the Aquatic Diseases Laboratory of Northwest A&F University, was used for in vitro antibacterial and in vivo infection tests. The whole genome of this strain has been sequenced and published on NCBI.

[0025] II. Culture Medium BHI medium is used for the culture of Nocardia amber.

[0026] III. Materials and Reagents Synthesis and purification reagents: 4-hydroxycoumarin, a raw material for synthesis; acetaldehyde, a raw material for synthesis; diethylamine, a raw material for synthesis; anhydrous ethanol, a solvent; dilute hydrochloric acid, used to adjust the pH of the reaction system; cold water, used for product precipitation and filter cake washing; the molar ratio of 4-hydroxycoumarin, acetaldehyde, and diethylamine is 2:1:2.

[0027] Drugs and preparation reagents: 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin), synthesized in Example 1, as the active ingredient; DMSO (dimethyl sulfoxide), used to prepare high-concentration drug stock solution and dissolve drug for spraying on feed; resazurin solution, 0.1% concentration, prepared with sterile PBS, used to determine the survival status of strains in in vitro antibacterial experiments; sterile PBS, used to prepare resazurin solution.

[0028] Laboratory animals and feed: Largemouth bass, 4-6 cm in length and 3±1 g in weight, were purchased from the Xianyang Aquatic Products Market in Shaanxi Province for in vivo safety evaluation and survival rate testing; basic feed was used to mix with drugs to prepare medicated feed.

[0029] IV. Instruments and Equipment Synthesis and characterization instruments: round-bottom culture flasks (reaction vessels); thin-layer chromatography (TLC) apparatus for monitoring the synthesis reaction process; vacuum filtration apparatus for product filtration and separation; vacuum drying equipment for product drying; 400MHz nuclear magnetic resonance spectrometer for product analysis. 1The structure was characterized by 1H NMR in DMSO-d6 solvent.

[0030] In vitro testing equipment: 24-well plate, reaction carrier for antibacterial experiments; 28℃ constant temperature culture equipment for Nocardia amberjack culture and antibacterial experiment incubation; In vivo testing equipment: fish tank for raising largemouth bass, with water temperature controlled at 27℃; feed drying equipment for drying feed after mixing with drugs.

[0031] Example 1 This example describes the synthesis of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin).

[0032] I. Experimental Methods Preparation of raw materials and reagents: Take 4-hydroxycoumarin, acetaldehyde, diethylamine (the molar ratio of 4-hydroxycoumarin, acetaldehyde, and diethylamine is 2:1:2), anhydrous ethanol, dilute hydrochloric acid, and cold water for later use.

[0033] Reaction process: 16.215 g of 4-hydroxycoumarin was placed in a 250 mL round-bottom culture flask, and 100 mL of anhydrous ethanol was added. The mixture was stirred until the 4-hydroxycoumarin was completely dissolved. Then, 2.81 mL of acetaldehyde and 10.35 mL of diethylamine were added dropwise to the round-bottom culture flask in the above molar ratio. After stirring and mixing, the mixture was heated to reflux (80 °C) and stirred continuously. The reaction progress was monitored by thin-layer chromatography (TLC) until the 4-hydroxycoumarin was completely consumed (3-4 h), at which point the reaction was stopped.

[0034] Product post-treatment: The reaction mixture was naturally cooled to room temperature, and dilute hydrochloric acid was added dropwise to adjust the pH of the system to 1-2 (strong acidity); then 50 mL of cold water was added, and the mixture was stirred continuously at room temperature to allow the product to precipitate in solid form.

[0035] Product purification: The precipitated solid mixture was filtered using a vacuum filtration device, and the filter cake was collected. The filter cake was washed with 200 mL of cold water for 4 hours to remove residual impurities, and then dried in a vacuum drying device (-80℃, 4h) to obtain a yellowish-brown crude solid. The washing, filtration and drying steps were repeated to obtain the refined 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin).

[0036] II. Test Results Product appearance and yield: After the above synthesis and purification steps, the final product 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) was a yellowish-brown solid with a yield of approximately 94%.

[0037] like Figure 1As shown in the chemical structural formula of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin), the target compound molecule contains two 4-hydroxycoumarin cores connected by ethane-1,1-diyl (-CH(CH3)-). Each 4-hydroxycoumarin core contains a hydroxyl group (-OH) at position 4. The molecular formula is C3. 20 H 14 O6.

[0038] Example 2 This embodiment describes the structural characterization of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin).

[0039] The obtained samples were analyzed using a 400MHz nuclear magnetic resonance spectrometer. 1 ¹H NMR was performed using DMSO-d6 as the detection solvent, and the chemical shifts and coupling constants of hydrogen atoms were recorded. Fourier transform infrared spectroscopy (FT-IR) was used to detect the obtained product using potassium bromide (KBr) pellet method, and the wavenumbers and absorption peaks of characteristic functional groups were recorded.

[0040] like Figure 2 As shown, 1 The H NMR (400MHz, DMSO-d6) detection data are as follows: δ7.84(dd,J=7.9,1.7Hz,2H),7.66(ddd,J=8.7,7.3,1.7Hz,2H),7.46–7.22(m,4H,H-8 / 8'),4.17(s,1H),1.45(d,J=7.2Hz).

[0041] like Figure 3 As shown, infrared radiation (KBr, cm) −1 The test data are as follows: 3076, 2976, 1701, 1661, 1616, 1273, 1244, 1190, 758.

[0042] Example 3 This embodiment describes the determination of the in vitro antibacterial activity of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) against aquatic pathogens.

[0043] I. Experimental Methods 1. Test materials Test strain: Nocardia amberjack ( Nocardia seriolae ).

[0044] Culture medium: BHI medium was used for the culture of test strains and antibacterial tests.

[0045] Drugs and reagents: The purified product of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) synthesized in Example 1 was dissolved in DMSO to prepare a stock solution of 20,000 μg / mL; resazurin solution (0.1%, prepared with sterile PBS) was used to determine the bacterial growth status.

[0046] Instruments and equipment: 24-well cell culture plates, 28℃ constant temperature incubator.

[0047] 2. Antibacterial test Preparation of bacterial suspension: Nocardia amberjack cultured at 28℃ for 5 days was centrifuged at 2000 rpm / min for 1 min to gently precipitate large aggregates. The presence of visible bacterial cells in the supernatant after centrifugation was observed. If no cells were found, the supernatant was transferred to a new sterile centrifuge tube to prepare the bacterial suspension.

[0048] Drug gradient setup: Set up drug gradient dilution wells in a 24-well plate sequentially (concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 μg / mL, respectively), and set up bacterial control wells (without drug) and blank control wells (DMSO, without bacterial solution).

[0049] Bacterial suspension inoculation: Add 50 μL of bacterial suspension to the drug gradient wells and bacterial control wells, respectively, and add an equal volume of DMSO to the blank control wells.

[0050] Culture and result determination: The 24-well plate was placed in a constant temperature incubator at 28℃ and incubated for 48 hours. After incubation, 100 μL of resveratrol solution was added to all wells and incubated for another 2 hours in the dark. The color change in the wells was observed by the naked eye (blue indicates no bacterial growth, which is negative; pink indicates bacterial growth, which is positive), and the minimum inhibitory concentration (MIC, i.e. the lowest drug concentration that completely inhibits bacterial growth) was recorded.

[0051] II. Test Results 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) exhibits clear antibacterial activity against Nocardia auriculata: such as Figure 4 As shown in the in vitro test results, when the drug concentration is ≥3.125 μg / mL, the solution in the corresponding well is blue (no bacterial growth); when the drug concentration is <3.125 μg / mL (i.e. 1.56, 0.78 μg / mL), the solution in the well is pink (normal bacterial growth). Therefore, the minimum inhibitory concentration (MIC) of this derivative against Nocardia auricula is approximately 3.125 μg / mL.

[0052] Example 4 This embodiment describes the safety evaluation of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) on largemouth bass.

[0053] I. Experimental Methods 1. Preparation of experimental materials Experimental animals: Healthy largemouth bass with a body length of 4-6 cm and a weight of 3±1 g were selected. They were acclimatized for 7 days before the experiment and were used for the experiment after confirming that they had no disease symptoms and were feeding normally.

[0054] Experimental feeds: 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) synthesized in Example 1 was dissolved in DMSO and sprayed evenly onto the surface of the base feed. The feed was then dried at 60°C to prevent degradation, resulting in experimental feeds with different drug concentrations. The blank control feed consisted of base feed without added drug but sprayed with an equal amount of DMSO.

[0055] Experimental environment: Aquariums of uniform size were used, the water temperature was controlled at 27℃, continuous aeration was maintained to keep the dissolved oxygen level ≥5mg / L, and recirculating aquaculture was used to maintain stable water quality.

[0056] 2. Experimental grouping and treatment Group setup: The experiment was divided into one blank control group and eight drug treatment groups, with 20 largemouth bass randomly released into each group. The drug concentrations added to the feed of the drug treatment groups were 100, 200, 400, 800, 1600, 3200, 6400, and 12800 mg / kg, respectively; the blank control group was fed a basal diet without added drugs.

[0057] Feeding and observation: All experimental groups were fed 3 times a day (at 8:00, 14:00 and 20:00 respectively), with a feeding amount of 3-5% of the fish's body weight (adjusted according to feeding situation), for 72 consecutive hours.

[0058] The number of surviving largemouth bass in each group was recorded as an observation indicator. At the same time, the clinical symptoms of the fish were observed, including swimming status (such as whether there is abnormal swimming or stillness), feeding behavior (such as whether they refuse to eat or their feeding enthusiasm), and the appearance of the body surface and internal organs (such as whether there is bleeding), to determine the toxic effect of the drug on the largemouth bass.

[0059] II. Test Results Survival status: In the blank control group, all largemouth bass survived without any discomfort symptoms during the 72-hour observation period. The survival status of the drug-treated groups varied with the concentration: when the feed drug concentration was 100-3200 mg / kg, there were no deaths among the largemouth bass in each group, and the number of survivors remained at 20; when the concentration was increased to 6400 mg / kg, one largemouth bass died within 72 hours, and the survival rate was 95%; when the concentration reached 12800 mg / kg, the number of deaths increased to 5 within 72 hours, and the survival rate dropped to 75%.

[0060] Clinical symptoms: In the drug treatment groups with concentrations of 100-3200 mg / kg, the largemouth bass swam flexibly, fed actively, and had smooth skin without bleeding points, showing no significant difference from the blank control group; in the 6400 mg / kg and 12800 mg / kg groups, the dead individuals showed symptoms of stopping feeding, while the surviving individuals showed a slight decrease in feeding activity compared to the control group, but their swimming behavior was basically normal.

[0061] Safe concentration range: Based on a comprehensive assessment of survival and clinical symptoms, the upper limit of the safe feed addition concentration of 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) for largemouth bass is 3200 mg / kg. The drug concentration in subsequent efficacy tests should be controlled within this range to avoid toxicity risks.

[0062] Example 5 This embodiment describes the efficacy test of the dicoumarin derivative in the treatment of nocardiosis in largemouth bass.

[0063] I. Experimental Methods 1. Preparation of experimental materials Experimental animals: Same as in Example 4.

[0064] Feed preparation: Referring to Example 4, therapeutic feeds with drug concentrations of 200 mg / kg (low dose) and 2000 mg / kg (high dose) were prepared. The blank control feed was a basic feed without drug addition, only sprayed with an equal amount of DMSO.

[0065] Preparation of pathogenic bacterial suspension: Nocardia auriculata ( Nocardia seriolae The cells were cultured in BHI medium at 28°C until the logarithmic growth phase. After centrifugation, the cells were resuspended in sterile physiological saline and the bacterial concentration was adjusted to 1.5 × 10⁻⁶. 7 CFU / mL ensures stable induction of nocardiosis after infection.

[0066] 2. Experimental grouping and treatment Grouping: Largemouth bass were randomly divided into 4 groups of 50 fish each, and the treatment for each group was as follows: Negative control group: Not infected with Nocardia auricula-judae, fed the blank control diet throughout the entire process; Infection control group: The above bacterial solution was inoculated via intraperitoneal injection (50 μL per fish), and the fish were fed a blank control diet throughout the entire process; Low-dose treatment group: Inoculated with bacterial solution in the same way as the infection control group, and fed with feed at a drug concentration of 200 mg / kg immediately after infection; High-dose treatment group: Inoculated with bacterial solution in the same way as the infection control group, and fed with feed containing 2000 mg / kg of drug immediately after infection.

[0067] Feeding and management: All groups were kept in aquariums with a water temperature of 27℃ and dissolved oxygen of ≥5mg / L, and fed 3 times a day (the amount of food was 3~5% of the fish's body weight). They were observed for 28 days, and the water quality was kept stable during the period.

[0068] 3. Observation and Indicator Calculation Daily records were kept of the mortality of largemouth bass in each group. The presence of typical symptoms of nocardiosis in the dead individuals was observed to confirm that the deaths were caused by the target disease. After the experiment, the survival rate of each group was calculated using the formula: Survival rate (%) = Number of surviving individuals in the treatment group / Total number in each group × 100%.

[0069] II. Test Results Survival rates for each group are as follows: Negative control group: No largemouth bass died during the 28-day observation period, with a survival rate of 100%. The fish maintained normal swimming and feeding behavior throughout, without exhibiting any disease symptoms. Infected control group: Mortality began to occur on day 7 post-infection, gradually increasing over time. By day 28, only 8 fish survived, a survival rate of 16%. Autopsies of the deceased individuals revealed distinct white nodules on the liver, spleen, and other internal organs, consistent with the typical pathological features of Nocardia amberjack disease, confirming that the deaths were caused by this disease. Low-dose (200 mg / kg) treatment group: A small number of deaths began on day 14 post-infection, significantly fewer than the infected control group. By day 28, 25 fish survived, a survival rate of 50%. High-dose (2000 mg / kg) treatment group: Mortality began on day 14 post-infection, with a further decrease in mortality frequency. By day 28, 28 fish survived, a survival rate of 56% (see...). Figure 5 ).

[0070] The above results indicate that 3,3'-(ethane-1,1-diyl)bis(4-hydroxycoumarin) has a clear therapeutic effect on nocardiosis in largemouth bass: both low-dose (200 mg / kg) and high-dose (2000 mg / kg) treatments significantly reduced the mortality rate after infection, and the high-dose group had a better therapeutic effect than the low-dose group, confirming that this derivative can effectively control the progression of nocardiosis in largemouth bass.

[0071] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

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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