Fish antiviral active drug and application thereof

Abstract

The invention relates to the crossing field of aquatic disease prevention and control and natural product antiviral drug research, in particular to a fish antiviral active drug and application thereof. The antiviral activity of the prodigiosin on the IHNV and the SVCV is defined for the first time, the application field of the prodigiosin is expanded, and a new lead compound can be provided for development of drugs for resisting the IHNV and the SVCV.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of aquatic disease prevention and control and natural product antiviral drug research, in particular to a fish antiviral active drug and its application. BACKGROUND

[0002] Prodigiosin (PG) is a natural secondary metabolite synthesized by bacteria such as Serratia marcescens. Its chemical structure is C 20 H 25 N3O, with a molecular weight of 323.43 and a core skeleton of a tripyrrole ring structure. One of the pyrrole rings is substituted with a methyl group at C-2 and a pentyl group at C-3, and the other pyrrole ring has a methoxy modification. It is a dark red solid and is soluble in organic solvents such as methanol, ethanol, and acetonitrile. It has good chemical modification and preparation basis.

[0003] In recent years, the biological activity of prodigiosin has attracted much attention, and it has shown significant potential in the fields of antibacterial, antitumor, immunomodulation, and antiviral. As a natural monomer compound with a clear structure, prodigiosin has multi-target antiviral activity, environmental compatibility, and industrial production feasibility, which can break through the limitations of existing prevention and control technologies and provide innovative solutions for the green prevention and control of infectious hematopoietic necrosis virus (IHNV) and spring viremia of carp virus (SVCV).

[0004] In the field of aquaculture, IHNV and SVCV are members of the Rhabdoviridae family and are important pathogens that harm the aquaculture industry. IHNV mainly infects salmonids such as rainbow trout and salmon, and is particularly pathogenic to fry and juvenile fish, with a mortality rate of 80-100%, resulting in serious economic losses to the aquaculture industry. SVCV has a wide host range and can infect various cyprinids such as carp, crucian carp, and grass carp, causing symptoms such as systemic hemorrhage and edema, with a mortality rate of over 70%, and is listed as an important aquatic animal disease.

[0005] Currently, the prevention and control of IHNV and SVCV have significant technical bottlenecks: 1) Fish vaccine development is limited by factors such as viral serotype diversity and rapid mutation rate, and cross-protection effect is not good; 2) Chemical synthesis of antiviral drugs can cause water pollution, drug residues, and viral resistance, which violates the concept of ecological aquaculture; 3) Existing plant extract preparations have complex components, making quality control difficult, and the antiviral effect is unstable.

[0006] In the field of antiviral, it has been confirmed that prodigiosin has high inhibitory effect on MSRV, PVY and other viruses, and plays an antiviral effect through mechanisms such as stabilizing host p53 protein, inducing lysosome alkalization or regulating host immune pathways, and has the characteristics of natural source, low toxicity and low residue, good environmental compatibility, which meets the demand of green development of aquaculture.

[0007] However, the systematic study and application of prodigiosin in the prevention and control of important pathogenic viruses IHNV and SVCV in fish are still blank. Therefore, the research on the resistance of prodigiosin to IHNV and SVCV has important significance for breaking through the bottleneck of existing technology and developing green antiviral drugs. SUMMARY

[0008] The present application aims to provide a new use of prodigiosin, i.e. fish antiviral active drugs and their applications.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions: The present application provides the use of prodigiosin related substances in the preparation of a drug for improving the survival rate of fish infected with infectious hematopoietic necrosis virus and / or spring viremia of carp virus, wherein the administration concentration of the prodigiosin related substances is 0.1-0.8 mg / kg (based on the weight of the fish).

[0010] The present application provides the use of prodigiosin related substances in the preparation of a drug for reducing the viral load of fish infected with infectious hematopoietic necrosis virus and / or spring viremia of carp virus, wherein the administration concentration of the prodigiosin related substances is 0.1-0.8 mg / kg (based on the weight of the fish).

[0011] Preferably, the administration concentration of the prodigiosin related substances is 0.2 mg / kg (based on the weight of the fish).

[0012] Preferably, the administration concentration of the prodigiosin related substances is 0.8 mg / kg (based on the weight of the fish).

[0013] Preferably, the prodigiosin related substances include prodigiosin and pharmaceutically acceptable salts of prodigiosin.

[0014] In the present application, the prodigiosin related substances include two categories, one is prodigiosin itself (prodigiosin), and the other is its pharmaceutically acceptable salt.

[0015] Preferably, the dosage form of the drug is selected from at least one of injection, soaking agent and oral preparation.

[0016] This invention provides a pharmaceutical composition for combating infectious hematopoietic necrosis virus and / or carp viremia virus, comprising a safe and effective amount of styraxone-related substances, said styraxone-related substances comprising styraxone and pharmaceutically acceptable salts of styraxone.

[0017] Preferably, the dosage of the lecithin-related substance is 0.1-0.8 mg / kg (based on fish body weight).

[0018] Preferably, the dosage of the lecithin-related substance is 0.2 mg / kg or 0.8 mg / kg (based on fish body weight).

[0019] The present invention provides a pharmaceutical composition for treating fish rhabdovirus, comprising a safe and effective amount of an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is selected from styraxone and a pharmaceutically acceptable salt of styraxone.

[0020] Preferably, the pharmaceutical composition is administered by injection, immersion, or oral administration.

[0021] Preferably, the pharmaceutically acceptable carrier is adapted to the administration needs of aquatic animals and is selected from at least one of buffer solutions, solubilizers, stabilizers, and dispersants.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The antiviral activity of styraxone against IHNV and SVCV was first clearly demonstrated, which expanded the application field of styraxone and can provide new lead compounds for the development of anti-IHNV and SVCV drugs.

[0023] (2) Lemon rubesin works by inhibiting viral infection, replication and regulating the host immune response. Its mechanism of action is diverse, it is not easy to induce viral drug resistance, and its prevention and control effect is stable.

[0024] (3) Lingjunhongxin is derived from microbial metabolism and has the characteristics of low toxicity, low residue and easy degradation. It will not cause pollution to the water environment and meets the development needs of green aquaculture.

[0025] (4) The drug composition containing styraxin has a flexible dosage form, is suitable for different aquaculture scenarios and fish species, is easy to operate, and is easy to promote and apply on a large scale. Attached Figure Description

[0026] Figure 1 This is the absolute quantitative standard curve of styraxin against IHNV; the horizontal axis represents the logarithmic value of IHNV copy number, and the vertical axis represents the Ct value. Figure 2 This is the absolute quantitative standard curve of styraxanthin against SVCV; the horizontal axis represents the logarithm of SVCV copy number, and the vertical axis represents the Ct value. Figure 3 The inhibition rate of IHNV in rainbow trout treated with different concentrations of styraxin (0.2 and 0.8 mg / kg) was determined. Figure 4 Cumulative survival curves of rainbow trout treated with different concentrations of styraxone (0.2 and 0.8 mg / kg); Figure 5 The inhibition rate of SVCV in carp treated with different concentrations of styraxone (0.2 and 0.8 mg / kg); Figure 6 Cumulative survival rate curves of carp treated with different concentrations of levofloxacin (0.2 and 0.8 mg / kg). Detailed Implementation

[0027] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0028] As a naturally derived secondary metabolite, squalene possesses a unique tripyrrole ring structure that endows it with highly efficient antiviral activity. Furthermore, it has abundant chemical modification sites, allowing for further enhancement of bioavailability through structural optimization. This invention, through in vitro and in vivo experiments, verifies that squalene can significantly inhibit the replication of IHNV and SVCV, reduce viral pathogenicity to the host, improve the survival rate of infected fish, and demonstrates high safety for farmed fish with no significant toxic side effects.

[0029] Example 1 This example illustrates the determination of the anti-IHNV activity of styrax rubigin. (1) Test materials Virus material: IHNV was isolated, identified and preserved by the Aquatic Diseases Laboratory of Northwest A&F University; Preparation of test solutions: Accurately weigh the styrax rubigin standard, prepare a stock solution of 100 mg / mL with chromatographic grade DMSO, and then dilute it to 10, 20, 40, 60, and 80 mg / mL working solutions. After filtration through a 0.22 μm organic filter, store the solution in a brown chromatographic bottle protected from light. Experimental animals: Rainbow trout juveniles (weight 6±0.3 g, used for IHNV infection test), purchased from Shihezi Reservoir in Gansu Province, were temporarily raised for 1 week, and healthy, disease-free individuals were selected for the experiment.

[0030] (2) Safety evaluation Healthy rainbow trout juveniles were randomly selected and divided into 8 groups of 15 fish each, and placed in a recirculating aquaculture tank at 16℃ (rainbow trout). A blank control group was prepared with a buffer solution (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5), and drug treatment groups were prepared by injection of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg / kg of styraxin, with each fish receiving 100 μL of injection. Fish were observed for 72 hours, and survival and any abnormal symptoms were recorded.

[0031] The results showed that within the concentration range of 0.1-0.8 mg / kg, none of the rainbow trout juveniles died, and there were no abnormal swimming or loss of appetite or other discomfort symptoms. At a concentration of 1.6 mg / kg, the survival rate of rainbow trout was 84.6%, indicating that the upper limit of the safe concentration of styraxone for experimental fish is higher than 0.8 mg / kg, but should not exceed 1.6 mg / kg. The concentration range of 0.1-0.8 mg / kg will be used in subsequent experiments.

[0032] (3) Antiviral activity detection Group design: Rainbow trout were divided into 4 groups of 33 each: a blank control group (injected with buffer, i.e., Control group), a positive control group (injected with virus solution, i.e., IHNV group), a low-dose styraxin group (0.2 mg / kg), and a high-dose styraxin group (0.8 mg / kg), with 3 replicates per group. The buffer for the blank control group was TM buffer (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5).

[0033] Treatment: Inject 100 μL of virus solution into the abdomen of each fish (IHNV dose is 5.2 × 10⁻⁶). 6 (Copies / each), 100 μL of erythromycin was injected intramuscularly from the back.

[0034] Sample collection and testing: At 48 h and 72 h post-injection, three animals were randomly selected from each group, and kidney and spleen tissues were collected. Nucleic acid was extracted using a genomic DNA / RNA extraction kit, and viral copy number was detected by RT-qPCR (primer sequences are shown in Table 1). An absolute quantitative standard curve was used. Figure 1 ) Calculate viral load.

[0035] Table 1 Primer sequences for quantitative detection of IHNV

[0036] RT-qPCR reaction system: 7.5 μL 2×SYBR Green Master Mix, 0.3 μL each of forward and reverse primers (10 μM), 1 μL template, and ddH2O to a final volume of 15 μL. Reaction program: 95℃ pre-denaturation for 5 min; 95℃ for 10 s, 56℃ for 30 s, 40 cycles.

[0037] The results show that ( Figure 3 ): 72 h after injection, the low- and high-dose groups of styraxin showed inhibition rates of 60.6% and 73.6% against IHNV, respectively, significantly reducing the replication level of IHNV in fish. The control group, however, showed an inhibition rate of 0% against IHNV.

[0038] (4) Survival rate detection The fish in the above groups were observed continuously for 14 days, and the number of deaths was recorded daily to calculate the cumulative survival rate.

[0039] The results show that ( Figure 4 ): Positive control group: Rainbow trout had a survival rate of 0% at 14 days; Low-dose styraxone group: Rainbow trout survival rate was 14.8%; High-dose styraxone group: Rainbow trout survival rate was 25.9%; The survival rate of the blank control group was 100%.

[0040] Example 2 This example illustrates the determination of the anti-SVCV activity of styraxone. (1) Test materials Viral material: SVCV was isolated, identified and preserved by the Aquatic Diseases Laboratory of Northwest A&F University; Preparation of test solutions: Accurately weigh the styrax rubigin standard, prepare a stock solution of 100 mg / mL with chromatographic grade DMSO, and then dilute it to 10, 20, 40, 60, and 80 mg / mL working solutions. After filtration through a 0.22 μm organic filter, store the solution in a brown chromatographic bottle protected from light. Experimental animals: juvenile carp (weight 8.3±0.4 g, used for SVCV infection test), purchased from the Zhuque Road Flower, Bird, Fish and Insect Market in Xi'an, Shaanxi Province. After being temporarily raised for 1 week, healthy and disease-free individuals were selected for the experiment.

[0041] (2) Safety evaluation Healthy juvenile carp were randomly selected and divided into 8 groups of 15 fish each, and placed in a recirculating aquaculture tank at 18℃ (carp). A blank control group was prepared using a buffer solution (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5), and drug treatment groups were prepared by injection of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg / kg of styraxin, with each fish receiving 100 μL of the solution. Fish were observed for 72 hours, and their survival and any abnormal symptoms were recorded.

[0042] The results showed that within the concentration range of 0.1-0.8 mg / kg, none of the juvenile carp died, and there were no abnormal swimming or loss of appetite or other discomfort symptoms. At a concentration of 1.6 mg / kg, the survival rate of juvenile carp was 79.3%, indicating that the upper limit of the safe concentration of styraxanthin for experimental fish is higher than 0.8 mg / kg, but should not exceed 1.6 mg / kg. The concentration range of 0.1-0.8 mg / kg will be used in subsequent experiments.

[0043] (3) Antiviral activity detection Group design: Carp were divided into 4 groups of 33 each, including a blank control group (injected with buffer, i.e., Control group), a positive control group (injected with virus solution, i.e., SVCV group), a low-dose styraxin group (0.2 mg / kg), and a high-dose styraxin group (0.8 mg / kg), with 3 replicates per group. The buffer for the blank control group was TM buffer (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5).

[0044] Treatment: Inject 100 μL of virus solution (SVCV dose of 3.7 × 10⁻⁶) into the abdomen of each carp. 6 (Copies / each), 100 μL of erythromycin was injected intramuscularly from the back.

[0045] Sample collection and testing: At 48 h and 72 h post-injection, three animals were randomly selected from each group, and kidney and spleen tissues were collected. Nucleic acid was extracted using a genomic DNA / RNA extraction kit, and viral copy number was detected by RT-qPCR (primer sequences are shown in Table 2). An absolute quantitative standard curve was used. Figure 2 ) Calculate viral load.

[0046] Table 2 Primer sequences for SVCV quantitative detection

[0047] RT-qPCR reaction system: 7.5 μL 2×SYBR Green Master Mix, 0.3 μL each of forward and reverse primers (10 μM), 1 μL template, and ddH2O to a final volume of 15 μL. Reaction program: 95℃ pre-denaturation for 5 min; 95℃ for 10 s, 56℃ for 30 s, 40 cycles.

[0048] The results show that ( Figure 5 ): 48 h after injection, the low- and high-dose groups of styraxin showed inhibition rates of 62.2% and 75.5% against SVCV, respectively, significantly reducing the replication level of SVCV in fish. The control group, however, showed an inhibition rate of 0% against SVCV.

[0049] (4) Survival rate detection The above-mentioned grouped fish were observed continuously for 14 days, with the number of deaths recorded daily and the cumulative survival rate calculated. The results showed that ( Figure 6 ): Positive control group: The survival rate of carp at 14 days was 11.1%.

[0050] Low-dose group of styraxone: The survival rate of carp was 29.6%; High-dose group of lecithin: The survival rate of carp was 37%; The survival rate of the blank control group was 100%.

[0051] In summary, styraxin can significantly inhibit the replication of IHNV and SVCV in fish and improve the survival rate of infected fish in a dose-dependent manner, demonstrating good antiviral activity against both rhabdoviruses.

[0052] The results of the embodiments of the present invention fully demonstrate that styraxin can effectively prevent and control IHNV and SVCV infections in fish. Its low toxicity, green and high efficiency make it an ideal candidate preparation to replace chemical drugs in aquaculture, providing a new technical means for the prevention and control of two important aquatic viral diseases, and laying the foundation for the application of styraxin in the prevention and control of other aquatic viruses.

[0053] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Claims

1. The use of styrax-related substances in the preparation of a drug for improving the survival rate of fish infected with infectious hematopoietic necrosis virus and / or carp spring viremia virus, characterized in that, The lecithin-related substances include lecithin and pharmaceutically acceptable salts of lecithin, and the dosage concentration of the lecithin-related substances is 0.1-0.8 mg / kg.

2. The use of styrax-related substances in the preparation of drugs for reducing viral load in fish infected with infectious hematopoietic necrosis virus and / or carp spring viremia virus, characterized in that, The lecithin-related substances include lecithin and pharmaceutically acceptable salts of lecithin, and the dosage concentration of the lecithin-related substances is 0.1-0.8 mg / kg.

3. The application according to claim 1 or 2, characterized in that, The dosage concentration of the lecithin-related substance is 0.2 mg / kg.

4. The application according to claim 1 or 2, characterized in that, The dosage concentration of the lecithin-related substance is 0.8 mg / kg.

5. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is selected from at least one of injections, soaking solutions, and oral preparations.

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