Application of epigallocatechin gallate in preparation of drugs for resisting nervous necrosis viruses of fishes

Abstract

The invention provides an application of epigallocatechin gallate in preparation of a medicine for resisting nervous necrosis viruses of fishes. Related experimental results show that the EGCG is used for preparing the medicine for resisting the nervous necrosis viruses of the fishes, infection of the nervous necrosis viruses of the fishes, especially groupers, can be effectively prevented and treated, the economic benefits of cultivation are improved, and a good ecological environment is maintained.

Description

Technical Field

[0001] The invention belongs to the field of aquatic animal disease prevention and treatment, and particularly relates to application of epigallocatechin gallate in the preparation of a drug for resisting fish neuronecrosis virus. Background Art

[0002] Nervous necrosis virus (NNV) is one of the major pathogens that endanger the aquaculture industry. It can infect a variety of marine fish such as grouper, sea bass and redfin pufferfish, seriously damaging the visual system, nervous system, and immune function of fish, leading to the death of fish and causing huge economic losses to the global marine fish farming industry.

[0003] Neurological necrosis virus (NNV) affects a wide range of fish species, and grouper is one of the most severely affected. Grouper is highly susceptible to NNV. Because their immune systems are not yet fully developed in juvenile grouper, the risk of infection is greatly increased, and the mortality rate for infected juvenile grouper can reach over 90%.

[0004] Currently, there are no commercially available drugs that effectively inhibit the Nervous Necrosis Virus (NNV), a serious constraint on the sustainable development of grouper farming and the aquaculture industry as a whole. To ensure the healthy development of the aquaculture industry, we are actively exploring new methods and approaches to develop highly effective NNV drugs for fish, particularly those targeting grouper species.

[0005] my country is rich in medicinal plant resources, and medicinal plants contain a wide variety of natural active ingredients. Active ingredients such as alkaloids, polysaccharides, and flavonoids have rich biological functions, especially showing great potential in antiviral activities. Studies have shown that medicinal plants and their active ingredients can affect the structure of viral particles and interfere with the life cycle of the virus. Compared with traditional antiviral drugs, the natural active ingredients in medicinal plants have gradually become a new direction in the research and development of antiviral drugs due to their significant advantages such as natural origin, low resistance to drug resistance, and environmental friendliness. The development of new plant-based fishery drugs can not only ensure the healthy growth of fish and improve the quality and safety of aquatic products, but also protect the ecological environment and promote the sustainable development of the aquaculture industry. It has broad application prospects. Summary of the Invention

[0006] In order to solve the problems and shortcomings in the prior art, the present invention provides an application of epigallocatechin gallate in the preparation of an anti-fish neuronecrosis virus drug.

[0007] According to a first aspect of the present invention, there is provided a use of epigallocatechin gallate in the preparation of an anti-fish neuronecrosis virus drug.

[0008] Fish Nervous Necrosis Virus (FNNV) can damage the nervous system, restrict organ function, and reduce immune function in fish, leading to various problems and mortality. This has severely impacted the healthy development of the fish farming industry. Effective prevention and control of FNNV infection and outbreaks is crucial to ensuring healthy fish farming. As one of the viral pathogens responsible for a high mortality rate in grouper, FNNV has caused significant economic losses to the grouper farming industry.

[0009] Epigallocatechin gallate (EGCG) is a natural polyphenol compound primarily found in green tea. EGCG exhibits a wide range of pharmacological activities. Studies have shown that, through its antioxidant effects, EGCG can protect normal cellular function and reduce the risk of disease. EGCG can also inhibit β-amyloid aggregation and Tau protein hyperphosphorylation, mitigate neuronal damage, activate related signaling pathways, promote neuronal repair, and improve motor function. Furthermore, EGCG has inhibitory effects against pathogens such as Streptococcus mutans, Helicobacter pylori, influenza virus, and hepatitis B virus. However, limited research has been conducted on the application of EGCG in the treatment of fish neuronecrosis virus.

[0010] In particular, relevant experimental results show that the present invention uses EGCG to prepare drugs against fish nervous necrosis virus, which can effectively prevent and treat fish, especially grouper, from being infected with nervous necrosis virus, improve the economic benefits of aquaculture, and maintain a good ecological environment.

[0011] Here, it should be noted that there are many viruses related to fish, and there are many differences between fish neuronecrosis virus and other fish viruses such as herpes virus, reovirus, and rhabdovirus. That is, different fish viruses have great differences in virus structure, infection mechanism, pathological characteristics, antiviral mechanism, etc., just like human viral infections or various diseases also have differences in many aspects, mainly reflected in virus structure, infection mechanism, pathological characteristics, clinical manifestations, transmission routes, prevention and treatment, etc.

[0012] Therefore, for different fish viruses, it is necessary to develop drugs suitable for different fish viruses by focusing on the structure, infection mechanism, pathological characteristics, antiviral mechanism, etc. Generally speaking, different fish viruses vary greatly in various aspects, so the drugs suitable for them also vary greatly. Generally speaking, simple analogies cannot be made, otherwise adverse reactions or side effects may occur.

[0013] The present invention has been verified through relevant experiments that the use of EGCG to prepare drugs against fish nervous necrosis virus can effectively prevent and treat infection of fish, especially grouper, with nervous necrosis virus. This is mainly due to the fact that EGCG can have a significant inhibitory effect on the replication stage of fish nervous necrosis virus at the cellular level, and its biological toxicity is extremely low. In-depth research has found that EGCG can significantly inhibit fish nervous necrosis virus. Its mechanism of action is to destroy the viral particle structure of fish nervous necrosis virus and interfere with the adsorption, invasion and replication stages of the virus in the process of infecting host cells. Moreover, EGCG is the component with the highest content in catechins, and has multiple effects such as enhancing immunity, anti-oxidation and anti-inflammatory, and also has the characteristic of extremely low biological toxicity. Based on these characteristics, EGCG has extremely high application value in the field of prevention and treatment of fish nervous necrosis virus, and can effectively improve the immune ability of fish themselves. It has important economic significance for promoting the healthy development of fish, increasing fish yield and quality, and further developing aquaculture.

[0014] Preferably, the fish neuronecrosis virus includes grouper neuronecrosis virus. Grouper is a marine fish with important economic and edible value and a large market demand, resulting in the rapid development of artificial breeding. However, artificial breeding is affected by the breeding environment and large production quantities, making it more prone to various diseases during the breeding process. Among them, grouper neuronecrosis virus is a disease that is more prone to occur during artificial breeding. EGCG can have a good therapeutic effect on grouper neuronecrosis virus and greatly improve the grouper's own immune capacity, thereby reducing the occurrence of diseases in grouper during the breeding process, especially infection with neuronecrosis virus.

[0015] Preferably, the source of epigallocatechin gallate includes green tea. EGCG extracted from green tea can better ensure a healthy and harmless source, thus being more beneficial for preventing and treating fish neuronecrosis virus, while also reducing side effects on fish and improving fish survival rates.

[0016] Preferably, the dosage form of the drug includes at least one of liquid, suspension, tablet, granule, and powder.

[0017] Preferably, the dosage form of the drug includes a liquid agent, in which the concentration of epigallocatechin gallate is 6 to 17 μg / mL. At this relatively low concentration, it can effectively prevent and treat fish neuronecrosis virus. At the same time, if the EGCG concentration is too low, the prevention and treatment effect on fish neuronecrosis virus is limited, which can easily cause repeated infection of fish neuronecrosis virus and reduce the survival rate of fish. If the EGCG concentration is too high, it can easily cause fish poisoning, causing adverse side effects in fish, and is also not conducive to the survival rate of fish.

[0018] Preferably, the solvent in the above-mentioned liquid agent includes dimethyl sulfoxide (DMSO). EGCG is preferably dissolved in DMSO to a concentration of 100 mg / ml as a stock solution, and the stock solution is diluted with L15 culture medium to act as an anti-fish neural necrosis virus agent. This agent prevents and inhibits the fish neural necrosis virus.

[0019] Preferably, the dosage form of the drug comprises a liquid agent, in which the concentration of epigallocatechin gallate is 12.5 μg / mL.

[0020] According to a second aspect of the present invention, there is provided an anti-fish nerve necrosis virus drug comprising epigallocatechin gallate.

[0021] According to a third aspect of the present invention, a method for preparing fish feed resistant to fish necrosis virus (FNV) is provided. Furthermore, the use of EGCG in preparing fish feed can effectively prevent and treat FNV, while not reacting adversely with other ingredients in the feed. Instead, it facilitates the absorption, digestion, and conversion of the feed by fish, thereby enhancing the fish's immunity and thereby increasing the yield and quantity of fish.

[0022] Preferably, the fish nervous necrosis virus includes grouper nervous necrosis virus.

[0023] Preferably, the mass proportion of epigallocatechin gallate in the fish feed is 0.01-0.05%.

[0024] In summary, epigallocatechin gallate can effectively prevent and treat fish nervous necrosis virus, especially grouper nervous necrosis virus. It also meets the requirements of safe, environmentally friendly and sustainable development, providing strong support for the healthy development of high-quality ecological aquaculture models for fish, especially grouper. Therefore, the use of epigallocatechin gallate to prepare drugs or feeds against fish nervous necrosis virus is of great significance for reducing nervous necrosis virus infection during fish farming, improving fish's own immune capacity, and optimizing the health and economic development of the fish farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram showing the effect of EGCG on the cytotoxicity of spleen tissue in Example 1; wherein, Figure 1 (A) The results of incubation of TOSF cells with EGCG; Figure 1 (B) The survival rate of TOSF cells.

[0026] Figure 2This is a diagram showing the inhibitory effect of EGCG on fish neural necrosis virus at different concentrations in Example 2.

[0027] Figure 3 This is a diagram showing how EGCG destroys the structure of fish neural necrosis virus particles and inhibits neural necrosis virus infection in Example 3.

[0028] Figure 4 This is a diagram showing the effect of EGCG interfering with the adsorption of fish neural necrosis virus on the host cell surface in Example 4, thereby exerting an anti-neural necrosis virus infection effect.

[0029] Figure 5 This is a diagram showing the effect of EGCG interfering with the invasion of fish neural necrosis virus into host cells and thus exerting an anti-neural necrosis virus infection effect in Example 5.

[0030] Figure 6 This is a diagram showing the effect of EGCG interfering with the replication process of fish nervous necrosis virus in host cells and thus exerting an anti-nerve necrosis virus infection effect in Example 6. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0032] The quantitative experimental data involved in the following examples are expressed as mean ± standard deviation (±s), and the inter-group comparison data were statistically processed using SPSS 17.0 statistical software using the one-way level variance analysis method.

[0033] Spleen tissue cell line of Pomfret ovata ( Trachinotus ovatus splenic fibroblasts (TOSF) are stored in this laboratory and are available to the public from the applicant for use only in repeating the experiments of the present invention.

[0034] The fish neurological necrosis virus (NNV) was isolated from the artificially cultured pearl grouper in Guangxi and stored in the applicant's laboratory. The public can obtain it from the applicant for limited use in repeating the experiments of the present invention.

[0035] EGCG: product of Shanghai MacLean Biochemical Technology Co., Ltd. (analytical purity >98%), product number E808891, CAS: 989-51-5.

[0036] Primers for the fish neuronecrosis virus capsid protein (CP) gene were as follows: forward primer (CP-F) 5'-CAACTGACAACGATCACACCTTC -3', reverse primer (CP-R) 5'-CAATCGAACACTCCAGCGACA -3'. Primers for the internal reference gene β-actin were as follows: forward primer (β-actin-F) 5'-GCTACGTCGCCCTGGACTTC -3', reverse primer (β-actin-R) 5'-CTCATGGATTCCGCAGGACTC -3'. These primers were synthesized by Shanghai Bioengineering.

[0037] Example 1 1. Main instruments and reagents Optical microscope, microplate reader, Cell Counting Kit-8 solution (CCK-8 solution).

[0038] 2. Experimental Methods TOSF cells (0.9 × 10 6 / mL) were inoculated into 96-well plates and cultured at 28°C for 18 h. The EGCG stock solution was diluted to 12.5 μg / mL with L15 medium, and then 100 μL of the drug solution was added to the well plate. The drug solution and TOSF cells were incubated together at 28°C. The experimental group set three time points of 24 h, 48 h and 72 h. The TOSF cells were observed under a light microscope to confirm whether 12.5 μg / mL of EGCG would cause damage to TOSF cells. The control group only added L15 medium. Observe the cell morphology and take pictures to record. Discard the supernatant and add CCK-8 solution. Incubate at room temperature in the dark for 4 h. Set the wavelength of the microplate reader to 450 nm and measure the absorbance value of each well. The formula for calculating cell viability is as follows: .

[0039] 3. Experimental Results The results of incubation of TOSF cells with EGCG were as follows Figure 1 As shown in (A), it can be seen that 12.5 μg / mL EGCG had no significant effect on TOSF cells at 24 h, 48 h, and 72 h in the experiment, and the cell morphology was consistent with that of the control group, without any obvious changes.

[0040] Figure 1 (B) is the survival rate of TOSF cells. When EGCG at a concentration of 12.5 μg / mL acted on TOSF cells at three time points, EGCG did not produce obvious cytotoxicity compared with the control group, and the cell survival rate exceeded 95%.

[0041] The above results indicate that EGCG at an appropriate concentration has little toxicity to TOSF cells.

[0042] Example 2 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0043] 2. Experimental Methods TOSF cells (0.9 × 10 6 The cells were inoculated into 12-well plates and cultured at 28°C for 18 h. The culture medium was discarded, and only 800 μL of L15 culture medium was added to the control group, and 8 μL (10 6 TCID 50 / mL) NNV + 800 μL L15 medium. The experimental group added 800 μL of three concentrations (12.5, 6.25, and 3.125 μg / mL) of EGCG + 8 μL NNV. After NNV infection for 48 h, TOSF cells were collected and total RNA was extracted. RT-qPCR was used to detect NNV. CP Relative gene expression. Each sample was replicated three times.

[0044] 3. Experimental Results Figure 2 The results of RT-qPCR show that compared with the NNV group, the TOSF cells inoculated with NNV+EGCG showed that NNV CP The relative expression level of the gene was significantly reduced, confirming that EGCG has a good antiviral effect on fish neural necrosis virus.

[0045] Example 3 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0046] 2. Experimental Methods TOSF cells (0.9 × 10 6 / mL) were inoculated into 12-well plates and cultured at 28°C for 18 h. 16 μL of NNV was incubated with 12.5 μg / mL of EGCG at 4°C for 2 h, centrifuged at low speed (4°C, 25,000 × g) for 1 h 30 min, the supernatant was discarded to obtain NNV virus particles, and 100 μL of TN buffer was added to mix the NNV virus particles; the control group was NNV and L15 culture medium, and the rest of the operations were the same as before. The L15 culture medium in the 12-well plate was replaced, and the NNV+EGCG suspension and NNV suspension were added to TOSF cells at 20 μL / well and cultured at 28°C for 48 h. The cells were collected and total RNA was extracted, and NNV was detected by RT-qPCR. CPThe relative expression levels of genes were calculated, and three replicates were set for each sample.

[0047] 3. Experimental Results RT-qPCR results are as follows Figure 3 It showed that TOSF cells inoculated with NNV+EGCG suspension CP The relative expression of the gene was significantly lower than that of TOSF cells inoculated with NNV suspension. It can be speculated that EGCG may have affected the structure of NNV virus particles, reduced the virus infectivity, and made the drug group cells CP The relative expression of genes was significantly lower than that of the control group.

[0048] Example 4 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0049] 2. Experimental Methods TOSF cells (0.9 × 10 6 800 μL of EGCG (12.5 μg / mL) + 8 μL of NNV were thoroughly mixed and inoculated into TOSF cells. The control group was inoculated with 800 μL of serum-free L15 medium + 8 μL of NNV. After incubation at 4°C for 30 min, the supernatant was discarded, the cells were washed twice with serum-free L15 medium, and 800 μL of fresh L15 medium was added. The cells were cultured at 28°C for another 12 h. Cell samples from each group were collected, total RNA was extracted, and NNV was detected by RT-qPCR. CP Relative gene expression. Each sample was replicated three times.

[0050] 3. Experimental Results RT-qPCR results are as follows Figure 4 As shown in the figure, compared with the control group, after EGCG and NNV were incubated for 30 min, NNV CP The relative expression of the gene was significantly reduced. From this, we can speculate that during the process of NNV infecting TOSF cells, EGCG will affect the binding process of NNV to the host cell surface, making it impossible for NNV to successfully complete the adsorption stage at the beginning of viral infection.

[0051] Example 5 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0052] 2. Experimental Methods TOSF cells (0.9 × 10 6 / mL) were inoculated into 12-well plates and cultured at 28°C for 18 h. 800 μL of serum-free L15 medium was mixed with 8 μL of NNV, inoculated into TOSF cells, and incubated at 4°C for 1 h to allow NNV to bind to the TOSF cell surface. The supernatant was discarded, and 800 μL of EGCG (12.5 μg / mL) was added to the well plate. The control group was added with serum-free L15 medium. After culturing at 28°C for 2 h, the supernatant was discarded, the L15 medium was washed twice, and 800 μL of fresh L15 medium was added again, and the cells were cultured for another 10 h. Cell samples from each group were collected, total RNA of the samples was extracted, and NNV was detected by RT-qPCR. CP Relative gene expression. Each sample was replicated three times.

[0053] 3. Experimental Results RT-qPCR results are as follows Figure 5 As shown, compared with the control group, after NNV was combined with TOSF cells for 1 h and then re-introduced with EGCG for 2 h, the NNV CP The relative expression of the gene was significantly reduced. From this, we can infer that during the process of NNV infection of TOSF cells, EGCG can interfere with NNV invasion of host cells, thereby effectively preventing NNV virus from infecting host cells.

[0054] Example 6 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0055] 2. Experimental Methods TOSF cells (0.9 × 10 6 / mL) were inoculated into 12-well plates and cultured at 28°C for 18 h. 800 μL of serum-free L15 medium was mixed with 8 μL of NNV and inoculated into TOSF cells. The cells were incubated at 4°C for 1 h. The supernatant was discarded and fresh serum-free L15 medium was added. The cells were cultured at 28°C for 2 h. After NNV invaded the TOSF cells, the supernatant was discarded and 800 μL of EGCG (12.5 μg / mL) was added. The control group was added with serum-free L15 medium. After culturing for 8 h, the supernatant was discarded, the cells were washed twice with L15 medium, and 800 μL of fresh L15 medium was added again. The cells were cultured for another 2 h. Cell samples from each group were collected, total RNA was extracted from the samples, and NNV was detected by RT-qPCR. CP Relative gene expression. Each sample was replicated three times.

[0056] 3. Experimental Results RT-qPCR results are as follows Figure 6As shown, compared with the control group, after NNV infection of TOSF cells for 3 h and EGCG treatment for 8 h, the CP The relative expression of the gene was significantly reduced, indicating that the replication ability of NNV in host cells was reduced. From this, we can infer that during the process of NNV infection of TOSF cells, EGCG limits the proliferation of NNV and can effectively inhibit the replication of the virus inside the host cells.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention.

Claims

1. Application of epigallocatechin gallate in the preparation of drugs against fish neuronecrosis virus.

2. The use of epigallocatechin gallate as claimed in claim 1 in the preparation of an anti-fish nerve necrosis virus drug, characterized in that: Fish neuronecrosis viruses include grouper neuronecrosis viruses.

3. The use of epigallocatechin gallate as claimed in claim 1 in the preparation of an anti-fish nerve necrosis virus drug, characterized in that: Sources of epigallocatechin gallate include green tea.

4. The use of epigallocatechin gallate as claimed in claim 1 in the preparation of an anti-fish nerve necrosis virus drug, characterized in that: The dosage form of the drug includes at least one of liquid, suspension, tablet, granule, and powder.

5. The use of epigallocatechin gallate as claimed in claim 4 in the preparation of a drug for resisting fish nervous necrosis virus, characterized in that: The dosage form of the drug includes a liquid agent, in which the concentration of epigallocatechin gallate is 6-17 μg / mL.

6. The use of epigallocatechin gallate as claimed in claim 4 in the preparation of a drug for resisting fish nervous necrosis virus, characterized in that: The dosage form of the drug includes a liquid agent, in which the concentration of epigallocatechin gallate is 12.5 μg / mL.

7. A drug for resisting fish neural necrosis virus, characterized in that: Includes epigallocatechin gallate.

8. Application of epigallocatechin gallate in the preparation of fish feed resistant to fish neuronecrosis virus.

9. The use of epigallocatechin gallate as claimed in claim 8 in preparing fish feed resistant to fish nervous necrosis virus, characterized in that: Fish neuronecrosis viruses include grouper neuronecrosis viruses.

10. The use of epigallocatechin gallate as claimed in claim 8 in preparing fish feed resistant to fish nervous necrosis virus, characterized in that: The mass proportion of epigallocatechin gallate in fish feed is 0.01~0.05%.

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