Application of alkannin in resisting fish ichthyophthiriasis

Abstract

The invention discloses an application of alkannin in resisting fish ichthyophthiriasis. The alkannin has remarkable killing activity on ichthyophthirius multifilis in an in-vitro stage, the 4-hour half effective insecticidal concentration (4 h-EC50) of the alkannin on a grazing body is 0.048 mg / L, and the 22 h-EC50 of the alkannin on a cyst is 1.275 mg / L. Besides, the shikonin has a 96-hour median lethal concentration (96h-LC50) of 2.229 mg / L to goldfish and a treatment index (TI) of 46.4 to a grazing body, and can be safely applied to fishes in an effective insecticidal concentration range. The shikonin disclosed by the invention has the characteristics of high insecticidal activity and low toxicity to fishes, has the potential of being developed into novel green fish ichthyophthiriasis-resistant aquatic products such as fish medicines, feed additives and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic disease prevention and control, relates to the prevention and control of fish parasitic diseases, and specifically relates to the application of shikonin in killing Ichthyophthirius punctatus, preparing drugs against Ichthyophthirius punctatus disease in fish, feed additives and other aquatic inputs. Background Art

[0002] Ichthyophthirius multifiliis, or Ichthyophthirius multifiliis for short, is a ciliated protozoan that parasitizes the gills and body surface of freshwater fish. It has low host specificity and can therefore infect most freshwater fish, causing fish deaths and bringing huge economic losses to the aquaculture industry. The life cycle of Ichthyophthirius multifiliis includes three main stages: the predator (Theront) and cyst (Tomont) in the in vitro stage, and the trophozoite (Trophont) that parasitizes on the surface of the fish. Since the trophozoite is embedded in the skin, general drugs cannot penetrate, so killing Ichthyophthirius in the in vitro stage is the main way of drug control. For a long time, chemical drugs such as malachite green, mercury preparations and formalin have been widely used to prevent and control the disease, but these chemical drugs have food safety risks and environmental pollution problems, and have been gradually banned in production. Therefore, the development of efficient and green anti-insect drugs has become an urgent need for the industry.

[0003] Shikonin is a dry root extract of the Boraginaceae plant Xinjiang Lithospermum or Inner Mongolia Lithospermum, and is a natural product with a naphthoquinone structure. Modern pharmaceutical research has found that the chemical composition of Lithospermum is complex and contains a variety of active ingredients. The most valuable and reported naphthoquinone compounds are represented by shikonin, whose molecular formula is C 16 H 16 O 5 Shikonin has multiple pharmacological effects such as antibacterial, anti-inflammatory, anti-tumor, anti-allergic, and liver protection. At present, there are no reports on the research and application of shikonin in killing fish parasitic ciliates. The structural formula of shikonin is:

[0004] Summary of the invention

[0005] Purpose of the invention: In order to solve the above technical problems, the purpose of the present invention is to provide an application of shikonin in killing Ichthyophthirius affine; another purpose of the present invention is to provide an application of shikonin in the preparation of aquatic inputs for killing Ichthyophthirius affine in fish.

[0006] Technical solution: On the one hand, the application of shikonin described in the present invention in killing Ichthyophthirius punctatus.

[0007] On the other hand, the invention provides an application in the preparation of aquatic inputs resistant to Ichthyophthirius scoparia.

[0008] Furthermore, the shikonin is levorotatory or dextrorotatory shikonin.

[0009] Furthermore, the aquatic inputs for resisting Ichthyophthirius scoparia include drugs for resisting Ichthyophthirius scoparia or feed additives for resisting Ichthyophthirius scoparia.

[0010] Furthermore, the 96h-LC of shikonin on goldfish 50 The value is 2.229mg / L.

[0011] Furthermore, the shikonin kills Ichthyophthirius gracilis in the cyst stage and / or the predator stage.

[0012] Furthermore, the shikonin is used to kill Ichthyophthirius trichosanthis by soaking, and when killing Ichthyophthirius trichosanthis at the cyst stage, the effective concentration of shikonin is 1-2 mg / L.

[0013] Furthermore, the effective concentration of the shikonin which has a killing effect of more than 90% on cysts is not less than 2 mg / L.

[0014] Furthermore, the shikonin has an effect on the encapsulated 22h-EC 50 It is 1.275 mg / L, and the 95% confidence interval is 1.148-1.402 mg / L.

[0015] Furthermore, the shikonin is used to kill Ichthyophthirius trichosanthis by soaking, and when killing Ichthyophthirius trichosanthis at the predatory stage, the effective concentration of shikonin is 0.05-1 mg / L.

[0016] Furthermore, the effective concentration of the shikonin having a 100% killing effect on predators is not less than 0.2 mg / L.

[0017] Furthermore, the shikonin has an effect on killing the 4h-EC of the predators of Ichthyophthirius spp. 50 It is 0.048 mg / L, and the 95% confidence interval is 0.035-0.052 mg / L.

[0018] Furthermore, shikonin soaking liquid is used to kill Ichthyophthirius gracilis, wherein the shikonin soaking liquid comprises a main active ingredient and a solvent, wherein the main active ingredient is shikonin, and the solvent is dimethyl sulfoxide.

[0019] Furthermore, the preparation method of the shikonin soaking solution is: weigh shikonin powder, add dimethyl sulfoxide and mix well to form a mother solution, and then dilute it for standby use.

[0020] Furthermore, the diluent used in the dilution and standby process is water.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the shikonin prepared by the present invention has a significant killing effect on Ichthyophthirius spp., and the 4h-EC50 The 96h-LC of shikonin on goldfish is 0.048mg / L, which is the strongest known natural compound with insecticidal activity. 50 It is 2.229 mg / L, which can be safely used on fish within the effective insecticidal concentration range. It has the potential for application in aquatic inputs such as anti-fish Ichthyophthirius disease drugs and feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the 4h-EC of shikonin on predators 50 and R 2 value.

[0023] Figure 2 The morphology of predators after shikonin treatment, where A is the control group predator, B is the 0.1 mg / L treatment group predator, the morphology changed from spindle to round, C and D are the 0.2 mg / L treatment group predators, cell membrane rupture, content outflow and other phenomena.

[0024] Figure 3 The effect of shikonin on the encapsulated 22h-EC 50 and R 2 value.

[0025] Figure 4 These are hatched and unhatched cysts after shikonin treatment, where A is the cysts in the control group that have entered the division stage, B is the cysts in the 0.1 mg / L treatment group, with cell membrane rupture and content outflow, and C and D are the cysts in the 0.5 mg / L treatment group and the 2 mg / L treatment group, respectively, with cyst cell membrane rupture.

[0026] Figure 5 The 96h-LC of shikonin on goldfish 50 . DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0029] Preparation method of shikonin solution: accurately weigh 10 mg of L-shikonin powder using an electronic balance, fully dissolve it with 1 mL of dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL stock solution.

[0030] Collection and counting method of Ichthyophthirius cysts: Place the goldfish infected with Ichthyophthirius cysts in a beaker filled with 100 mL of distilled water, stimulate it slightly with a glass rod and let it stand for 10 minutes. The mature Ichthyophthirius cysts will fall off and settle at the bottom of the beaker. Suck out the Ichthyophthirius cysts with a rubber-tipped pipette, place them in a culture dish, and wash them repeatedly with distilled water for 3 times. Count the cysts under a microscope and collect the cysts for subsequent experiments.

[0031] Collection and counting method of Ichthyophthirius punctatus predators: Place cleaned Ichthyophthirius cysts in a 96-well plate, with 5-10 cysts and 200 μL of distilled water in each well. Then place the 96-well plate in a constant temperature incubator at 22°C. After 22 hours of incubation, stir the distilled water containing the predators evenly, pipette 100 μL and fix it with formaldehyde solution, and count it with a plankton counting plate and microscope after gradient dilution. The number of predators in the mother solution is calculated based on the average number of predators and the dilution multiple. The calculation formula for the number of predators is: average number × dilution multiple.

[0032] Example 1: Application of shikonin in killing Ichthyophthirius punctatus predators

[0033] The predators were placed in a 96-well plate, with 100 predators in each well. Different concentrations of shikonin solution were added to the 96-well plate in sequence, with the final concentrations of each well being 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / L, respectively. Only distilled water was added to the control group, and the 96-well plate was placed in a constant temperature incubator at 22°C. After 4 hours, the morphology of the Ichthyophthirius predators was observed under a microscope, and the survival of the predators was recorded. The predators were considered dead when the following conditions occurred: the insect body became round, the structure was blurred, the original transparency was lost, the cilia stopped moving, or the insect body was broken. The experiment was repeated 3 times.

[0034] The insecticidal results of shikonin are shown in Table 1. After 4 hours, the average mortality rate of the Ichthyophthirius predators in the control group was 12.7%. As the concentration of shikonin solution increased, the mortality rate of the Ichthyophthirius predators showed a clear upward trend. When the concentration of shikonin was 0.05 mg / L, the mortality rate of the predators was 53.6%, significantly higher than that of the control group. When the concentration of shikonin was 0.1 mg / L, the mortality rate of the Ichthyophthirius predators reached 97.7%, and when the concentration reached 0.2 mg / L, the mortality rate of the predators was 100%. After calculation ( Figure 1 ), 4h EC of shikonin killing Ichthyophthirius punctatus predators 50 It is 0.048 mg / L (95% confidence interval is 0.035-0.052 mg / L).

[0035] Table 1 Average mortality of Ichthyophthirius punctatus predators after 4 h of shikonin treatment

[0036]

[0037] Note: One-way ANOVA was used to analyze the differences among the groups. There were significant differences among the groups with different letters.

[0038] Example 2: Application of shikonin in killing Ichthyophthirius cysts

[0039] Cysts were collected manually and placed in a 24-well plate at a rate of 10 per well. Shikonin solutions with concentrations of 0, 0.1, 0.5, 1.0, 2.0, and 4.0 mg / L were added to the 24-well plate, respectively. Only distilled water was added to the control group.

[0040] The 24-well plate was placed in a constant temperature incubator. After 22 hours, the survival of Ichthyophthirius cysts was observed under a microscope and counted. The cyst death was determined by the absence of cyst division to hatch predators or cyst membrane rupture. The experimental temperature was 22.0±1.0℃. The experiment was repeated 3 times.

[0041] The results of shikonin insecticide are shown in Table 2. When the concentration of shikonin was 1 mg / L, the mortality rate of cysts was 21.7%, which was significantly different from the control group. As the concentration of shikonin increased, the mortality rate of cysts increased. When the concentration of shikonin was 2 mg / L, the mortality rate reached 91.7%. After calculation ( Figure 3 ), 22h EC of shikonin killing cysts 50 It is 1.275mg / L.

[0042] Table 2 Average mortality of cysts after 22h of shikonin treatment

[0043]

[0044] Note: One-way ANOVA was used to analyze the differences among the groups. There were significant differences among the groups with different letters.

[0045] Example 3: 96h acute toxicity of shikonin to goldfish

[0046] Healthy goldfish were placed in fish tanks containing different concentrations of shikonin, with 5 goldfish in each tank. According to the results of the previous pre-experiment, the concentrations of shikonin were set to 1, 2, 3, 4, 6, and 8 mg / L. At the same time, aerated tap water was set as the control group. During the experiment, the goldfish were observed every 12 hours, the number of deaths was recorded, and the mortality rate of goldfish was calculated at 24, 48, 72, and 96 hours. In the experiment, a glass rod was used to touch the tail of the goldfish. If the fish did not react, it was judged to be completely dead. The goldfish were fished out in time after death to prevent it from affecting the water quality.

[0047] Table 3 Mortality of goldfish after 96h of shikonin treatment

[0048]

[0049] Table 4 Data of acute toxicity test of shikonin on goldfish

[0050]

[0051] The results of acute toxicity of shikonin showed that the mortality rate of goldfish was less than 50% after treatment with 2mg / L shikonin. In the group treated with 6mg / L shikonin, the goldfish swam violently, and some fish gradually became restless. As time went on, the mortality rate of goldfish was 100% after 96h (Table 3). After calculation (Table 4), the 96h-LC of shikonin on goldfish 50 It is 2.229 mg / L, and the 95% confidence interval is 1.606-2.874 mg / L.

Claims

1. Application of shikonin in killing Ichthyophthirius punctatus.

2. Application of shikonin in the preparation of aquatic inputs to resist Ichthyophthirius scoparia disease in fish.

3. The use according to claim 1 or 2, characterized in that: The shikonin is left-handed or right-handed shikonin.

4. The use according to claim 2, characterized in that: The aquatic inputs for resisting fish Ichthyophthirius spp. include drugs for resisting fish Ichthyophthirius spp. or feed additives for resisting fish Ichthyophthirius spp.

5. The use according to claim 1 or 2, characterized in that: The shikonin kills Ichthyophthirius gracilis at the cyst stage and / or the predator stage.

6. The use according to claim 5, characterized in that: The shikonin is used to kill Ichthyophthirius spp. by soaking. When killing Ichthyophthirius spp. in the cyst stage, the effective concentration of the shikonin is 1-2 mg / L.

7. The use according to claim 5, characterized in that: The shikonin is used to kill Ichthyophthirius trichosanthis by soaking. When killing Ichthyophthirius trichosanthis at the predatory stage, the effective concentration of the shikonin is 0.05-1 mg / L.

8. The use according to claim 5, characterized in that: The shikonin soaking liquid is used to kill Ichthyophthirius spp. The shikonin soaking liquid comprises a main active ingredient and a solvent, wherein the main active ingredient is shikonin and the solvent is dimethyl sulfoxide.

9. The use according to claim 8, characterized in that: The preparation method of the shikonin soaking solution is as follows: weigh shikonin powder, add dimethyl sulfoxide and mix well to form a mother solution, and then dilute and set aside.

10. The use according to claim 9, characterized in that: The diluent used in the dilution and standby process is water.

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