Fish virus resistant medicine and preparation method thereof

Abstract

The invention particularly discloses an anti-fish virus medicine and a preparation method thereof. The invention relates to an anti-fish virus drug, which comprises epicatechin and aucubin. The invention has the advantages of preventing and treating the infection of iridovirus on grouper, reducing the side effect of medication on the grouper body and realizing high-quality ecological breeding of aquatic products.

Description

Technical Field

[0001] The invention relates to the field of aquatic animal disease prevention and control, and in particular to an anti-fish virus drug and a preparation method thereof. Background Art

[0002] Grouper is characterized by fast growth, large size, tender meat, rich nutrition, high economic value, and is very popular in the market. It is regarded as auspicious in Hong Kong and Macao, China, and is known as a "superior delicacy". However, due to the degradation of germplasm resources, water pollution, and the breeding of pathogenic microorganisms, grouper diseases have occurred. Among them, grouper iridovirus (SGIV) can cause a mortality rate of more than 90% in grouper, which has become one of the main viral pathogens that causes economic losses to grouper and has seriously affected the global grouper aquaculture industry.

[0003] At present, there is no commercial antiviral drug that can effectively prevent and treat grouper iridovirus, and the grouper farming industry is in a dilemma where viral diseases are difficult to control. Therefore, it is urgent to explore new ideas and develop highly effective anti-grouper iridovirus drugs to promote the sustainable and healthy development of the grouper farming industry.

[0004] In recent years, the research hotspot of developing new plant-based antiviral drugs at home and abroad is to explore natural active ingredients with antiviral effects from medicinal plants. my country is rich in medicinal plant resources, so there is great potential in finding anti-aquatic animal virus fishery drugs from medicinal plants. Medicinal plants contain natural active ingredients such as terpenoids, saponins, flavonoids, polysaccharides, alkaloids, etc. These active ingredients have a variety of biological functions, including antiviral functions. In view of the safety and environmental protection of medicinal plants, and their antiviral and other functions, the research and development of medicinal plants for aquatic products will be able to solve the environmental pollution and food safety problems caused by chemical drugs such as antibiotics, and is an important development direction for achieving environmental protection, efficient disease prevention, and high-quality ecological aquaculture. Summary of the invention

[0005] In order to provide a drug that has preventive and therapeutic effects on iridovirus, reduce the adverse effects of the drug on fish, and achieve high-quality ecological aquaculture, the present invention provides an anti-fish virus drug and a preparation method thereof.

[0006] In a first aspect, the present invention provides an anti-fish virus drug, which adopts the following technical solution:

[0007] An antiviral drug for fish comprising epicatechin and aucubin.

[0008] In the present invention, epicatechin can effectively inhibit the infection of iridovirus in grouper cells and living bodies, and its biological toxicity is very low. In-depth research has found that epicatechin can not only directly interact with grouper iridovirus to exert a direct antiviral effect, but also interfere with the invasion and replication stages of grouper iridovirus infecting host cells to exert antiviral efficacy. Further, the combination of aucubin and epicatechin can further improve the anti-fish virus drug to inhibit the replication of iridovirus in grouper, and can significantly improve the therapeutic effect of grouper infected with iridovirus; moreover, the combination of epicatechin and aucubin can also improve the immunity of fish, enhance the detoxification function of liver, improve liver function, increase enzyme activity, promote bile secretion, enhance liver metabolic function, promote the repair of liver damage, and reduce the degree of liver lesions, thereby achieving the prevention and treatment effect of grouper iridovirus, and after the grouper infected with iridovirus is treated with the anti-fish virus drug in the present invention, its liver function recovery effect is good, reducing the risk of secondary infection in the subsequent breeding process. In summary, the anti-grouper virus drug provided by the present invention can safely and efficiently prevent and treat grouper iris virus, meets environmental protection requirements, can promote high-quality ecological breeding in the grouper breeding industry, and bring considerable economic benefits.

[0009] Preferably, the fish virus is a fish iridovirus.

[0010] Preferably, the fish iridovirus is grouper iridovirus.

[0011] The grouper iridovirus is tropic to the epithelial and endothelial tissues of the fish body, and is particularly destructive to the hematopoietic organs and tissues of fish such as the spleen and kidneys, thereby causing anemia, multiple organ failure and death of the sick fish; by using the anti-fish virus drug in the present application, not only can the intestinal health of grouper be improved, the immunity of grouper groups be enhanced, and the infection rate of the iridovirus of stonefish be reduced, but also the combination of epicatechin and aucubin can significantly improve the antiviral effect of the anti-fish virus drug on the iridovirus, especially the healing effect on grouper infected with the iridovirus in the early stage is significant.

[0012] In a second aspect, the present invention provides a method for preparing an anti-fish virus drug, using the following technical solution:

[0013] A method for preparing an anti-fish virus drug comprises the following steps: selecting one of green tea, polygonum multiflorum and millettia spatholobi as the plant source to extract epicatechin; and selecting one of eucommia ulmoides, plantain and rehmannia root as the plant source to extract aucubin.

[0014] Preferably, green tea is used as the plant source to extract the epicatechin; and plantain is used as the plant source to extract the aucubin.

[0015] The product uses plant-derived epicatechin and plant-derived peach leaf juglone, which are safe ingredients and easily available raw materials. They can avoid the toxic side effects of anti-fish virus drugs in fish bodies after use to the greatest extent, and improve the health of fish bodies.

[0016] Preferably, the epicatechin is used to prepare an epicatechin solution, and the aucubin solution is used to prepare an aucubin solution, and the epicatechin solution and the aucubin solution are mixed for preventing or treating fish viruses;

[0017] The concentration of the epicatechin solution is 12.5 μg / mL-50 μg / mL, and the concentration of the aucubin solution is 2.5 μg / mL-10 μg / mL.

[0018] Preferably, the preparation process of the epicatechin solution is as follows: dissolving the epicatechin in 50 mg / mL dimethyl sulfoxide (DMSO), and diluting with culture medium to obtain the epicatechin solution;

[0019] The preparation process of the aucubin solution is as follows: dissolving the aucubin in 50 mg / mL dimethyl sulfoxide (DMSO), and diluting with culture medium to obtain the aucubin solution.

[0020] The present invention utilizes epicatechin and aucubin to prepare an anti-fish virus drug. Relevant tests show that the combination of epicatechin and aucubin can effectively and safely achieve the prevention and treatment effects on grouper iridovirus, and the liver function of fish infected with iridovirus is slightly damaged after recovery, which meets the requirements of green and sustainable development and helps to achieve high-quality ecological breeding in the aquaculture industry.

[0021] Preferably, the volume ratio of the epicatechin solution to the aucubin solution is 1-2.5:0.3-0.8.

[0022] By selecting epicatechin solution and aucubin solution with appropriate concentration and volume ratio, on the one hand, the combination of the two can maximize the preventive and therapeutic effects of anti-fish virus drugs on grouper iridovirus infection. On the other hand, it can also avoid side effects or drug toxicity accumulation in the fish body after use, which is beneficial to the effective prevention and treatment of grouper iridovirus by anti-fish virus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The toxic effect of epicatechin (Figure ab) on grouper spleen cells.

[0024] Figure 2 The results show the inhibitory effects of epicatechin solution and aucubin solution at different concentrations on grouper iridovirus.

[0025] Figure 3 These are the test results of the combination of epicatechin and aucubin in inhibiting grouper iridovirus infection.

[0026] Figure 4 Epicatechin and aucubin cooperate to interfere with the invasion of grouper iridovirus into host cells and inhibit the infection of grouper iridovirus.

[0027] Figure 5 Epicatechin and aucubin cooperate to interfere with the replication stage of grouper iridovirus in host cells and inhibit the infection of grouper iridovirus.

[0028] Figure 6 These are the test results of the effect of epicatechin and aucubin on the survival rate of live grouper infected with grouper iridovirus. DETAILED DESCRIPTION

[0029] For better understanding and implementation, the technical solution of the present invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as modified by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties desired to be obtained.

[0032] As used herein, "and / or" means one or all of the mentioned elements.

[0033] As used herein, “including” and “comprising” encompasses the case where only the stated elements are present as well as the case where there are other elements other than the stated elements.

[0034] All percentages in the present invention are by weight, unless otherwise specified.

[0035] Unless otherwise indicated, "a", "an", "an" and "the" used in this specification are intended to include "at least one" or "one or more". For example, "a component" means one or more components, so more than one component may be considered and may be employed or used in the implementation of the embodiment.

[0036] 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 horizontal variance analysis method.

[0037] Grouper spleen cells (GS) are stored in the applicant's laboratory and are available to the public from the applicant for use only in repeating the experiments of the present invention.

[0038] Grouper iridovirus Guangxi strain, SGIV, was isolated from diseased pearl grouper (Epinephelus fuscoguttatus♀×E.lanceolatus♂) in Beihai, Guangxi, and is stored in the applicant's laboratory. The public can obtain it from the applicant for use only in repeating the experiments of the present invention. Before use in the following examples, the SGIV virus was diluted to 10 in cell culture medium. 5 TCID 50 / mL.

[0039] Epicatechin EC: a product of Chengdu Ruifensi Biotechnology Co., Ltd. (analytical purity>97%), product number Q817146.

[0040] Aucubin AU: Product of Chengdu Ruifensi Biotechnology Co., Ltd. (analytical purity > 97%).

[0041] Primers for the major capsid protein (MCP) gene of grouper iridovirus: forward primer (qMCP-F) 5'-GCACGCTTCTCTCACCTTCA-3', reverse primer (qMCP-R) 5'-AACGGCAACGGGAGCACTA-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 Bioengineering.

[0042] Example 1

[0043] 1. Main instruments and reagents

[0044] Optical microscope, microplate reader, 5-(2,4-disulfophenyl)-3-(2-methoxy-4-nitrophenyl)-2H-tetrazolium sodium inner salt solution (WST-8 solution).

[0045] 2. Experimental Methods

[0046] GS cells were cultured at 1×105 Each well was transferred into a 96-well plate and cultured at 28°C for 18 hours. After 18 hours, the experimental group used L15 culture medium to dilute the EC solution by half to different concentrations (50μg / mL, 100μg / mL, 250μg / mL, 500μg / mL and 1000μg / mL), and the control group (Control) was cultured with L15 culture medium, with 3 wells in each group and 100μL of solution added to each well. Cultured at 28°C for 48 hours. The cell morphology was observed under an optical microscope for 48 hours. Then the supernatant was removed, the cells were washed with phosphate buffered saline (PBS), the CCK-8 solution was diluted 10 times with PBS, and 100μL of the diluted CCK-8 solution was added to each well, and kept at room temperature and protected from light for 4 hours. Then the absorbance of each group of cells at 450nm was detected using an enzyme reader to determine the effect of EC on the viability of GS cells. The measurement results are expressed as the mean ± SD of three wells. The cell viability is calculated according to formula 2-1:

[0047]

[0048] 3. Experimental results

[0049] GS cells were treated with different concentrations of EC (50μg / mL, 100μg / mL, 250μg / mL, 500μg / mL and 1000μg / mL) for 48h. The results showed that compared with the control group cells using only L15 medium ( Figure 1 a) When EC concentration is greater than 50 μg / mL, GS cells will show obvious cytopathic effects (CPEs), including cell shrinkage, rounding, and vacuoles. When EC concentration is ≤50 μg / mL, cell morphology changes significantly compared with the control group. CCK-8 was then used to detect cell viability ( Figure 1 b) Compared with the control group, the cell viability decreased significantly when the EC concentration was greater than 50 μg / mL, indicating that the EC concentration greater than 50 μg / mL had toxic side effects on GS cells. When the EC concentration was 50 μg / mL, the cell viability did not decrease significantly compared with the control, indicating that the EC concentration ≤ 50 μg / mL had no toxic side effects on GS cells.

[0050] Example 2

[0051] 1. Main instruments and reagents

[0052] Real-time fluorescence quantitative PCR technology (qRT-PCR).

[0053] 2. Experimental Methods

[0054] GS cells were cultured at 8×10 512-well plates were transferred per well and cultured at 28°C for 18 h. After 18 h, EC was diluted to a working concentration (50 μg / mL) using L15 medium, and the aucubin solution was diluted to a working concentration (10 μg / mL) in the high-concentration experimental group and mixed with 0.5 μL SGIV (10 6.3 TCID50 / mL) were incubated at 4℃ for 2h before co-treatment of GS cells. The volume ratio of EC solution to aucubin solution was 2:0.5 (total system was 800μL); the EC concentration used in the medium concentration experimental group was 1 / 2 of the working concentration (25μg / mL), the aucubin solution concentration was 1 / 2 of the working concentration (5μg / mL), and the volume ratio of EC solution to aucubin solution was 1:0.3 (total system was 800μL). The EC concentration used in the low concentration experimental group was 1 / 4 of the working concentration (12.5μg / mL), the aucubin solution concentration was 1 / 4 of the working concentration (2.5μg / mL), and the volume ratio of EC solution to aucubin solution was 2.5:0.8 (total system was 800μL); the rest of the operations were the same as the high concentration experimental group.

[0055] Two control groups were set up: the negative control group (Control) used only L15 medium, and the positive control group (SGIV) used 0.5 μL SGIV (10 6.3 TCID50 / mL) and L15 medium, and the rest of the operation was the same as the experimental group (total system was 800 μL). After culturing at 28°C for 48 hours, cells in each group were collected to extract RNA and reverse transcribed into cDNA. RT-qPCR was used to detect the relative expression of SGIV MCP gene in each group of samples, and 3 parallels were set for each sample.

[0056] 3. Experimental results

[0057] The antiviral effect of EC on SGIV was detected and analyzed using RT-qPCR technology. The results are as follows Figure 2 As shown, compared with the group treated with L15 medium + SGIV only (SGIV), the relative expression of SGIV MCP gene in GS cells in the group treated with different concentrations of EC solution and aucubin solution (high: 50μg / mL EC solution + 10μg / mL aucubin solution, medium: 25μg / mL EC solution + 5μg / mL aucubin solution, low: 12.5μg / mL EC solution + 2.5μg / mL aucubin solution) + SGIV (experimental group) was significantly lower than that in the control group. Among them, the relative expression of SGIVMCP gene in the group treated with 50μg / mL EC solution + 10μg / mL aucubin solution decreased the most. This further indicates that EC can inhibit SGIV infection, and the inhibitory effect is concentration-dependent.

[0058] Example 3

[0059] 1. Main instruments and reagents

[0060] Real-time fluorescence quantitative PCR technology (qRT-PCR).

[0061] 2. Experimental Methods

[0062] GS cells were cultured at 8×10 5 The EC solution and aucubin solution were diluted to the working concentration (50 μg / mL EC solution + 10 μg / mL aucubin solution, the volume ratio of EC solution to aucubin solution was 2:0.5) using L15 medium in the experimental group and mixed with 1 μL SGIV (10 6.3 TCID50 / mL) were incubated at 4°C for 2 h, and the control group (SGIV) was treated with 1 μL SGIV (10 6.3 TCID50 / mL) and L15 medium were incubated at 4°C for 2h, then centrifuged at 25000g for 1h at 4°C, the supernatant was discarded, and the virus particles were resuspended. 20μL of resuspended virus particles and L15 medium were added to GS cells (total system was 800μL) and cultured at 28°C for 48h. After 48h, the total RNA of the sample was extracted and reverse transcribed into cDNA. RT-qPCR was used to detect the relative expression of the SGIV MCP gene in each group of samples. The results are expressed as the mean ± SD of three independent samples.

[0063] 3. Experimental results

[0064] The results of RT-qPCR detection technology showed that the relative expression of MCP gene of SGIV in cells of SGIV group treated with EC (50 μg / mL) was significantly downregulated compared with that of SGIV group infected with SGIV only, as shown in Figure 3 This suggests that EC and aucubin may interact with SGIV, thereby reducing the infectivity of SGIV.

[0065] Example 4

[0066] 1. Main instruments and reagents

[0067] Flow cytometry.

[0068] 2. Experimental Methods

[0069] First, SGIV was labeled with cyanine dye (Cy5) to prepare Cy5-SGIV. 20 μL SGIV (10 6.3TCID50 / mL) was added with 0.5 μL Cy5 dye and incubated at room temperature in the dark for 2 h using a horizontal shaker. After 2 h, the virus particles were collected by centrifugation at 25000 g for 1 h at 4 °C, the supernatant was discarded, and the virus particles were washed once with 100 μL TN Buffer. The cells were centrifuged at 25000 g for 1 h at 4 °C, the supernatant was discarded, and the virus particles were mixed with 20 μL TN Buffer to obtain Cy5-SGIV. The cells were placed at 4 °C in the dark for later use. GS cells were cultured at 8 × 10 5 1 μL of L15 medium was added to 12-well plates and cultured at 28°C for 18h. The experimental group and the control group (Cy5-SGIV) were mixed with 1 μL of L15 medium and added to GS cells (total system 800 μL) and treated at 4°C in the dark for 2h. After 2h, the supernatant was discarded and washed twice with L15 medium. EC solution (50 μg / mL) and aucubin solution (10 μg / mL) diluted to working concentrations with L15 medium were added to the experimental group, and the volume ratio of EC solution to aucubin solution was 2:0.5 (total system 800 μL) and cultured at 28°C for 2h. The control group was cultured with 800 μL of L15 medium, and the rest of the operations were the same as the experimental group. After 2h, the supernatant was discarded, and the cells in each well were collected by digestion with 0.1 mg / mL of proteinase K, washed twice with 300 μL of PBS solution, and finally resuspended in 200 μL PBS and analyzed by flow cytometry. The results are expressed as the mean ± SD of three independent samples.

[0070] 3. Experimental results

[0071] The results of flow cytometry detection of Cy5 fluorescence signals showed that the fluorescence signal intensity of GS cells in the groups treated with EC solution (50 μg / mL) and aucubin solution (10 μg / mL) was significantly lower than that in the group treated with L15 medium only (Cy5-SGIV), indicating that EC and aucubin treatment would reduce the number of SGIV invading host cells. Figure 4 These results suggest that EC and aucubin can exert antiviral effects by interfering with the invasion of SGIV into host cells.

[0072] Example 5

[0073] 1. Main instruments and reagents

[0074] Real-time fluorescence quantitative PCR technology (qRT-PCR).

[0075] 2. Experimental Methods

[0076] GS cells were cultured at 8×10 5 Each well was transferred into a 12-well plate and cultured at 28°C for 18 h. Both the experimental group and the control group (SGIV) were treated with 800 μL L15 medium and 1 μL SGIV (106.3 GS cells were treated with TCID50 / mL) at 4°C for 30 minutes, then the supernatant was discarded, washed twice, and then 800μL of new L15 medium was added. Incubate at 28°C for 2h, discard the supernatant, and wash twice. EC (50μg / mL) and aucubin solution (10μg / mL) diluted to working concentrations using L15 medium were added to the experimental group. The volume ratio of EC solution to aucubin solution was 2:0.5 (total system 800μL). The control group was cultured with 800μL L15 medium. The rest of the operations were the same as the experimental group. After culturing at 28°C for 10h, the total RNA of each group of cells was extracted and reverse transcribed into cDNA. RT-qPCR was used to detect the relative expression of SGIV MCP genes in each group of samples. The results are expressed as the mean ± SD of three independent samples.

[0077] 3. Experimental results

[0078] The results of RT-qPCR detection technology showed that compared with the control group (SGIV) with only L15 culture medium added, the relative expression of MCP gene of SGIV in GS cells treated with EC solution (50 μg / mL) and aucubin solution (10 μg / mL) was significantly downregulated, as shown in Figure 5 As shown. This indicates that the presence of EC and aucubin will weaken the replication of SGIV in host cells. This indicates that the combination of EC and aucubin can play an antiviral role by inhibiting the replication stage of SGIV in host cells.

[0079] Example 6

[0080] 1. Experimental Methods

[0081] 100 pearl groupers with a body length of 4-6 cm were divided into 5 groups, each with 20 fish. They were kept in the laboratory for one week before being used in the experiment. According to the results of the preliminary experiment, each fish was injected with 8 μL / g SGIV (10 6.3TCID50 / mL) virus was infected at 25℃ water temperature for 24h, and the negative control group (Control) was injected with the corresponding volume of PBS solution. After 24h of infection, the experimental groups were injected with 80μL of 16mg / mL EC solution + 20μL of 3.125mg / mL aucubin solution (recorded as group A), 80μL of 8mg / mL EC solution + 20μL of 1.56mg / mL aucubin solution (recorded as group B), and 80μL of 1.6mg / mL EC solution + 20μL of 0.32mg / mL aucubin solution (recorded as group C). Each fish in the negative control group (Control) and the positive control group (SGIV) was injected with 100μL PBS solution. The mortality of fish in each group was recorded daily, and the number of fish deaths in each group within 7 days was counted and the survival curve was drawn.

[0082] The injection concentrations of EC solution and aucubin solution in the living pearl grouper were calculated using the following formula: in vivo injection concentration (mg / kg) = cell administration dose (mg / mL) × liquid intake (mL) / animal body weight (kg).

[0083] 2. Experimental results

[0084] like Figure 6 The cumulative mortality results showed that the survival rate of grouper infected with SGIV treated with different concentrations of EC solution and aucubin solution within 7 days was higher than that of grouper infected with SGIV not treated with EC solution and aucubin solution, indicating that EC and aucubin also have a good inhibitory effect on SGIV at the in vivo level, and the inhibition rate is concentration-dependent.

[0085] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. An anti-fish virus drug, characterized in that: Includes epicatechin and aucubin.

2. The anti-fish virus drug according to claim 1, characterized in that: The fish virus is fish iridovirus.

3. The method for preparing the anti-fish virus drug according to any one of claims 1 to 2, characterized in that: Selecting one of green tea, Polygonum multiflorum and Millettia reticulata as the plant source to extract epicatechin; One of Eucommia ulmoides, Plantago asiatica and Rehmannia glutinosa is selected as the plant source to extract the aucubin.

4. The method for preparing the anti-fish virus drug according to claim 3, characterized in that: Green tea is used as the plant source to extract the epicatechin; plantain is used as the plant source to extract the aucubin.

5. The method for preparing the anti-fish virus drug according to claim 4, characterized in that: The epicatechin is used to prepare an epicatechin solution, the aucubin is used to prepare an aucubin solution, and the epicatechin solution and the aucubin solution are mixed for preventing or treating fish viruses; The concentration of the epicatechin solution is 12.5 μg / mL-50 μg / mL, and the concentration of the aucubin solution is 2.5 μg / mL-10 μg / mL.

6. The method for preparing the anti-fish virus drug according to claim 5, characterized in that: The volume ratio of the epicatechin solution to the aucubin solution is 1-2.5:0.3-0.8.

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