Application of naphthoquinone compound in prevention and / or treatment of aquatic animal endoparasitosis

Abstract

The invention provides application of naphthoquinone compounds in preparation of insecticides for preventing and / or treating parasitic diseases in aquatic animals, and belongs to the technical field of aquaculture. The naphthoquinone compound disclosed by the invention is 2-chloro-1, 4-naphthoquinone, and can be used for killing or preventing pyramidal bodies in aquatic animals. The invention also provides an insecticide for killing or preventing pyramidal worms in aquatic animals by using the naphthoquinone compound, and the insecticide comprises the following raw materials in percentage by weight: 20-30% of the naphthoquinone compound, 5-10% of a surfactant, 5-10% of a dissolving agent and the balance of water. An in-vitro insecticidal experiment and an in-vivo insecticidal experiment prove that the 2-chloro-1, 4-naphthoquinone can be used for efficiently killing insects; and the 2-chloro-1, 4-naphthoquinone has no obvious toxic or side effect on the fish body, and does not influence the growth and physiological functions of the fish body.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquaculture, and particularly relates to application of a naphthoquinone compound in prevention and / or treatment of parasitic diseases in aquatic animals, in particular, application in prevention and / or treatment of sphaeridium disease in aquatic animals. BACKGROUND

[0002] Sphaeridium is a general term for sphaeridium of the order of kinetoplastida of the class of dinoflagellate, and adult sphaeridium mainly parasitizes in the blood of vertebrates. Sphaeridium can infect freshwater fish and marine fish, but the harm to marine fish is significantly greater than that to freshwater fish. Sphaeridium disease breaks out in coastal large yellow croaker breeding areas, and diseased fish generally shows symptoms such as anemia, liver and spleen lesions, and skin ulceration, with a mortality rate of more than 50%, or will become a bottleneck restricting the development of the large yellow croaker industry. The infection process of sphaeridium disease can be divided into four stages: incubation period, chronic infection period, acute death period and post-infection period. When the water temperature is higher than 20℃, the infection intensifies, and the fish body will appear ulceration, scale shedding, muscle putrefaction and even expose a large area of red wounds. The blood parameters of diseased large yellow croaker change significantly, showing anemia, blood color lightening and significant reduction of red blood cell count.

[0003] At present, the prevention and control measures for sphaeridium disease mainly include: reducing breeding density, improving breeding environment; strengthening biological security measures, reducing the feeding of ice fresh bait; feeding full-nutrition functional feed to improve the disease resistance of large yellow croaker; strengthening daily detection to master the health level of fish body; and drug prevention and control measures such as using garlicin, pumpkin seeds, areca nuts and eucalyptus essential oil. However, these methods have limited effect and cannot effectively control the disease.

[0004] 2-Chloro-1,4-naphthoquinone is a chemically synthesized naphthoquinone compound, which is a yellow or orange crystalline powder. The compound has a melting point of 140-145℃ and a boiling point of about 310℃, and is soluble in organic solvents such as alcohol, ether and chloroform, but has low solubility in water. Previous studies mainly focused on its use as a pharmaceutical intermediate, especially for the synthesis of drugs such as atovaquone, but there has been no report on its use in resisting fish body parasites, especially sphaeridium. SUMMARY

[0005] The application provides application of a naphthoquinone compound in prevention and / or treatment of parasitic diseases in aquatic animals, which can prevent and / or treat sphaeridium disease in aquatic animals, has high biological safety and good treatment effect.

[0006] The application provides application of a naphthoquinone compound in preparation of an insecticide for killing or preventing parasites in aquatic animals, and the naphthoquinone compound is 2-chloro-1,4-naphthoquinone.

[0007] In a preferred mode of the present application, the parasite in the aquatic animal includes conoid worms.

[0008] In a preferred mode of the present application, the effective concentration of the insecticide, calculated based on 2-chloro-1,4-naphthoquinone, is not less than 10 mg / kg fish weight.

[0009] The present application also provides an insecticide for killing or preventing parasites in aquatic animals, comprising a naphthoquinone compound, a surfactant, a dissolving agent and water; the naphthoquinone compound is 2-chloro-1,4-naphthoquinone.

[0010] In a preferred mode of the present application, the insecticide comprises the following ingredients by weight: 20-30 parts by weight of the naphthoquinone compound, 5-10 parts by weight of the surfactant, 5-10 parts by weight of the dissolving agent, and the balance of water.

[0011] In a preferred mode of the present application, the dissolving agent comprises dimethyl sulfoxide or N,N-dimethylformamide. The surfactant comprises Tween-20 or Tween-80.

[0012] In a preferred mode of the present application, the dosage form of the insecticide comprises an oral dosage form.

[0013] The present application also provides a method for breeding aquatic animals, comprising feeding the above-mentioned insecticide during the breeding process of the aquatic animals.

[0014] In a preferred mode of the present application, when preventing the parasite disease in the aquatic animals, the insecticide is fed once every other day, for a total of 7-10 times; the amount of each feeding, calculated based on 2-chloro-1,4-naphthoquinone, is 10-20 mg / kg fish weight.

[0015] In a preferred mode of the present application, when treating the parasite disease in the aquatic animals, the insecticide is fed once a day, for a total of 5 times; the amount of each feeding, calculated based on 2-chloro-1,4-naphthoquinone, is 10 mg / kg fish weight.

[0016] Beneficial effects: The present application first discovers that 2-chloro-1,4-naphthoquinone has a significant parasite killing effect and is highly safe for fish bodies. The present application proves that 2-chloro-1,4-naphthoquinone can efficiently kill parasites through in-vitro and in-vivo experiments in the examples; and the 2-chloro-1,4-naphthoquinone has no obvious toxic side effects on fish bodies and does not affect the growth and physiological functions of fish bodies; the insecticide is simple to prepare, easy to use, and low in production cost. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Relationship between the concentration of the insecticide of Example 4 and the mortality rate of large yellow croakers; Figure 2The contrast chart of the big yellow croaker infected with Trypanosoma before and after the drug administration of Example 5 is shown in the figure, in which A: before the drug administration; and B: after the drug administration. DETAILED DESCRIPTION

[0018] The application provides application of a naphthoquinone compound in preparation of an insecticide for killing or preventing parasites in aquatic animals, and the naphthoquinone compound is 2-chloro-1,4-naphthoquinone.

[0019] The 2-chloro-1,4-naphthoquinone in the application is a chemically synthesized naphthoquinone compound, which is a yellow or orange crystalline powder. The compound has a melting point of 140-145 DEG C, a boiling point of about 310 DEG C, and can be dissolved in organic solvents such as alcohol, ether, chloroform and the like, but has a low solubility in water. The molecular formula of the 2-chloro-1,4-naphthoquinone is C 10 H5ClO2, and the molecular weight is 194.6. The source of the 2-chloro-1,4-naphthoquinone is not particularly limited in the application, and a conventional commercially available reagent in the art can be used, for example, the drug supplier in the example is Aladdin.

[0020] In one embodiment of the application, it is found through in-vitro experiments that the mortality rate of Trypanosoma reaches 100% after the 2-chloro-1,4-naphthoquinone is used at a concentration of 1.0 μg / mL for 4 hours, and the 2-chloro-1,4-naphthoquinone has a high insecticidal effect.

[0021] In one embodiment of the application, the in-vivo insecticidal effect of the 2-chloro-1,4-naphthoquinone is also verified, and the results show that the 2-chloro-1,4-naphthoquinone has an insecticidal effect when the effective concentration of the 2-chloro-1,4-naphthoquinone is not less than 10 mg / kg of fish weight, and the insecticidal effect has a concentration dependence, that is, the higher the concentration, the better the in-vivo Trypanosoma killing effect, and the lower the mortality rate of the big yellow croaker.

[0022] The application further provides an insecticide for killing or preventing parasites in aquatic animals, which comprises a naphthoquinone compound, a surfactant, a dissolving agent and water, and the naphthoquinone compound is 2-chloro-1,4-naphthoquinone.

[0023] The insecticide comprises the following components in parts by weight: 20-30 parts by weight of the naphthoquinone compound, 5-10 parts by weight of the surfactant, 5-10 parts by weight of the dissolving agent, and the balance of water. The dissolving agent comprises dimethyl sulfoxide or N,N-dimethylformamide, and the surfactant comprises Tween-20 or Tween-80.

[0024] The preparation method of the insecticide is not particularly limited in the application, and the insecticide can be prepared into an oral preparation through a conventional method.

[0025] The application further provides a breeding method of aquatic animals, which comprises feeding the above-mentioned insecticide in the breeding process of the aquatic animals.

[0026] The insecticide is used for preventing parasitic diseases in aquatic animals, and is fed once every other day for 7 to 10 times. The amount of the insecticide fed each time is 10 to 20 mg / kg of fish weight, calculated based on 2-chloro-1,4-naphthoquinone. The insecticide is used for treating parasitic diseases in aquatic animals, and is fed once a day for 5 times. The amount of the insecticide fed each time is 10 mg / kg of fish weight, calculated based on 2-chloro-1,4-naphthoquinone.

[0027] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not intended to limit the scope of the present application, but to explain certain aspects, features and embodiments of the present application.

[0028] It is to be understood that the terms used in the present application are merely descriptive, rather than limiting, of the particular embodiments. In addition, for numerical ranges in the present application, it is to be understood that every intermediate value of the upper and lower limits of the range is specifically disclosed. Each intermediate value between any stated value or stated range, as well as any other stated value or intermediate value in the stated range, is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the ranges.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. Any and all

[0030] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the present application. Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the present application. The specification and examples are illustrative only.

[0031] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

[0032] The insecticide of the present application can be directly mixed in the feed for feeding. The dosage is 10 to 20 mg / kg of fish weight.

[0033] Example 1 Insecticide formulation A: 25 g of 2-chloro-1,4-naphthoquinone was weighed and dissolved in 10 g of N,N-dimethylformamide. 10 g of surfactant Tween-20 was weighed and mixed with 55 g of water to obtain 25% insecticide.

[0034] B: Take 2-chloro-1, 4-naphthoquinone 30g, stir and dissolve in 10g N, N- dimethylformamide, take surfactant Tween-80 5g, add 55g of water and mix well to obtain 30% insecticide.

[0035] C: Take 2-chloro-1, 4-naphthoquinone 28g, stir and dissolve in 10g dimethyl sulfoxide, take surfactant Tween-20 10g, add 52g of water and mix well to obtain 28% insecticide.

[0036] D: Take 2-chloro-1, 4-naphthoquinone 20g, stir and dissolve in 10g dimethyl sulfoxide, take surfactant Tween-80 10g, add 60g of water and mix well to obtain 20% insecticide.

[0037] To test the effect of surfactant and dissolving agent on the insecticide, the surfactant of the four groups of insecticides with different proportions is replaced with water, and the rest of the ingredients remain unchanged. The research results show that the killing effect and mortality of the cone worm have no obvious change, which shows that the surfactant in the application only has a dissolving effect. The dissolving agent of the four groups of insecticides with different proportions is replaced with surfactant, and the rest of the ingredients remain unchanged. The research results show that the killing effect and mortality of the cone worm have no obvious change, which shows that the dissolving agent in the application only has a dissolving effect, and the substance that plays the role of insecticide is 2-chloro-1, 4-naphthoquinone.

[0038] Example 2 Insecticidal experiment of 2-chloro-1, 4-naphthoquinone in vitro After the large yellow croaker seriously infected with the cone worm is anesthetized, the venous blood is extracted and added to the culture medium with an anticoagulant, and the number and activity of the cone worm are observed under a microscope for subsequent drug efficacy test. The test is carried out on a 24-well cell culture plate, about 0.2mL of the culture medium containing the cone worm is added to each cell well, and about 1.8mL of the insecticide with different final concentrations of the effective component 2-chloro-1, 4-naphthoquinone (the concentration of the insecticide in this example is calculated based on the effective component 2-chloro-1, 4-naphthoquinone) is added, and the cone worm is observed under a microscope at 15min, 1h, 2h, 3h and 4h after the drug is added, and the mortality of the cone worm in each well is counted after 4h. The test is set and fully aerated with natural water as a control group, and each test is repeated three times.

[0039] The judgment standard for the death of the cone worm is that the cilia of the worm body do not move.

[0040] The 2-chloro-1, 4-naphthoquinone insecticide has a strong killing effect on the cone worm, and the killing effect gradually increases with the increase of the concentration, and the half lethal effective concentration (EC 50) were 0.71, 0.61, 0.49, 0.37 and 0.26 mg / L, respectively. The results of the in vitro insecticidal test of 2-chloro-1, 4-naphthoquinone insecticide on Trypanosoma sp. are shown in Table 1. There was no difference in the insecticidal effect of the four insecticides. When the concentration of the insecticide was 1 mg / L, the Trypanosoma sp. could be killed within 4 h at a rate of 100%.

[0041] Table 1. Killing rate of 2-chloro-1, 4-naphthoquinone insecticide on Trypanosoma sp. (4 h)

[0042] Note: Different letters represent significant differences (p < 0.05) Example 3 In vivo efficacy test of 2-chloro-1, 4-naphthoquinone The Trypanosoma sp. infected Pseudosciaena crocea was injected intraperitoneally. Immediately after infection, the Pseudosciaena crocea was grouped with 10 fish per group and placed in a 100 L fish tank, which was separated by a partition. According to the in vitro insecticidal test, considering the loss of insecticide in water, the concentration of the insecticide was set to 6.0, 8.0, 10.0 and 12.0 mg / kg fish weight. Different concentrations of insecticide were added to the water according to the set concentration, and the fish were continuously fed for 5 days, and it was ensured that all the test fish could eat the bait.

[0043] On the 5th day of the test, 3 Pseudosciaena crocea were taken out from each concentration group, and the blood was collected according to the method of the in vitro insecticidal test, and the death of Trypanosoma sp. in the blood was observed.

[0044] On the 10th day of the test, all Pseudosciaena crocea in the group were taken out, the blood was collected, the number of Trypanosoma sp. in the blood of each Pseudosciaena crocea was counted, and the average insecticidal rate was calculated.

[0045] Insecticidal rate (%) = (average number of Trypanosoma sp. in the control group per field of view / average total number of Trypanosoma sp. in the control group per field of view - average number of Trypanosoma sp. in the test group per field of view / average total number of Trypanosoma sp. in the test group per field of view) x 100% Average fish mortality rate (%) = (number of fish released - number of fish alive at the time of microscopic examination) / number of fish released x 100%.

[0046] The results of the in vitro insecticidal test of 2-chloro-1, 4-naphthoquinone insecticide are shown in Table 2. 2-Chloro-1, 4-naphthoquinone insecticide has strong killing effect on Trypanosoma sp. After using the insecticide, the number of Trypanosoma sp. in the blood of Pseudosciaena crocea decreased significantly. When the concentration of the insecticide was 10.0 mg / kg, the mortality rate of Pseudosciaena crocea decreased to 10%, and the number of Trypanosoma sp. in the blood decreased to 1.2 ± 0.2 per mL. The mortality rate of the control group of Pseudosciaena crocea was 100%, and the number of Trypanosoma sp. in the blood was 1.6 x 10 7 ±4.5 x 10 6The results show that 2-chloro-1,4-naphthoquinone insecticide has strong protective effect on the test fish infected with Trypanosoma.

[0047] Table 2 In vivo insecticidal effect of 2-chloro-1,4-naphthoquinone insecticide on Trypanosoma

[0048] Note: Different letters represent significant difference (p<0.05) Example 4 Safety evaluation test of 2-chloro-1,4-naphthoquinone insecticide The test was carried out in a glass tank with a volume of 80L, 10 healthy large yellow croakers were placed in each tank, and the insecticide was fed at a concentration of 100.0, 200.0, 300.0, 400.0, 500.0 and 600.0 mg / kg, and the number of dead large yellow croakers was counted after 96h, and the half lethal concentration of 2-chloro-1,4-naphthoquinone on large yellow croakers was calculated. Each concentration group was repeated 3 times, and a control group was set up.

[0049] The toxicity results of 2-chloro-1,4-naphthoquinone insecticide on large yellow croakers are as follows Figure 1 . The mortality rate of large yellow croakers was 0 after 96h of 2-chloro-1,4-naphthoquinone insecticide at a concentration of 100mg / kg, and the activity of large yellow croakers was normal, and no abnormal reaction was found. After 4h of 2-chloro-1,4-naphthoquinone insecticide at a concentration of 600mg / kg, the large yellow croakers showed abnormal reactions such as rapid breathing and jumping, and all the large yellow croakers died after 96h. The 96h LD50 of 2-chloro-1,4-naphthoquinone insecticide on large yellow croakers was 396.1mg / kg.

[0050] Example 5 Clinical application effect A clinical test was carried out in a large yellow croaker farm in Zhejiang. The farm broke out Trypanosoma disease in May 2025, and the daily mortality rate reached about 2% at the early stage of the outbreak. After 5 days of continuous feeding of the insecticide (10mg / kg) provided by the present application, the daily mortality rate of the sick fish was significantly reduced to below 0.1%, and the health status of the fish was significantly improved (Fig. 1), and the number of Trypanosoma in the blood was reduced by more than 70%. Figure 2

[0051] Although the above examples have made a detailed description of the present application, it is only a part of the examples of the present application, not all the examples, and other examples can be obtained under the premise of no creativity according to the present examples, which all belong to the protection scope of the present application.​

Claims

1. Use of a naphthoquinone compound for the manufacture of a parasiticide for killing or preventing parasites in an aquatic animal, characterized in that, The naphthoquinone compound is 2-chloro-1,4-naphthoquinone.

2. Use according to claim 1, characterized in that, The internal parasite of the aquatic animal includes trypanosome.

3. Use according to claim 2, characterized in that, The effective concentration of the insecticide is not less than 10 mg / kg fish weight in terms of 2-chloro-1,4-naphthoquinone.

4. A parasiticide for killing or preventing parasites in an aquatic animal, characterized in that, The insecticide comprises the naphthoquinone compound, the surfactant, the dissolving agent and water; the naphthoquinone compound is 2-chloro-1,4-naphthoquinone.

5. The insecticide of claim 4, wherein, The insecticide comprises the naphthoquinone compound, the surfactant, the dissolving agent and water; the naphthoquinone compound is 2-chloro-1,4-naphthoquinone.

6. The insecticide according to claim 4 or 5, characterized in that, The insecticide comprises the naphthoquinone compound, the surfactant, the dissolving agent and water; the naphthoquinone compound is 2-chloro-1,4-naphthoquinone. The dissolving agent comprises dimethyl sulfoxide or N,N-dimethylformamide.

7. The insecticide of claim 4, wherein, The surfactant comprises Tween-20 or Tween-80.

8. A method for aquaculture of aquatic animals, characterized by, The dosage form of the insecticide comprises oral dosage.

9. The method of claim 8, wherein the method further comprises, The insecticide is fed to the aquatic animal during the breeding process of the aquatic animal.

10. The method of claim 9, wherein, In the prevention of the internal parasite disease of the aquatic animal, the insecticide is fed once every other day, totally 7-10 times; the amount of each feeding is 10-20 mg / kg fish weight in terms of 2-chloro-1,4-naphthoquinone. In the treatment of the internal parasite disease of the aquatic animal, the insecticide is fed once a day, totally 5 times; the amount of each feeding is 10 mg / kg fish weight in terms of 2-chloro-1,4-naphthoquinone.

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