Application of green tea extract in preparation of medicine for resisting grouper nervous necrosis virus

Abstract

The invention provides application of a green tea extract in preparation of a medicine for resisting grouper nervous necrosis virus. Related experiments prove that the green tea extract provided by the invention can prevent and treat grouper nervous necrosis virus infection, so that the healthy development of the grouper culture industry can be promoted.

Description

Technical Field

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

[0002] Grouper, a prized aquaculture product in my country, possesses both high economic value and abundant edible value. Its firm texture, delicious flavor, and nutritious properties make it highly sought after by consumers, and its market prospects are extremely promising. With continuous innovations in aquaculture technology and improvements in grouper's reproductive efficiency and growth rate, my country's grouper production has increased significantly, significantly driving the prosperity of the grouper aquaculture industry.

[0003] In recent years, grouper aquaculture has experienced rapid growth and expansion. However, the discharge of aquaculture wastewater and the accumulation of feed residues have led to severe water pollution. As water quality deteriorates, disease problems have become increasingly prominent. Among them, grouper nervous necrosis virus (NNV), a rapidly spreading and highly lethal infectious viral pathogen, has severely impacted the sustainable development of grouper aquaculture in recent years. Currently, NNV can be prevented through vaccination, but factors such as vaccine production, transportation, and storage can affect its effectiveness and, consequently, the health of the fish population. Therefore, the development of effective and practical fishery drugs to prevent, control, and treat NNV outbreaks in grouper aquaculture is an urgent need.

[0004] Currently, botanical antiviral drugs, due to their safety, abundant resources, and environmental friendliness, have become a research hotspot for screening antiviral drugs for aquatic animals. With the continuous development of the aquaculture industry, the advantages of medicinal plants with antiviral properties have become even more significant. They not only effectively address aquatic animal diseases but also contribute to maintaining aquatic ecological balance, and their future application prospects are promising. The diverse active ingredients in medicinal plants, such as alkaloids, flavonoids, and polysaccharides, possess diverse biological functions, playing important roles in antibacterial, anti-inflammatory, antiviral, and immune regulation. Numerous research data demonstrate that natural active ingredients can influence viral structure, interfere with different stages of viral infection, regulate cellular immune factors, and enhance immunity to viral infections, thereby producing antiviral effects and effectively reducing morbidity and mortality in aquatic animals. Unlike traditional aquaculture drugs, medicinal plants can avoid the development of drug resistance. Their low toxicity and minimal side effects make them an effective solution to the problems of aquaculture water pollution and ecological imbalance caused by antibiotics, providing strong support for the healthy development of the aquaculture industry. Summary of the Invention

[0005] To address the problems and shortcomings of the existing technology, the present invention provides a method for preparing a drug for treating grouper neural necrosis virus (ENV). Experimental studies have demonstrated that the green tea extract provided by the present invention can prevent and treat Epinephelus neural necrosis virus infection, thereby promoting the healthy development of the grouper aquaculture industry.

[0006] According to a first aspect of the present invention, there is provided a use of a green tea extract in the preparation of a drug for resisting grouper neuronecrosis virus.

[0007] Green tea extract contains a variety of active ingredients, such as tea polyphenols (especially catechins), caffeine, and theanine. These ingredients have diverse pharmacological effects, including antioxidant, antibacterial, anti-inflammatory, anti-cancer, cardiovascular, neuroprotective, cholesterol-lowering, blood sugar-lowering, and weight-loss benefits. Furthermore, the appropriate ratio of active ingredients in a specific green tea extract can be beneficial for the prevention or treatment of specific diseases.

[0008] Experimental studies have shown that green tea extract can effectively inhibit the replication of Epinephelus necrotic virus in host cells, with minimal biological toxicity. Further research has shown that green tea extract can also disrupt the structure of Epinephelus necrotic virus particles, interfering with the virus's adsorption, invasion, and replication processes during cell infection, effectively combating Epinephelus necrotic virus.

[0009] Furthermore, green tea, a health supplement known for its heat-clearing, detoxifying, and refreshing properties, has negligible biotoxicity. Therefore, green tea is recommended for the prevention and treatment of grouper neuronecrosis virus. Combined with relevant experimental results, the anti-grouper neuronecrosis virus drug (green tea extract) provided by the present invention can safely and effectively prevent and treat grouper neuronecrosis virus infection. Furthermore, based on the pursuit of green health and sustainable development, it can actively promote the steady progress of high-quality ecological grouper farming.

[0010] Preferably, the green tea extract is a green tea alcohol extract. The combination of the active ingredients in the green tea alcohol extract has a more pronounced effect on the prevention or treatment of grouper neural necrosis virus. That is, the combination of the active ingredients in the green tea alcohol extract can produce a more pronounced synergistic effect, particularly for the prevention or treatment of grouper neural necrosis virus, with a more targeted therapeutic effect and lower biological toxicity to grouper.

[0011] Preferably, the green tea alcohol extract is a green tea methanol extract. Furthermore, the green tea alcohol extract obtained by methanol extraction has a better ratio and combination of active ingredients, and has a more excellent effect on the prevention or treatment of grouper nervous necrosis virus.

[0012] Preferably, the preparation method of the green tea extract includes the following steps: S1. Washing and drying the green tea, and crushing it to obtain green tea powder; mixing the green tea powder and the petroleum ether, soaking them at 55~65°C for 0.5~2h, and then continuing to ultrasonicate at 55~65°C for 2~5 times, with the ultrasonication time being 3~8 min / time, and collecting the first filter residue; adding the petroleum ether to the first filter residue 1~2 times and repeating the above operation to collect the second filter residue; S2. Adding methanol to the second filter residue, ultrasonicate at 55~65°C for 2~5 times, with the ultrasonication time being 3~8 min / time, and collecting the first filtrate; adding the methanol to the remaining third filter residue 1~2 times and repeating the above operation to collect the second filtrate, combining the first filtrate and the second filtrate, vacuum distilling, and drying to obtain the green tea extract. Through the above steps, not only can the impurities and adverse substances in the green tea be effectively removed and the biological toxicity of the obtained green tea extract be reduced to the greatest extent, but also the effective active ingredients in the green tea can be fully extracted and the various effective active ingredients in the green tea can be matched in a better ratio, thereby exerting a better effect of preventing or treating grouper nervous necrosis virus.

[0013] Preferably, in S1, the green tea powder and the petroleum ether are mixed at a material-liquid ratio of 1:8 to 15. Preferably, in S1, the green tea powder and the petroleum ether are mixed at a material-liquid ratio of 1:10.

[0014] Preferably, in S2, the amount of methanol added is calculated based on a solid-liquid ratio of the green tea powder to the methanol of 1:8 to 15. Preferably, in S2, the amount of methanol added is calculated based on a solid-liquid ratio of the green tea powder to the methanol of 1:10.

[0015] Preferably, a working solution prepared with the green tea extract is used to prevent and / or treat grouper neurological necrosis virus at a concentration of 6.25 to 25 μg / L. This concentration effectively prevents or treats grouper neurological necrosis virus while ensuring that the relatively low concentration of green tea extract has low toxicity to grouper, thereby improving the yield and quality of grouper aquaculture.

[0016] Preferably, the concentration of the working solution is 12.5-25 μg / L.

[0017] Preferably, the specific operation of preparing the working solution is as follows: using a solvent to dissolve the green tea extract to 90-110 mg / mL, using this as the mother solution, and then diluting it to a cell-safe concentration using L15 culture medium as the working solution, the concentration of the working solution is 6.25-25 μg / L.

[0018] Preferably, the solvent comprises dimethyl sulfoxide.

[0019] According to a second aspect of the present invention, there is provided a use of a green tea extract in preparing fish feed resistant to grouper nervous necrosis virus.

[0020] Preferably, the mass proportion of green tea extract in fish feed is 0.02-0.1%.

[0021] In summary, green tea extract, through the appropriate proportions of its various active ingredients, can effectively prevent or treat grouper NNV virus. Furthermore, green tea extract has extremely low biotoxicity, making it a safe and effective treatment for grouper NNV virus infection, significantly promoting the sustainable and healthy development of grouper. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of the green tea extract on the cytotoxicity of the spleen tissue of the oval pomfret in Example 1 is shown; wherein, Figure 1 (A) Observation results of TOSF cells co-incubated with green tea extract; Figure 1 (B) The results of TOSF cell survival rate.

[0023] Figure 2 The figure shows the inhibitory effect of green tea extract at different concentrations on grouper nervous necrosis virus in Example 2.

[0024] Figure 3 This is the effect of the green tea extract in Example 3 on destroying the structure of grouper nervous necrosis virus particles and thus inhibiting the infection of nervous necrosis virus.

[0025] Figure 4 The green tea extract in Example 4 interferes with the process of grouper nervous necrosis virus adsorbing on the cell surface, thereby achieving the effect of inhibiting nervous necrosis virus infection.

[0026] Figure 5 The green tea extract in Example 5 interferes with the process of grouper nervous necrosis virus invading host cells, thereby achieving the effect of inhibiting nervous necrosis virus infection.

[0027] Figure 6 The green tea alcohol extract in Example 6 interferes with the replication process of grouper nervous necrosis virus in host cells, thereby achieving the effect of inhibiting nervous necrosis virus infection. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 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. The grouper neurological necrosis virus (NNV) was isolated from the artificially cultured pearl grouper in Guangxi and is 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.

[0031] Green tea: purchased from Yixin Pharmacy in Nanning, Guangxi (origin: Anhui).

[0032] Primers for the grouper 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.

[0033] The green tea extract used in the following examples was prepared by the following steps: S1. Wash and dry the green tea leaves, crush them to obtain green tea powder; mix the green tea powder and petroleum ether at a material-to-liquid ratio of 1:10 (25 g of green tea powder and 250 mL of petroleum ether), soak for 1 hour, and then sonicate at 60°C for 5 minutes each. Collect the first filter residue; repeat the above steps by adding 250 mL of new petroleum ether to the first filter residue, and collect the second filter residue. S2. Add 250 mL of methanol to the second filter residue, perform ultrasonication at 60°C for 3 times, with an ultrasonication time of 5 min / time, and collect the first filtrate; add 250 mL of new methanol to the remaining third filter residue and repeat the above operation once, collect the second filtrate, combine the first filtrate and the second filtrate, vacuum distill and dry to obtain the green tea extract.

[0034] The green tea extract obtained above was dissolved in DMSO reagent to 100 mg / mL as a mother solution, and diluted to a cell-safe concentration using L15 culture medium as a working solution. The concentration of the working solution was 6.25~25 μg / L.

[0035] Example 1 1. Main instruments and reagents Optical microscope, microplate reader, Cell Counting Kit-8 (CCK-8) solution.

[0036] 2. Experimental Methods 0.9×10 6 TOSF cells at a concentration of 1 μg / mL were inoculated into a 96-well plate and cultured at 28°C for 18 h. The green tea extract stock solution was diluted to a concentration of 25 μg / mL using serum-free L15 medium. Subsequently, 100 μL of the drug solution was added to the well plate and incubated with the TOSF cells at 28°C. Three time points of 24 h, 48 h, and 72 h of incubation with green tea extract and TOSF cells were set as experimental groups. Light microscopy was used to observe whether 25 μg / mL of green tea extract harmed TOSF cells. The control group was serum-free L15 medium without green tea extract. After observing the cell morphology under a light microscope, the drug solution in each well was discarded and CCK-8 solution was added. The cells were incubated in the dark at room temperature for 4 h, and the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. The formula for calculating cell viability is as follows: .

[0037] 3. Experimental Results The results of the co-incubation of TOSF cells with green tea extract were as follows Figure 1 As shown in (A), it can be seen that the green tea extract at a concentration of 25 μg / mL had no significant effect on the TOSF cells co-incubated at 24 h, 48 h, and 72 h. The TOSF cell morphology at the three time points did not change significantly, and the cell morphology was consistent with that of the control group. The results of TOSF cell survival rate were as follows Figure 1 As shown in (B), it can be seen that when the green tea extract at a concentration of 25 μg / mL acts on TOSF cells at three time points in the experiment, the green tea extract does not produce significant cytotoxicity to the cells, and the cell survival rate can exceed 95%.

[0038] Example 2 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0039] 2. Experimental Methods 0.9×106 TOSF cells were seeded at 100 μg / mL in 12-well plates and cultured at 28°C for 18 h. The culture medium was discarded, and 800 μL serum-free L15 medium was added to the control group, and 8 μL (10 6 TCID 50 / mL) NNV + 800 μL serum-free L15 medium. In the experimental group, 800 μL of three concentrations (25, 12.5, and 6.25 μg / mL) of green tea extract + 8 μL NNV were added. After 48 h of NNV infection, 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.

[0040] 3. Experimental Results RT-qPCR results are as follows Figure 2 As shown, compared with the NNV group, TOSF cells inoculated with NNV + green tea extract (25, 12.5 and 6.25 μg / mL) CP The relative expression level of the gene was significantly reduced, indicating that green tea extract has a significant antiviral effect on grouper neural necrosis virus.

[0041] Example 3 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0042] 2. Experimental Methods 0.9×10 6 TOSF cells at a concentration of 1 μg / mL were inoculated into 12-well plates and cultured at 28°C for 18 h. 16 μL of NNV was incubated with 25 μg / mL of green tea extract at 4°C for 2 h, centrifuged at 4°C and 25,000 × g for 1 h 30 min, the drug solution was discarded to obtain NNV virus particles, and the NNV virus particles were resuspended and mixed with 100 μL of TN buffer; the control group was incubated with NNV and L15 culture medium at 4°C for 2 h, and the rest of the operations were the same as before. NNV + green tea extract suspension and NNV suspension were inoculated into TOSF cells at 20 μL / well and continued to be cultured at 28°C. After 48 h, the cells were collected and total RNA was extracted, and NNV was detected by RT-qPCR. CP Relative gene expression. Each sample was replicated three times.

[0043] 3. Experimental Results RT-qPCR results are as follows Figure 3 As shown, compared with TOSF cells inoculated with NNV suspension, TOSF cells inoculated with NNV+green tea extract suspension CPThe relative expression of the gene was significantly reduced, which indicates that green tea extract may have changed the structure of NNV virus particles, weakened the virus infectivity, and thus led to the CP The relative expression of genes was significantly lower than that of the control group.

[0044] Example 4 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0045] 2. Experimental Methods 0.9×10 6 TOSF cells (100 μg / mL) were seeded into 12-well plates and cultured at 28°C for 18 h. 800 μL of green tea extract (25 μg / mL) was mixed with 8 μL of NNV and then inoculated into TOSF cells. The control group was inoculated with 800 μL of serum-free L15 medium + 8 μL of NNV and incubated 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 for another 12 h at 28°C. Cell samples were collected and total RNA was extracted. NNV was detected by RT-qPCR. CP Relative gene expression. Each sample was replicated three times.

[0046] 3. Experimental Results RT-qPCR results are as follows Figure 4 As shown, after green tea extract and NNV were co-incubated in TOSF cells for 30 min, the CP The relative expression of the gene was significantly lower than that of the control group, indicating that green tea extract can affect the binding of NNV to the host cell surface during NNV infection of TOSF cells.

[0047] Example 5 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0048] 2. Experimental Methods 0.9×10 6TOSF cells (100 μg / mL) were seeded into 12-well plates and cultured at 28°C for 18 h. First, 8 μL of NNV+serum-free L15 medium was inoculated into the TOSF cells and incubated at 4°C for 1 h to allow NNV to adsorb to the TOSF cell surface. The supernatant was discarded and 800 μL of green tea extract (25 μg / mL) was added to the well plate; the control group only replaced the serum-free L15 medium. After culturing at 28°C for 2 h, the supernatant was discarded, the L15 medium was washed twice, 800 μL of fresh L15 medium was added, and the culture continued at 28°C for 10 h. Cell samples were collected and total RNA was extracted, and NNV was detected by RT-qPCR. CP Relative gene expression. Each sample was replicated three times.

[0049] 3. Experimental Results RT-qPCR results are as follows Figure 5 As shown, after NNV adsorbed TOSF cells for 1 h, TOSF cells were re-inoculated with green tea extract for 2 h. CP The relative expression of the gene was significantly lower than that of the control group, indicating that green tea extract can interfere with the invasion of NNV into host cells during NNV infection of TOSF cells.

[0050] Example 6 1. Main instruments and reagents Fluorescence quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0051] 2. Experimental Methods 0.9×10 6 TOSF cells (100 μg / mL) were seeded into 12-well plates and cultured at 28°C for 18 h. 8 μL of NNV+serum-free L15 medium was inoculated into TOSF cells and 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 to allow NNV to invade the interior of the TOSF cells. The supernatant was discarded and 800 μL of green tea extract (25 μg / mL) was added. The control group only replaced the serum-free L15 medium. After culturing at 28°C for 8 h, the supernatant was discarded, the L15 medium was washed twice, 800 μL of fresh L15 medium was added, and the cells were cultured at 28°C for another 2 h. Cell samples were collected and total RNA was extracted. NNV was detected by RT-qPCR. CP Relative gene expression. Each sample was replicated three times.

[0052] 3. Experimental Results RT-qPCR Figure 6 As shown, after NNV infection in TOSF cells for 3 h, green tea extract was treated in TOSF cells for 8 h. CPThe relative expression of the gene was significantly lower than that of the control group, indicating that the replication ability of NNV in TOSF cells was significantly weakened. This shows that during the process of NNV infection of TOSF cells, green tea extract can affect the replication of NNV in host cells.

[0053] 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 green tea extract in the preparation of drugs against grouper neuronecrosis virus.

2. The use of the green tea extract as claimed in claim 1 in the preparation of a drug for resisting grouper neural necrosis virus, characterized in that: The green tea extract is green tea alcohol extract.

3. The use of the green tea extract in the preparation of an anti-grouper nervous necrosis virus drug as claimed in claim 2, characterized in that: The preparation method of the green tea extract comprises the following steps: S1. Wash and dry the green tea leaves, crush them to obtain green tea powder; mix the green tea powder and petroleum ether, soak them at 55-65°C for 0.5-2 hours, and then sonicate them at 55-65°C for 2-5 times, each time for 3-8 minutes, collecting a first filter residue; repeat the above steps by adding petroleum ether to the first filter residue once or twice, and collect a second filter residue; S2. Add methanol to the second filter residue, perform ultrasonication at 55-65°C for 2-5 times, with an ultrasonication time of 3-8 min / time, and collect the first filtrate; repeat the above operation by adding methanol to the remaining third filter residue 1-2 times, collect the second filtrate, combine the first filtrate and the second filtrate, perform vacuum distillation, and dry to obtain the green tea extract.

4. The use of the green tea extract as claimed in claim 2 in the preparation of a drug for resisting grouper nervous necrosis virus, characterized in that: In S1, the green tea powder and the petroleum ether are mixed in a material-liquid ratio of 1:8-15.

5. The use of the green tea extract as claimed in claim 2 in the preparation of a drug for resisting grouper nervous necrosis virus, characterized in that: In S2, the amount of methanol added is calculated based on a material-liquid ratio of the green tea powder to the methanol of 1:8-15.

6. The use of the green tea extract as claimed in claim 1 in the preparation of a drug for resisting grouper nervous necrosis virus, characterized in that: The working solution prepared with the green tea extract is used to prevent and / or treat grouper nervous necrosis virus, and the concentration of the working solution is 6.25-25 μg / L.

7. The use of the green tea extract as claimed in claim 6 in the preparation of a drug for resisting grouper nervous necrosis virus, characterized in that: The concentration of the working solution is 12.5-25 μg / L.

8. The use of the green tea extract in the preparation of a drug for resisting grouper nervous necrosis virus according to claim 6, characterized in that: The specific operation of preparing the working solution is as follows: using a solvent to dissolve the green tea extract to 90-110 mg / mL, using this as the mother solution, and then diluting it to a cell-safe concentration using L15 culture medium as the working solution. The concentration of the working solution is 6.25-25 μg / L.

9. Application of green tea extract in the preparation of fish feed resistant to grouper nervous necrosis virus.

10. The use of the green tea extract according to claim 9 in preparing fish feed resistant to grouper nervous necrosis virus, characterized in that: The mass proportion of green tea extract in fish feed is 0.02~0.1%.

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