Application of gallnut extract in preparation of medicine for resisting Micropterus salmoides virus

Abstract

According to the application of the gallnut extract to preparation of the medicine for resisting the Micropterus salmoides virus, related experiments verify that the gallnut extract can effectively prevent and / or treat Micropterus salmoides rhabdovirus, and sustainable and stable development of the Micropterus salmoides culture industry is guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal disease prevention and control, specifically relating to the application of a gallnut extract in the preparation of drugs against largemouth bass viruses. Background Technology

[0002] Largemouth bass, belonging to the family Sunfishidae in the order Perciformes, is characterized by rapid growth, delicate flesh, and high nutritional value, making it one of the important economic fish species in my country's freshwater aquaculture. Driven by increasing market demand and advancements in aquaculture technology, the largemouth bass farming industry has experienced rapid development. In 2023, my country's annual bass production exceeded 880,000 tons, with the industry's economic benefits continuing to grow. Largemouth bass rhabdovirus (LRV) Micropterus salmoides rhabdovirus (MSRV) is one of the most infectious viral pathogens, with a mortality rate of over 80%, posing a serious threat to the sustainable development of largemouth bass farming.

[0003] Currently, there are no effective drugs on the market for treating largemouth bass rhabdovirus. Traditional antibiotics are not only ineffective against viral infections, but also cause environmental pollution and drug residues. Therefore, developing safe, efficient, and environmentally friendly new antiviral fish medicines is an important task to ensure the healthy and sustainable development of largemouth bass aquaculture. Summary of the Invention

[0004] To address the problems and shortcomings of existing technologies, this invention provides an application of gallnut extract in the preparation of drugs against largemouth bass viruses. Relevant experimental verification shows that gallnut extract can effectively prevent and / or treat largemouth bass rhabdovirus, ensuring the sustainable and stable development of the largemouth bass aquaculture industry.

[0005] According to a first aspect of the present invention, there is an application of gallnut extract in the preparation of a drug for treating largemouth bass virus.

[0006] Largemouth bass rhabdovirus (LAV) is characterized by rapid transmission, high virulence, and high mortality, primarily infecting juvenile largemouth bass. The virus can cause large-scale mortality within a short period, resulting in significant economic losses to the largemouth bass aquaculture industry. As mentioned in the background section, there are currently no effective drugs against LAV. Therefore, developing a novel, environmentally friendly, and highly effective anti-LAV drug is of great significance for the healthy and ecological development of the largemouth bass aquaculture industry.

[0007] my country boasts abundant natural medicinal resources. Due to their safe and natural origin, low toxicity and side effects, and multiple benefits such as promoting growth, enhancing immunity, and antibacterial and antiviral capabilities, they represent a key area for research into novel antiviral aquaculture drugs. Natural medicinal plants contain various active ingredients, primarily flavonoids, alkaloids, polysaccharides, and saponins. Numerous studies have shown that natural medicinal plants and their active ingredients can not only interact with viral particles to disrupt viral structure and interfere with viral adsorption, invasion, and replication stages to reduce infectivity, but also regulate the expression of host immune-related factors, thereby achieving antiviral effects. Based on the unique advantages and antiviral activity of natural medicinal plants, in-depth research into green and safe novel antiviral aquaculture drugs can effectively prevent outbreaks of viral diseases in aquatic animals and address antibiotic resistance and environmental pollution problems, representing an important direction for promoting high-quality and ecological development in aquaculture.

[0008] Gallnut (Gallus chinensis) is a gall formed on the leaves of plants in the Anacardiaceae family after being parasitized by the gall aphid. It possesses various pharmacological effects, including antibacterial, antiviral, antioxidant, and anti-inflammatory properties. Through long-term research and related experimental verification, this invention has discovered that gallnut extract can interact with largemouth bass rhabdovirus (GBV), disrupting the virus particle structure and thus effectively preventing and controlling GBV. In other words, gallnut extract can be used as a preventative drug against GBV. Furthermore, in-depth research has revealed that gallnut extract can also inhibit the adsorption, invasion, and replication of GBV on host cells, effectively reducing its infectivity and thus exerting an antiviral effect. Moreover, as an anti-GBV drug, gallnut extract exhibits almost no biotoxicity and is unlikely to induce drug resistance, thus promoting the healthy and green development of largemouth bass farming and effectively improving its economic benefits.

[0009] Preferably, the gallnut extract is a gallnut alcohol extract. Gallnut alcohol extract, obtained using alcohol as a solvent, ensures the full extraction of effective active ingredients from gallnuts while reducing the content of undesirable impurities, thus improving the efficacy of the gallnut extract against largemouth bass rhabdovirus.

[0010] Preferably, the gallnut alcohol extract is a gallnut methanol extract. Gallnut methanol extract, obtained using methanol as a solvent, not only allows for more complete extraction of the active ingredients from gallnuts and reduces the content of undesirable impurities, but more importantly, it results in a more balanced ratio of various active ingredients, making it easier to achieve a better synergistic effect. This further enhances the antiviral effect against largemouth bass rhabdovirus while reducing the side effects of gallnut extract on largemouth bass.

[0011] Preferably, the preparation method of the gallnut extract includes the following steps: S1. Crushing gallnuts to obtain gallnut powder; mixing the gallnut powder and the petroleum ether, soaking at 55~65℃ for 0.5~2h, and then continuing to sonicate at 55~65℃ for 2~5 times, with a sonication time of 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, and collecting the second filter residue; S2. Adding methanol to the second filter residue, sonicating at 55~65℃ for 2~5 times, with a sonication time of 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, collecting the second filtrate, combining the first filtrate and the second filtrate, vacuum distilling, and drying to obtain the gallnut extract. The above preparation method can not only effectively remove impurities and other unfavorable components from gallnuts, but also fully extract the effective active ingredients from gallnuts. At the same time, it can make the active ingredients have a good synergistic effect, which is more conducive to improving the prevention and control effect against largemouth bass rhabdovirus.

[0012] Preferably, in step S1, the gallnut powder and the petroleum ether are mixed at a material-to-liquid ratio of 1:8 to 15.

[0013] Preferably, in step S2, the amount of methanol added is calculated based on a material-to-liquid ratio of 1:8 to 15 for the gallnut powder and the methanol.

[0014] Preferably, a working solution prepared from the gallnut extract is used to prevent and / or treat grouper neuronecrosis virus, wherein the concentration of the working solution is 3.125 μg / mL to 12.5 μg / mL. Within this concentration range, a sufficient concentration of gallnut extract can be ensured as a drug to achieve good antiviral effects. Preferably, the concentration of the working solution is 12.5 μg / mL.

[0015] Preferably, the specific operation for preparing the working solution is as follows: the gallnut extract is dissolved in a solvent to a concentration of 90-110 mg / mL, which is used as the stock solution. Then, it is diluted with L15 culture medium to a cell-safe concentration to obtain the working solution, wherein the concentration of the working solution is 3.125 μg / mL to 12.5 μg / mL.

[0016] According to a second aspect of the present invention, there is an application of gallnut extract in the preparation of fish feed resistant to largemouth bass virus.

[0017] Preferably, the gallnut extract accounts for 0.01~0.05% of the fish feed by mass. Attached Figure Description

[0018] Figure 1 The effect of gallnut alcohol extract on the toxicity of fathead carp muscle cells in Example 1 is shown.

[0019] Figure 2 This describes the inhibitory effect of gallnut alcohol extract at different concentrations on largemouth bass rhabdovirus in Example 2.

[0020] Figure 3 This demonstrates the effect of the gallnut alcohol extract in Example 3 on reducing the infectivity of the largemouth bass rhabdovirus by interacting with its particles.

[0021] Figure 4 The effect of the gallnut alcohol extract in Example 4 on the adsorption of largemouth bass rhabdovirus to the cell surface stage, thereby reducing the infectivity of largemouth bass rhabdovirus.

[0022] Figure 5 The effect of the gallnut alcohol extract in Example 5 on the largemouth bass rhabdovirus's invasion of cells, thereby reducing the infectivity of the largemouth bass rhabdovirus.

[0023] Figure 6 The effect of the gallnut alcohol extract in Example 6 on the largemouth bass rhabdovirus to interfere with the intracellular replication stage of the virus, thereby reducing the infectivity of the largemouth bass rhabdovirus. Detailed Implementation

[0024] To enable those skilled in the art to better understand 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 some embodiments of the present invention, and not all embodiments.

[0025] The quantitative experimental data involved in the following examples are expressed as mean ± standard deviation (±s). The data for comparison between groups were statistically processed using SPSS 17.0 statistical software with one-way ANOVA.

[0026] Fathead flounder ( Pimephales promelas The muscle cell line (Fathead minnow, FHM) is preserved in this laboratory and is available to the public from the applicant for use only to replicate the experiments of this invention. Largemouth bass rhabdovirus ( Micropterus salmoides The rhabdovirus (MSRV) was isolated from the artificially bred largemouth bass in Guangxi and is preserved in the applicant's laboratory. It is available to the public from the applicant and is used only for repeating the experiments of this invention.

[0027] Gallnut: Purchased from Yixin Pharmacy in Nanning, Guangxi (originating from Sichuan).

[0028] All primers used in the examples were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Largemouth bass rhabdovirus nucleoprotein (Nucleoprotein, N Gene primers: Forward primer ( N -F) 5'-GCCCACATCGCATCATTCAC-3'; Reverse primer ( N -R) 5'-GTGGCAGAGTAAGGGGACAC-3'.

[0029] Internal reference gene β -actin primer: Forward primer ( β -actin-F) 5'-TCTTCCAGCCATCCTTCCTTGG-3'; Reverse primer ( β -actin-R) 5'-CTGCATACGGTCAGCAATGCC-3'. The gallnut extract used in the following examples was prepared through the following steps: S1. After crushing the gallnuts, obtain gallnut powder; mix the gallnut powder and the petroleum ether at a material-to-liquid ratio of 1:10 (the amount of gallnut powder used is 25g, and the amount of petroleum ether used is 250mL), soak for 1 hour, and then perform ultrasonication 5 times at 60℃, with an ultrasonication time of 5 min / time, and collect the first filter residue; add 250 mL of the new petroleum ether to the first filter residue and repeat the above operation to collect the second filter residue. S2. Add 250 mL of methanol to the second filter residue and sonicate it three times at 60°C for 5 min each time. Collect the first filtrate. Add 250 mL of fresh methanol to the remaining third filter residue and repeat the above operation. Collect the second filtrate. Combine the first filtrate and the second filtrate, vacuum distill, and dry to obtain the gallnut extract.

[0030] The gallnut extract obtained above was dissolved in DMSO to a concentration of 100 mg / mL as a stock solution, and then diluted with L15 culture medium to a cell-safe concentration as a working solution. The concentration of the working solution was 3.125 μg / mL - 12.5 μg / mL.

[0031] Example 1 1. Main instruments and reagents Optical microscope, microplate reader, PBS buffer, Cell Counting Kit solution (CCK-8 solution).

[0032] 2. Experimental Methods 1×10 6 FHM cells were seeded at 100 μL / well in 96-well plates and cultured at 28°C for 18 h. Gallnut extract was diluted to different concentrations (100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL) with L-15 medium, and these concentrations were added to the 96-well plates and co-incubated with FHM cells at 28°C. The control group was cultured in L-15 medium. After 48 h, the effect of gallnut extract on FHM cells was observed under an optical microscope. After discarding the medium, cells in each well were washed with PBS buffer, and then 10% (v / v) CCK-8 solution was added and incubated at room temperature in the dark for 4 h. The absorbance of each well was measured using a microplate reader at 450 nm, and cell viability was calculated.

[0033] The formula for calculating cell viability is as follows: .

[0034] 3. Experimental Results Light microscopic observation results of different concentrations of gallnut extract incubated with FHM cells are as follows: Figure 1 As shown in Figure A, it can be seen that the morphology of FHM cells in the control group showed no significant changes. When the concentration of gallnut extract was ≤12.5 μg / mL, the morphology of FHM cells did not change significantly, and the cell morphology was consistent with that of the control group cells. The cell viability results are as follows: Figure 1 As shown in Figure B, when the concentration of gallnut extract was ≤12.5 μg / mL, the survival rate of FHM cells was not significantly different from that of the control group. This indicates that 12.5 μg / mL is the safe working concentration of gallnut extract on FHM cells.

[0035] Example 2 1. Main instruments and reagents Real-time quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0036] 2. Experimental Methods 1×10 6 FHM cells at a density of 10 cells / mL were seeded at 800 μL / well into 12-well culture plates and cultured at 28°C for 18 h. The culture medium in each well was discarded. The negative control group received only L-15 medium, while the positive control group received 8 μL of MSRV (1×10⁻⁶ cells / mL). 6 TCID 50Cells in the experimental groups were treated with three concentrations (12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL) of gallnut extract plus 8 μL of MSRV. After 48 h, cells from each group were collected, RNA was extracted, reverse transcribed into cDNA, and MSRV was detected by RT-qPCR. N The relative expression levels of genes were used to determine the inhibitory effect of gallnut extract on largemouth bass rhabdovirus. All samples were tested in triplicate.

[0037] 3. Experimental Results RT-qPCR results are as follows Figure 2 As shown, MSRV in cells treated with different concentrations (12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL) of gallnut extract... N The relative expression level of the gene was significantly lower than that of the positive control with MSRV infection alone. This indicates that gallnut extracts at concentrations of 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL all have inhibitory effects on largemouth bass rhabdovirus, and the antiviral effect is dose-dependent. Among them, the high concentration (12.5 μg / mL) of gallnut alcohol extract has the best effect against largemouth bass rhabdovirus.

[0038] Example 3 1. Main instruments and reagents Real-time quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0039] 2. Experimental Methods 1×10 6 FHM cells (12.5 μg / mL) were seeded at 800 μL / well into 12-well plates and cultured at 28°C for 18 h. Gallnut extract (12.5 μg / mL) and 8 μL MSRV (1×10⁻⁶) were added. 6 TCID 50 MSRV virus particles were obtained by incubating (8 μL / mL) at 4°C for 2 h, followed by centrifugation at 25000 ×g for 2 h at the same temperature, discarding the supernatant, and resuspending the MSRV virus particles in 100 μL of TN buffer and mixing thoroughly. The control group consisted of only L-15 medium and 8 μL of MSRV incubated at 4°C for 2 h, with centrifugation and resuspending performed as before. The medium in the 12-well plates was replaced, and 20 μL / well of the experimental and control group suspensions were inoculated into FHM cells and cultured at 28°C for 48 h. Cells from each group were collected, RNA was extracted, reverse transcribed into cDNA, and MSRV was detected by RT-qPCR. N The relative expression levels of genes were assessed to evaluate the effect of gallnut extract on largemouth bass rhabdovirus particles. All samples were tested in triplicate.

[0040] 3. Experimental Results RT-qPCR results are as follows Figure 3 As shown, compared with the control group (MSRV + L-15 medium), the experimental group (MSRV + gallnut extract) cells contained MSRV... N The relative expression levels of genes were significantly reduced. This suggests that the MSRV viral particles may have undergone structural changes after treatment with gallnut extract, leading to a decrease in MSRV infectivity. N The relative expression level of the gene was significantly lower than that of the control group that was not treated with gallnut extract.

[0041] Example 4 1. Main instruments and reagents Real-time quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0042] 2. Experimental Methods 1×10 6 FHM cells were seeded at 800 μL / well in 12-well plates and cultured at 28°C for 18 h. Gallnut extract (12.5 μg / mL) was incubated and then thoroughly mixed with 8 μL MSRV before being inoculated into FHM cells; the control group was inoculated with only 8 μL MSRV and L-15 medium. All groups were incubated at 4°C. After 30 min, the supernatant was discarded, and FHM cells were washed twice with L-15 medium. Cells were then transferred and cultured at 28°C for 12 h. Cells were collected, RNA was extracted, reverse transcribed into cDNA, and MSRV was detected by RT-qPCR. N Relative gene expression levels. Each sample was prepared in triplicate.

[0043] 3. Experimental Results RT-qPCR results are as follows Figure 4 As shown, compared with the control group (MSRV + L-15 medium), the experimental group (MSRV + gallnut extract) cells N The relative expression level of the gene was significantly reduced. This indicates that during the MSRV infection of FHM cells, gallnut extract can inhibit the adsorption phase of MSRV, affect the binding of MSRV to the surface of host cells, and weaken the infectivity of MSRV.

[0044] Example 5 1. Main instruments and reagents Real-time quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0045] 2. Experimental Methods 1×10 6FHM cells were seeded at 800 μL / well in 12-well plates and cultured at 28°C for 18 h. 8 μL of MSRV was added to pre-chilled L-15 medium and mixed thoroughly, then inoculated into FHM cells and incubated at 4°C for 30 min. After MSRV adsorbed onto the FHM cell surface, the supernatant was discarded. The experimental group received gallnut extract (12.5 μg / mL); the control group only received fresh L-15 medium. Cells were transferred to 28°C and cultured for another 2 h. The supernatant was discarded from each group, and FHM cells were washed twice and cultured for another 10 h. Cells were collected, RNA was extracted, reverse transcribed into cDNA, and MSRV was detected by RT-qPCR. N Relative gene expression levels. Each sample was prepared in triplicate.

[0046] 3. Experimental Results RT-qPCR results are as follows Figure 5 As shown, after MSRV adsorbed into cells for 30 min, compared with the control group (with L-15 medium), the experimental group (with gallnut extract) showed a decrease in cell count. N The relative expression levels of the gene were significantly reduced. This indicates that during MSRV infection of FHM cells, gallnut extract can affect the invasion phase of MSRV, inhibit MSRV from entering host cells, and reduce MSRV infectivity.

[0047] Example 6 1. Main instruments and reagents Real-time quantitative PCR instrument (Hangzhou Langji, Q2000B).

[0048] 2. Experimental Methods 1×10 6 FHM cells were seeded at 800 μL / well in 12-well plates and cultured at 28°C for 18 h. 8 μL of MSRV was added to pre-chilled L-15 medium and mixed thoroughly, then inoculated into FHM cells and incubated at 4°C for 30 min. The L-15 medium containing MSRV was discarded, and fresh L-15 medium was added. Cells were transferred and cultured at 28°C for 2 h. After MSRV invaded FHM cells, the supernatant was discarded, and gallnut extract (12.5 μg / mL) was added; the control group received fresh L-15 medium. Cells were cultured for another 8 h, the supernatant was discarded, and FHM cells were washed twice, followed by another 2 h of culture. Cells were collected, RNA was extracted, reverse transcribed into cDNA, and MSRV was detected by RT-qPCR. N Relative gene expression levels. Each sample was prepared in triplicate.

[0049] 3. Experimental Results RT-qPCR results showed that ( Figure 6After MSRV invaded the cells, compared with the control group (with L-15 medium), the experimental group (with gallnut extract) showed higher cell counts. N The relative expression level of the gene was significantly reduced, indicating that MSRV replication in FHM cells was disrupted. This suggests that gallnut extract can affect the MSRV replication phase during MSRV infection of FHM cells, and even if MSRV successfully enters the host cell, gallnut extract can still inhibit MSRV replication within the host cell.

[0050] 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 modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. Application of gallnut extract in the preparation of drugs against largemouth bass virus.

2. The application of the gallnut extract as described in claim 1 in the preparation of drugs against largemouth bass viruses, characterized in that: The gallnut extract is a gallnut alcohol extract.

3. The application of the gallnut extract as described in claim 1 in the preparation of drugs against largemouth bass viruses, characterized in that: The preparation method of the gallnut extract includes the following steps: S1. Crush gallnuts to obtain gallnut powder; mix the gallnut powder and the petroleum ether, soak at 55~65℃ for 0.5~2h, and then continue to sonicate at 55~65℃ 2~5 times, with a sonication time of 3~8 min / time, and collect the first filter residue; add the petroleum ether to the first filter residue 1~2 times and repeat the above operation, and collect the second filter residue; S2. Add methanol to the second filter residue and sonicate at 55-65°C 2-5 times, with a sonication time of 3-8 min / time. Collect the first filtrate. Add methanol to the remaining third filter residue 1-2 times and repeat the above operation. Collect the second filtrate. Combine the first filtrate and the second filtrate, vacuum distill, and dry to obtain the gallnut extract.

4. The application of the gallnut extract as described in claim 2 in the preparation of drugs against largemouth bass viruses, characterized in that: In step S1, the gallnut powder and the petroleum ether are mixed at a material-to-liquid ratio of 1:8 to 15.

5. The application of the gallnut extract as described in claim 2 in the preparation of drugs against largemouth bass viruses, characterized in that: In step S2, the amount of methanol added is calculated with a material-to-liquid ratio of gallnut powder to methanol of 1:8~15.

6. The application of the gallnut extract as described in claim 1 in the preparation of drugs against largemouth bass viruses, characterized in that: The working solution prepared from the gallnut extract is used to prevent and / or treat neuronecrosis virus of the grouper genus, wherein the concentration of the working solution is 3.125 μg / mL to 12.5 μg / mL.

7. The application of the gallnut extract as described in claim 6 in the preparation of drugs against largemouth bass viruses, characterized in that: The concentration of the working solution is 12.5 μg / mL.

8. The application of the gallnut extract as described in claim 6 in the preparation of a drug against largemouth bass virus, characterized in that, The specific steps for preparing the working solution are as follows: Dissolve the gallnut extract in a solvent to a concentration of 90-110 mg / mL, using this as the stock solution. Then, dilute it with L15 culture medium to a cell-safe concentration to obtain the working solution, which has a concentration of 3.125 μg / mL to 12.5 μg / mL.

9. Application of gallnut extract in the preparation of fish feed resistant to largemouth bass virus.

10. The application of the gallnut extract as described in claim 9 in the preparation of fish feed resistant to largemouth bass virus, characterized in that: The mass percentage of gallnut extract in fish feed is 0.01~0.05%.

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