A composition against fish viruses and its use

Abstract

To provide a highly effective antiviral drug for fish, this invention provides an antiviral composition and its application. Further research revealed that the combination of ellagic acid and baicalin exhibits a synergistic effect in combating fish viruses, significantly enhancing the inhibitory effect. Compared to single components, the combination forms a more effective antiviral strategy for fish, demonstrating significant application value in the field of fish virus prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of aquatic animal disease prevention and control, and particularly relates to a composition for fighting fish viruses and its application. Background Technology

[0002] As aquaculture develops towards intensification, viral diseases have gradually become a significant factor affecting the stability of fish farming. Fish viral diseases are characterized by rapid transmission, long incubation periods, and high mortality rates, often causing substantial economic losses once an outbreak occurs. Furthermore, the prevalence of viral diseases can lead to increased drug use and heightened risks to the quality and safety of aquatic products, posing a potential threat to the safety of the entire industry chain. Therefore, conducting research on fish virus prevention and control technologies is of great importance for ensuring the stability of aquaculture production and the sustainable development of the industry.

[0003] The prevention and control of viral diseases in fish still faces certain technical challenges. As strict intracellular parasites, viruses' life cycle depends on the host cell's metabolic mechanisms, which means that antiviral drug development must ensure both inhibition of viral replication and low toxicity to host cells. Furthermore, the open nature of aquaculture environments leads to complex viral transmission routes, making it difficult for existing single-method control approaches to achieve complete control.

[0004] Regarding specific prevention and control measures, existing technologies mainly involve biosafety measures, vaccine immunization, immune enhancers, and chemical drug intervention. Among these, biosafety measures such as quarantine and disinfection are preventative measures, and their intervention effect is relatively limited after the virus has entered the aquaculture system; vaccine development cycles are relatively long, and the serotype diversity of the virus may make it difficult for a single vaccine to cover all circulating strains; although immune enhancers can improve non-specific immunity to some extent, their protective efficacy in emergency treatment scenarios needs further clarification; in terms of chemical drugs, there are currently relatively few antiviral drugs specifically for aquaculture, and commonly used clinical regimens mostly draw on human or veterinary experience, with insufficient data on their pharmacokinetic characteristics and safety in aquatic animals.

[0005] In conclusion, existing fish virus control technologies still have room for improvement in terms of targeting, effectiveness, and safety. Given the frequent occurrence of viral diseases, developing novel antiviral technologies is of significant research value for enhancing the prevention and control of aquatic animal diseases. Summary of the Invention

[0006] In order to provide a drug with specific antiviral effects against fish viruses, the present invention provides an antiviral composition and its application.

[0007] According to one aspect of the present invention, an anti-fish virus composition is provided, the composition comprising baicalin and ellagic acid.

[0008] Scutellaria baicalensis is a traditional Chinese herbal medicine. Its main active ingredient, baicalein, belongs to the flavonoid family. Studies have shown that baicalein possesses various pharmacological activities, including anti-inflammatory, antioxidant, and antiviral effects. It exerts its effects by regulating inflammatory signaling pathways, scavenging free radicals, and interfering with viral replication. Baicalein can reduce the production of various pro-inflammatory cytokines by inhibiting key inflammatory signaling pathways, such as NF-κB. It can act as an effective antioxidant, scavenging free radicals in the body, reducing oxidative stress-induced cell damage, inhibiting the proliferation of various cancer cells, and inducing apoptosis. Ellagic acid is a natural polyphenol compound with potential benefits in anti-tumor activity, improving metabolic syndrome (such as aiding in lowering blood sugar and lipids), and maintaining cardiovascular and nervous system health. Further research in this invention has found that the combination of ellagic acid and baicalein has a synergistic effect in combating fish viruses, significantly enhancing the inhibitory effect on fish viruses and synergistically blocking viral infection of host cells. Compared to single components, the combination of these two ingredients forms a more effective antiviral strategy for fish, demonstrating significant application value in the field of fish virus prevention and control. More importantly, both baicalin and ellagic acid are plant-derived natural components, friendly to host cells and the aquatic environment, avoiding food safety and ecological risks associated with chemical drug residues. Therefore, the composition provided by this invention possesses high safety, significant inhibitory effect, and environmental friendliness, making it suitable for virus control in healthy and ecological fish farming, and exhibiting excellent promotional value and application potential.

[0009] Preferably, the concentration of baicalein in the composition is 6.25 μg / mL to 8 mg / mL.

[0010] Preferably, the concentration of ellagic acid in the composition is 12.5 μg / mL to 50 μg / mL.

[0011] Preferably, the composition contains baicalein at a mass ratio of 6.25–12.5: 12.5–25 ellagic acid. Furthermore, this invention has found that when baicalein and ellagic acid are combined in a specific mass ratio, their synergistic effect on antiviral activity can be optimized. Within this ratio range, baicalein limits SGIV infection in cells by interfering with the invasion and replication phases of SGIV infection, while the introduction of ellagic acid further optimizes the composition's inhibitory effect on SGIV replication in cells, resulting in a complementary mechanism of action. The composition at this specific ratio exhibits a more balanced and stable effect in inhibiting fish viruses.

[0012] Preferably, the extraction method of baicalin includes the following steps: S1. Mixing Scutellaria baicalensis with a solvent and controlling the liquid-to-solid ratio at 10-15:1 to obtain a first reaction solution; S2. Performing a first reflux extraction on the first reaction solution at 80-85°C, followed by vacuum concentration and precipitation to obtain crude baicalin; S3. Mixing the crude baicalin with an acid solution and performing a second reflux extraction, precipitation, and filtration to obtain crude baicalin; S4. Mixing the crude baicalin with a crystallization solvent and performing crystallization, filtration, and drying to obtain baicalin. Furthermore, the baicalin obtained by the above method of the present invention has high purity, which helps to improve the accuracy of antiviral experiments and the reliability of efficacy evaluation.

[0013] Preferably, the solvent added in step S1 is 70% ethanol.

[0014] Preferably, the first reflux extraction process is as follows: after refluxing the first reaction solution at 80°C to 85°C for 1.5 to 2 hours, it is cooled and filtered to obtain a first filtrate and a first filter residue; then the first filter residue is mixed with a solvent, refluxed again at 80°C to 85°C for 1 hour, cooled and filtered to obtain a second filtrate and a second filter residue; the first filtrate and the second filtrate are mixed to obtain a second reaction solution.

[0015] Preferably, the vacuum concentration process involves concentrating the second reaction solution under reduced pressure at 60°C to 65°C until it reaches approximately 1 / 5 to 1 / 4 of its original volume.

[0016] Preferably, the acid solution added in step S3 is at least one of dilute sulfuric acid and dilute hydrochloric acid.

[0017] Preferably, the second reflux extraction step is as follows: the mixture of crude baicalin and acid solution is heated and refluxed in a boiling water bath (about 100°C) or oil bath for 3 to 4 hours.

[0018] Preferably, the crystallization solvent in step S4 is 95% ethanol.

[0019] Preferably, in step S4, the ratio of crude baicalein to crystallization solvent is calculated to be 1:20~30 by mass.

[0020] Preferably, the composition contains pharmaceutically acceptable excipients or auxiliary ingredients.

[0021] In another aspect, the present invention provides the use of the above-described composition in the preparation of a medicament for the prevention or treatment of viral diseases in fish.

[0022] Preferably, when the drug is applied in cells, the concentration range of baicalein is 6.25 μg / mL to 25 μg / mL.

[0023] Preferably, when the drug is applied in cells, the concentration of baicalein is 25 μg / mL.

[0024] Preferably, when the drug is applied in vivo, the concentration range of baicalein is 0.8 mg / mL to 8 mg / mL.

[0025] Preferably, when the drug is applied in vivo, the concentration of baicalein is 8 mg / mL.

[0026] Preferably, when the drug is applied in cells, the concentration of ellagic acid is in the range of 12.5 μg / mL to 25 μg / mL.

[0027] Preferably, when the drug is applied in cells, the concentration of ellagic acid is 25 μg / mL.

[0028] Preferably, when the drug is used in vivo, the concentration range of ellagic acid is 1.6 mg / mL to 8 mg / mL.

[0029] Preferably, when the drug is used in vivo, the concentration of ellagic acid is 8 mg / mL.

[0030] Preferably, the fish virus includes iridovirus.

[0031] Preferably, the iridovirus is an iridovirus that infects grouper. As a high-value marine aquaculture species, grouper farming is severely affected by viral diseases. Among them, grouper iridovirus (SGIV) is highly infectious, has a high pathogenicity, and a high mortality rate after infection, which has a significant impact on the grouper farming industry. Attached Figure Description

[0032] Figure 1 The graph shows the qualitative and quantitative detection results of different concentrations of baicalin inhibiting grouper iridovirus in Example 1 of the present invention. Figure 2 This is a flow cytometry result of different concentrations of baicalin inhibiting grouper iridovirus in Example 1 of the present invention; Figure 3 This is a graph showing the detection results of ellagic acid inhibiting the replication of grouper iridovirus in cells in Example 2 of the present invention.

[0033] Figure 4 The image shows the light microscopic observation and crystal violet staining results of the inhibition of grouper iridovirus infection by the combination of baicalin and different concentrations of ellagic acid in Example 3 of the present invention.

[0034] Figure 5 The image shows the light microscopic observation and crystal violet staining results of the inhibition of grouper iridovirus infection by the combination of ellagic acid and different concentrations of baicalin in Example 3 of the present invention. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of this invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0036] Unless otherwise specified, the technical terms and scientific jargon used in this invention shall have the same meaning as commonly understood by those skilled in the art. The terminology used in this specification is intended to describe specific implementations and is not intended to limit the scope of the invention.

[0037] Unless otherwise expressly stated, all numerical values ​​relating to composition, reaction conditions, etc., appearing in this specification and claims should be understood as approximate values ​​with reasonable deviations, and may be appropriately adjusted according to the desired target performance.

[0038] The term "and / or" as used in this invention refers to any one or all combinations of the elements involved.

[0039] The terms "include" and "contain" refer to either the case where only the specified elements are included, or the case where other unlisted elements may be included.

[0040] Unless otherwise specified, all percentages in this invention refer to weight percentages.

[0041] Furthermore, the terms “a,” “an,” “an,” and “the” used in this specification are intended to mean “at least one” or “one or more” unless the context otherwise requires. For example, “a component” should be understood as one or more types of components; therefore, in practice, a single component or a combination of multiple components may be used.

[0042] 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.

[0043] The grouper spleen cells (GS) are preserved in the applicant's laboratory and are available to the public from the applicant for use only to replicate the experiments of this invention.

[0044] The grouper iridovirus (Guangxi strain, SGIV) was isolated from diseased pearl grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) in Beihai, Guangxi, and is preserved in the applicant's laboratory. It is publicly available and used solely for replicating experiments of this invention. In the following examples, the SGIV virus was diluted to 10⁵ TCID₅₀ / mL in cell culture medium before use.

[0045] Primers for the main capsid protein MCP gene of grouper iridovirus: forward primer (qMCP-F) 5'-GCACGCTTCTCTCACCTTCA-3', reverse primer (qMCP-R) 5'-AACGGCAACGGGAGCACTA-3'; primers for the VP19 gene of grouper virus envelope protein: forward primer (qVP19-F) 5'-TCCAAGGGAGAAACTGTA AG-3', reverse primer (qVP19-R) 5'-GGGGTAAGCGTGAAGAC T-3'. Primers for the internal reference gene β-actin: forward primer (β-actin-F) 5'-TACGAGCTGCCTGACGGACA-3', reverse primer (β-actin-R) 5'-GGCTGTGATCTCCTTCTGCA-3'. Primers were synthesized by Shanghai Sangon Biotech.

[0046] Example 1 1. Preparation of baicalin The extraction method for baicalin is carried out according to the following steps: S1. Weigh 100g of Scutellaria baicalensis powder (purchased from Yixin Pharmacy), add 70% ethanol solution as solvent, and control the liquid-to-solid ratio (mL:g) at 10:1 to 15:1 to obtain the first reaction solution.

[0047] S2. In the reflux apparatus, set the water bath temperature to 80℃~85℃ to maintain the first reaction solution in a gentle reflux state. Then perform the first reflux extraction, continuing heating under reflux for 1.5~2 hours before stopping heating. After slight cooling, filter while hot using a Buchner funnel, retaining the first filtrate. Add an equal volume of fresh 70% ethanol to the first filter residue and perform a second reflux extraction under the same conditions for approximately 1 hour. Filter again while hot, and combine all the filtrates from both extractions. Pour the combined filtrate into a rotary evaporator and concentrate under reduced pressure in a 60℃-65℃ water bath, recovering the ethanol and concentrating to approximately 1 / 5 to 1 / 4 of the original volume (at this point, the solution should be relatively viscous, but no large amount of solids should have precipitated). Pour the concentrated solution into a large beaker while hot, and slowly, dropwise add concentrated hydrochloric acid while continuously stirring to adjust the pH of the solution to 1.5-2.0. At this time, a large amount of yellow flocculent precipitate will be formed. Place the beaker in a 4°C refrigerator or ice water bath and refrigerate for 24 hours to allow the precipitate to fully aggregate and precipitate completely, thus obtaining crude baicalin (the yellow precipitate mentioned above).

[0048] S3. Weigh 10 g of dried crude baicalin and add 150 mL of 5% dilute sulfuric acid solution as the hydrolysis medium. Heat under reflux in a boiling water bath (approximately 100°C) for 3-4 hours. The solution color will gradually deepen during this process. Stop heating and cool to room temperature. At this point, a large amount of brownish-yellow to yellowish-brown precipitate should precipitate in the solution; this is the crude baicalin. Filter using a Buchner funnel and collect the precipitate. Wash the precipitate with plenty of distilled water until the eluent is neutral. Dry the filter cake (crude baicalin) in a vacuum drying oven at 60°C.

[0049] S4. Dissolve the dried crude baicalein in approximately 20-30 times its volume of 95% ethanol in a water bath at 70-75°C with stirring. Allow the solution to cool naturally at room temperature to allow slow crystallization, or place it in a 4°C refrigerator to promote crystallization. Let it stand for 12-24 hours until crystallization is complete. Then, filter the solution and collect the pale yellow needle-like or powdery crystals. Quickly wash the crystals 1-2 times with a small amount of cold ethanol (4°C). Dry the crystals under vacuum at 60°C to obtain baicalein of higher purity.

[0050] 2. Effects of different concentrations of baicalin against grouper iridovirus The following study was used to verify the effect of baicalin on grouper iridovirus.

[0051] (1) Grouper spleen cells (GS cells) were seeded into 24-well culture plates at a concentration of 1×10⁻⁶ cells / well. 5 Cells / mL were incubated at 28°C for 18 hours, and the culture medium (Leibovitz's L-15, Seville Biosciences) was discarded. The negative control group received 400 μL of fresh L15 culture medium; the virus test group received 1 μL (10...6 The test included 400 μL of TCID50 / mL grouper iridovirus (SGIV) and 400 μL of fresh cell culture medium. The baicalin test group received 400 μL of baicalin at different concentrations (0.025 mg / mL, 0.0125 mg / mL, 0.00625 mg / mL) and 1 μL of 10... 6 SGIV (TCID50 / mL). Three wells were set up for both the negative control and experimental groups to serve as replicates. After culturing for 48 hours, the cell culture medium was removed, and the cells were washed once with PBS buffer. Then, 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 1 hour. After 1 hour, the cells were washed three times with PBS buffer, and 300 μL of 0.1% crystal violet staining solution was added, followed by staining at room temperature for 2 hours. After staining, the dye was discarded, and the cells were air-dried upside down. The intensity of cell color was observed to determine cell viability, and photographs were taken for recording.

[0052] (2) Seed GS cells into 12-well culture plates at a concentration of 9 × 10⁻⁶ cells / well. 5 Cells were cultured at 28°C for 18 hours at a density of 10 cells / mL, and the culture medium was discarded. 800 μL of fresh cell culture medium was added to the negative control group cells; 2 μL (10⁻⁶ cells / mL) of fresh cell culture medium was added to the virus test group cells. 6 TCID50 / mL) SGIV and 800 μL of fresh cell culture medium were added. The baicalin experimental groups were treated with 800 μL of different concentrations (0.025 mg / mL, 0.0125 mg / mL, 0.00625 mg / mL) of baicalin and 2 μL of 10 (TCID50 / mL) SGIV. 6 SGIV (TCID50 / mL). Three wells were set up for both the negative control group and the experimental group as replicates. After cells were cultured for 48 hours, RNA was extracted from each group, reverse transcribed into cDNA, and the relative expression levels of SGIV MCP and VP19 genes were detected using real-time quantitative PCR (RT-qPCR) as an internal reference gene to determine the inhibitory effect of baicalin on grouper iridovirus in this embodiment.

[0053] (3) Flow cytometry detection of different concentrations of baicalin against grouper iridovirus was performed using the following steps. Aptamer LYGV1 was labeled with 6-carboxyfluorescein (FAM) to obtain FAM-LYGV1. Cells were cultured at 8 × 10⁻⁶ cells / cells. 5 Each hole is fed into a 12-well plate, at 28... Cultured at C for 18 hours. The baicalin test group consisted of different concentrations of baicalin (0.025 mg / mL, 0.0125 mg / mL, 0.00625 mg / mL) mixed with 2 µL SGIV (10... 6 TCID50 / mL) at 4 After incubation at C for 2 hours, GS cells were co-treated (total volume 800 µL). The negative control group was incubated with only 800 µL L15 medium, while the virus test group was incubated with 2 µL SGIV (10... 6 After mixing TCID50 / mL and 800 µL of L15 medium, incubate at 4°C. After incubating at C for 2 hours, cells were incubated at 28°C. After culturing at 92°C for 48 hours, cells were collected from each well using a cell scraper. FAM-LYGV1 was cultured at 92°C. C-transformation takes 5 minutes, 4 C-refolding for 5 minutes, followed by each group of cells being incubated with 10 µL of FAM-LYGV1 at 4°C. Incubate at C for 30 minutes, gently tapping the cells every 5 minutes. Wash twice with 300 µL PBS, then resuspend the cells in 200 µL PBS and analyze using a flow cytometer with the FITC channel selected. Results are expressed as mean ± SD of three independent samples.

[0054] Example 2 This embodiment was used to investigate the effect of ellagic acid on grouper iridovirus. The ellagic acid used in this embodiment was purchased from Chengdu Ruifens Biotechnology Co., Ltd. (analytical purity > 97%), product number CAS: 476-66-4.

[0055] GS cells were seeded into 24-well culture plates at a concentration of 1×10⁶ cells / well. 5 Cells were cultured at 28°C for 18 hours at a density of 10 cells / mL, and the culture medium was discarded. 400 μL of fresh cell culture medium was added to the negative control group cells; 1 μL (10⁻⁶ cells / mL) was added to the virus test group cells. 6 400 μL of fresh cell culture medium was added to the SGIV (TCID50 / mL). The ellagic acid test group was treated with 400 μL of ellagic acid at different concentrations (0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL) and 1 μL of 10 mg / mL SGIV (TCID50 / mL). 6 SGIV (TCID50 / mL). Both the negative control and experimental groups were replicated in triplicate. After 48 hours of culture, the cell culture medium was removed, and the cells were washed once with PBS buffer. Then, 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 1 hour. After 1 hour, the cells were washed three times with PBS buffer, and 300 μL of 0.1% crystal violet solution was added for staining at room temperature for 2 hours. After staining, the dye was discarded, and the cells were air-dried upside down. The intensity of cell color was observed to determine cell viability, and photographs were taken for recording.

[0056] Example 3 This embodiment is used to investigate the effect of the combination of baicalin and ellagic acid on grouper iridovirus, wherein baicalin was provided in Example 1 and ellagic acid was provided in Example 2.

[0057] GS cells were seeded into 24-well culture plates at a concentration of 1×10⁶ cells / well. 5 Cells were cultured at 28°C for 18 hours at a density of 10 cells / mL, and the culture medium was discarded. 400 μL of fresh cell culture medium was added to the negative control group cells; 1 μL (10⁻⁶ cells / mL) was added to the virus test group cells. 6 TCID50 / mL) SGIV and 400 μL of fresh cell culture medium were added. The drug test groups were treated with 400 μL of 0.00625 mg / mL baicalein and different concentrations of ellagic acid (0, 0.0125 mg / mL and 0.025 mg / mL), and 1 μL (10) [unclear text - possibly a typo, should be 0.00625 mg / mL]. 6 TCID50 / mL) SGIV. In addition, in another experiment, the drug test group received 400 μL of 0.0125 mg / mL ellagic acid and different concentrations of baicalein (0, 0.00625 mg / mL and 0.0125 mg / mL), and 1 μL (10 6 SGIV (TCID50 / mL). Both the negative control and experimental groups were replicated in triplicate. After 48 hours of culture, the cytopathic effect of each group was observed under an optical microscope. The cell culture medium was then removed, and the cells were washed once with PBS buffer. 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 1 hour. After 1 hour, the cells were washed three times with PBS buffer, and 300 μL of 0.1% crystal violet solution was added for staining at room temperature for 2 hours. After staining, the dye was discarded, and the cells were air-dried upside down. The intensity of the cell color was observed to determine cell viability, and photographs were taken for recording.

[0058] Test Analysis Crystal violet staining results as follows Figure 1 As shown in Figure A, the virus test group (SGIV only) showed a significantly lighter color, indicating a significant decrease in the number of viable cells. The baicalin test group (SGIV + different concentrations of baicalin) showed a significantly darker color, indicating a significantly higher number of viable cells, demonstrating that baicalin can significantly inhibit SGIV virus infection. Real-time quantitative PCR results are shown below. Figure 1 B Figure 1 As shown in Figure C, the relative expression levels of SGIV MCP and VP19 genes in the baicalin experimental group were significantly lower than those in the virus experimental group, indicating that baicalin has an anti-SGIV effect.

[0059] Flow cytometry analysis results as follows Figure 2As shown, compared with the virus test group (SGIV+L15 medium), the fluorescence signal intensity of the baicalin test group (different concentrations of baicalin + SGIV) was significantly reduced and showed a concentration-dependent relationship, indicating that baicalin can inhibit SGIV infection.

[0060] Crystal violet staining results as follows Figure 3 As shown, the virus test group (SGIV only) had a significantly lighter color, indicating a significant decrease in the number of viable cells. The ellagic acid test group (SGIV + different concentrations of ellagic acid) had a significantly darker color, indicating a significantly higher number of viable cells, demonstrating that ellagic acid can significantly inhibit SGIV virus infection.

[0061] Furthermore, combined Figure 4 and Figure 5 The test results showed that the experimental group using both ellagic acid and baicalein had a better anti-SGIV effect than the experimental group using only ellagic acid or baicalein, indicating that the two complement each other in their mechanism of action in inhibiting SGIV.

[0062] Specifically, optical microscopy observations showed that cells treated with baicalin and different concentrations of ellagic acid exhibited significantly lower pathogenic effects than cells infected with SGIV alone without treatment (e.g., cells infected with SGIV but not treated with any drugs). Figure 4 (As shown in A). Crystal violet staining results showed that the pores of cells treated with baicalein and different concentrations of ellagic acid were significantly darker than those of cells infected with SGIV only without treatment, and the staining deepened with increasing ellagic acid concentration (e.g., ...). Figure 4 (As shown in B), this indicates that baicalin and ellagic acid synergistically inhibited SGIV infection in cells, and baicalin enhanced the anti-SGIV effect of ellagic acid.

[0063] Furthermore, the pathogenic effects of cells treated with ellagic acid and different concentrations of baicalin were significantly lower than those of cells infected with SGIV alone without treatment (e.g., cells infected with SGIV alone without drug treatment). Figure 5 (As shown in A). Crystal violet staining results showed that the pores of cells treated with ellagic acid and different concentrations of baicalin were significantly darker than those of untreated cells infected with SGIV only, and the staining deepened with increasing baicalin concentration (e.g., ...). Figure 5 As shown in B), this further confirms that baicalein and ellagic acid synergistically inhibit SGIV infection in cells, and ellagic acid can also enhance the anti-SGIV effect of baicalein.

[0064] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A composition for resisting fish viruses, characterized in that, The composition includes baicalin and ellagic acid.

2. The composition according to claim 1, characterized in that, In the composition, the concentration of baicalin is 6.25 μg / mL to 8 mg / mL.

3. The composition according to claim 1, characterized in that, In the composition, the concentration of ellagic acid is 12.5 μg / mL to 50 μg / mL.

4. The composition according to any one of claims 1 to 3, characterized in that, According to the mass ratio, in the composition, the baicalein : ellagic acid = 6.25~12.5 : 12.5~25.

5. The composition according to claim 1, characterized in that, The extraction method of baicalin includes the following steps: S1. Mix Scutellaria baicalensis with solvent and control the liquid-to-solid ratio to 10-15:1 to obtain the first reaction solution; S2. The first reaction solution is subjected to a first reflux extraction at 80℃~85℃, followed by vacuum concentration and precipitation to obtain crude baicalin. S3. The crude baicalin product is mixed with an acid solution and subjected to a second reflux extraction, precipitation, and filtration processes in sequence to obtain crude baicalin product; S4. The crude baicalein is mixed with a crystallization solvent and subjected to crystallization treatment, filtration and drying treatment in sequence to obtain the baicalein.

6. The composition according to claim 5, characterized in that, The first reflux extraction process is as follows: after refluxing the first reaction solution at 80℃~85℃ for 1.5 hours to 2 hours, it is cooled and filtered to obtain the first filtrate and the first filter residue; then the first filter residue is mixed with the solvent, and refluxed again at 80℃~85℃ for 1 hour, and then cooled and filtered to obtain the second filtrate and the second filter residue; the first filtrate and the second filtrate are mixed to obtain the second reaction solution.

7. The composition according to claim 1, characterized in that, The composition contains pharmaceutically acceptable excipients or auxiliary ingredients.

8. Use of the composition according to any one of claims 1 to 7 in the preparation of a medicament for the prevention or treatment of viral diseases in fish.

9. The application as described in claim 8, characterized in that, The fish viruses mentioned include iridoviruses.

10. The application as described in claim 8, characterized in that, The iridovirus mentioned is an iridovirus that infects grouper.

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