Application of epicatechin in preparation of medicine for resisting frog iridovirus

Abstract

The invention provides application of epicatechin in preparation of a medicine for resisting frog iridovirus. Related experiments prove that the epicatechin can efficiently prevent and control infection and transmission of the frog iridovirus, so that the harm of the frog iridovirus to healthy culture of frogs such as bullfrogs can be reduced to a great extent, and high-quality ecological culture and economic benefits of the frog culture industry are promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of aquatic animal disease prevention and control, and particularly relates to application of epicatechin in preparation of an anti-frog iridovirus drug. BACKGROUND

[0002] The bullfrog is an amphibian with delicious meat, rich in protein, vitamins and minerals, and low in fat, and has high nutritional value, and is deeply loved by the public in cooking. In addition, the bullfrog also has certain medicinal value, because there are many bioactive substances in the skin and some organs of the bullfrog, and these active substances can be used for the preparation of traditional Chinese medicines and medicinal health products. Because the bullfrog has economic, nutritional and medicinal values, the bullfrog breeding industry has been increasingly popular in recent years.

[0003] However, the intensification of water environmental pollution, the breeding of pathogenic microorganisms, and the increasingly serious disease problems of the bullfrog seriously threaten the healthy and sustainable development of the bullfrog breeding industry. Among them, the model species of frog virus type 3 (FV3) that can infect fish, amphibians and reptiles can cause widespread transmission and infection, thereby causing significant economic losses to the breeding industry of these species. At present, there is no effective commercial antiviral drug on the market that can effectively control the infection of frog iridovirus, and the bullfrog breeding industry is facing the situation that viral diseases are difficult to control. Therefore, it is urgent to find new ways and innovative ideas to develop efficient drugs against frog iridovirus in order to ensure the sustainable and healthy development of the bullfrog breeding industry.

[0004] One of the common means for preventing and treating diseases of aquatic animals in aquaculture is to use antibiotics and vaccines. Antibiotics have a significant effect on bacterial diseases, but the abuse of antibiotics can easily lead to water environmental pollution, the generation of drug-resistant pathogens, and food safety problems. Vaccines have good preventive effect on disease outbreaks, but their strict inactivation and storage conditions and the uncertainty of the dose to some extent affect the application of vaccines in aquaculture. More and more research and development of antiviral drugs for aquatic animals focus on medicinal plants, which are called “green drugs”, because of their rich resources, environmental friendliness, low price, and functions of antibacterial, antiviral, and immune enhancement. In view of the advantages and clear antiviral mechanism of medicinal plants, the development of medicinal plant fish medicines will solve the environmental pollution and food safety problems caused by the abuse of chemical drugs such as antibiotics, and can be used as an important development direction of high-quality ecological aquaculture. SUMMARY

[0005] In order to solve the problems and deficiencies in the prior art, the application provides an application of epicatechin in preparation of an anti-frog iridovirus drug. The relevant experiments prove that epicatechin can efficiently prevent and treat frog iridovirus infection and transmission, thus greatly reducing the harm of frog iridovirus to the healthy breeding of bullfrogs and the like, and promoting the high-quality ecological breeding and economic benefits of the frog breeding industry.

[0006] According to a first aspect of the application, an application of epicatechin in preparation of an anti-frog iridovirus drug is provided.

[0007] Frog iridovirus is a double-stranded DNA virus that can infect cold-blooded vertebrates including amphibians, fish and reptiles, and is a catastrophic pathogen in the bullfrog breeding industry. It can infect all stages of the life cycle of bullfrogs, has a very high mortality rate, and seriously threatens the healthy and sustainable development of the bullfrog breeding industry. At present, there are almost no commercial drugs that can effectively target frog iridovirus. Moreover, as mentioned earlier, the current drugs for frog iridovirus are traditional antibiotics and vaccines, and the use of antibiotics and vaccines has many problems as mentioned above. Therefore, the development of new medicinal plants to effectively prevent and / or treat frog iridovirus is of great significance to the healthy and ecological breeding of bullfrogs and the like.

[0008] Epicatechin is a polyphenolic compound mainly derived from green tea, and also has a high content in cocoa, black grapes, apples, raspberries and cherries. It has various activities, including antioxidant and anti-inflammatory activities, reduction and delay of muscle mass loss, improvement of symptoms of cardiovascular and cerebrovascular diseases, alteration of metabolic characteristics, rheological properties of blood and crossing of the blood-brain barrier, prevention of diabetes, protection of the nervous system, influence on the life cycle of tumor cells to inhibit tumor cell growth. However, there is little prior art using epicatechin to prevent or treat frog iridovirus.

[0009] However, through relevant experimental verification, the application proves that epicatechin can effectively inhibit the infection of frog iridovirus in cells, specifically, epicatechin can effectively inhibit the replication of frog iridovirus in cells, reduce the cytopathic effect caused by frog iridovirus infection, and inhibit the continuous infection of frog iridovirus in cells. Moreover, epicatechin has almost no biological toxicity, so as an anti-frog iridovirus drug, it can effectively reduce the side effects on frogs, promote the healthy and ecological development of frog aquaculture and environmental friendliness, and is also conducive to humans eating healthier bullfrogs and the like, and thus is conducive to improving human health.

[0010] Preferably, the frog iridovirus is Ranavirus 3 (FV3). Specifically, the experiments related to Ranavirus 3 (FV3) prove that epicatechin has a significant inhibitory effect on the infection of Ranavirus 3 (FV3) in cells, and has little toxicity to the cells.

[0011] Preferably, the frog includes bullfrog. Bullfrog is the main frog infected with Ranavirus 3 (FV3), and bullfrog farming has important economic, ecological and social significance. Epicatechin can have a significant inhibitory effect on bullfrog susceptible to Ranavirus 3 (FV3), that is, epicatechin has better targeted prevention and / or treatment for bullfrog infected with Ranavirus 3 (FV3), and has important economic and ecological significance for the aquaculture industry of bullfrog.

[0012] Preferably, a working solution prepared from the epicatechin is used to prevent and / or treat frog iridovirus, and the concentration of the epicatechin in the working solution is 3.125 μg / mL to 12.5 μg / mL. At the above concentration, the epicatechin at a lower concentration cannot sufficiently and effectively prevent and / or treat frog iridovirus, and the epicatechin at a higher concentration can cause adverse reactions to the frog, affecting the quality and survival rate of the frog in frog farming.

[0013] Preferably, the concentration of the epicatechin in the working solution is 6.25 μg / mL to 12.5 μg / mL. Preferably, the concentration of the epicatechin in the working solution is 12.5 μg / mL.

[0014] Preferably, the specific operation for preparing the working solution is as follows: the epicatechin is dissolved in a solvent to 40 to 60 mg / mL, which is used as a mother liquor, and then diluted with L15 culture solution to a cell safety concentration as the working solution, and the concentration of the epicatechin in the working solution is 3.125 μg / mL to 12.5 μg / mL.

[0015] According to a second aspect of the present application, the epicatechin is used in the preparation of frog feed for resisting frog iridovirus.

[0016] Preferably, the frog iridovirus is Ranavirus 3 (FV3).

[0017] Preferably, the frog includes bullfrog.

[0018] Preferably, the mass ratio of epicatechin in the frog feed is 0.01 to 0.05%.

[0019] In summary, epicatechin can effectively prevent and control the infection and transmission of frog iridovirus, and has little biological toxicity, is friendly to the environment, promotes high-quality ecological breeding of frog farming, and can bring considerable economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Toxicity of epicatechin on carp epithelioma cells in Example 1.

[0021] Figure 2 Optical microscope observation of inhibition of frog iridovirus infection in cells by epicatechin in Example 2.

[0022] Figure 3 Quantitative analysis of inhibition of frog iridovirus infection in cells by epicatechin in Example 3.

[0023] Figure 4 Optical microscope observation of inhibition of frog iridovirus infection in cells by epicatechin in Example 4.

[0024] Figure 5 Quantitative analysis of inhibition of frog iridovirus infection in cells by epicatechin in Example 5. DETAILED DESCRIPTION

[0025] In order to enable the personnel in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all.

[0026] The quantitative experimental data involved in the following examples are expressed as mean ± standard deviation (±s), and the statistical software SPSS 17.0 is used to statistically process the comparison data between groups by using the method of one-way square difference analysis.

[0027] The carp epithelioma cell line (epithelioma papulosum cyprini cells, EPCs) is stored in the laboratory of the applicant and is available from the applicant for use only for repeating the experiments of the present application.

[0028] The frog iridovirus (frog virus 3, FV3) is isolated from sick bullfrogs and stored in the laboratory of the applicant and is available from the applicant for use only for repeating the experiments of the present application.

[0029] The product of Chengdu Ruifenshi Biological Technology Co., Ltd. (analytical purity > 97%) has a product number of B-020.

[0030] Primers for the frog iridovirus ORF82 gene: forward primer (q82R-F) 5'-TGTGTCTGGAGAACCCTACA -3', reverse primer (q82R-R) 5'-TGGTGATCTTGACTTGAAACTC -3'. Primers for the internal reference gene β-actin: forward primer (β-actin-F) 5'-TCTCCAGCCATCCTTCCTTGG-3', reverse primer (β-actin-R) 5'-CTGCATACGGTCAGCAATGCC-3'. Primers were synthesized by Shanghai Sangon Biotech.

[0031] Example 1 1. Main instruments and reagents Optical microscope, microplate reader, 5-(2,4-disulfophenyl)-3-(2-methoxy-4-nitrophenyl)-2H-tetrazole sodium inner salt solution (WST-8 solution).

[0032] 2. Experimental Methods EPC cells (1×10) 5 Cells were inoculated into 96-well plates (number of cells / well) and cultured at 28°C for 18 h. Then, in the experimental group, epicatechin was diluted half-fold to 6.25 µg / mL, 12.5 µg / mL, 25 µg / mL, 50 µg / mL, and 100 µg / mL in L15 medium, respectively. Control group cells were cultured in L15 medium without epicatechin. Each group had 3 wells, with 100 µL of liquid added to each well. After 48 h, cell morphology was observed under an optical microscope. After removing the cell supernatant, cells were washed twice with phosphate-buffered saline (PBS), and 100 µL of CCK-8 solution diluted 10-fold with PBS was added to each well. Cells were incubated at room temperature in the dark for 4 h. The absorbance of each group of cells at 450 nm was then measured using a microplate reader to determine the effect of epicatechin on EPC cell viability. Results are expressed as the mean ± SD of the three wells. Cell viability is calculated using formula 2-1: Cell viability = 100% 3. Experimental Results Optical microscopy observations revealed that, compared to the control group cells (e.g. Figure 1 As shown in Figure A), EPC cells with epicatechin concentrations >12.5 µg / mL exhibited significant morphological changes such as cell shrinkage and rounding, while when the epicatechin concentration was ≤12.5 µg / mL, the cell morphology showed no significant difference compared to the control group. Cell viability was determined by CCK-8 assay (as shown in Figure A). Figure 1As shown in FIG. 2B, compared with the control group, the cell viability was significantly reduced when the epicatechin concentration was > 12.5 μg / mL, indicating that epicatechin concentrations > 12.5 μg / mL were toxic to EPC cells, and the cells were damaged, while when the epicatechin concentration was ≤ 12.5 μg / mL, the cell viability did not change significantly compared with the control. The above results show that epicatechin concentrations ≤ 12.5 μg / mL have no toxic effect on EPC cells.

[0033] Example 2 1. Main instruments and reagents Optical microscope.

[0034] 2. Experimental method EPC cells (1 x 10 5 After 18 h of culture at 28°C, the experimental group used L15 medium to prepare epicatechin at a concentration of 12.5 μg / mL, and then incubated with 20 μL of FV3 virus (EC+FV3) at 4°C for 2 h before adding to the EPC cells (total system 800 μL). After 12 h, 24 h, and 36 h of culture at 28°C, the virus infection of the cells in each group was observed by optical microscope. Cells treated only with L15 medium were used as the negative control group (Control), and cells infected only with FV3 virus were used as the positive control group (FV3). The average value of three wells ± SD was used to represent the determination results. 3. Experimental results The optical microscope observation results showed (as shown in FIG. 3B), compared with the control group, the cells in the FV3 group showed obvious cytopathic effect, while the cytopathic effect of the EPC cells in the epicatechin+FV3 group was significantly lower than that in the FV3 group, indicating that epicatechin can reduce the cytopathic effect caused by FV3 infection to a certain extent. Figure 2

[0035] Example 3 1. Main instruments and reagents Real-time fluorescent quantitative PCR technology (qRT-PCR).

[0036] 2. Experimental method EPC cells (1 x 10 5 ​Cells were inoculated into 12-well plates and cultured at 28°C for 18 h. Then, in the experimental group, epicatechin was prepared to a concentration of 12.5 µg / mL using L15 medium and incubated with 20 µL of FV3 virus (EC+FV3) at 4°C for 2 h before being added to EPC cells (total volume 800 µL). Cells from each group were then cultured at 28°C for 12 h, 24 h, and 36 h, after which cells were collected, RNA was extracted, and reverse transcribed into cDNA. Using cDNA as a template, the relative expression level of the FV3 ORF82 gene in each group was detected by RT-qPCR. Cells treated only with L15 medium served as the negative control (Control), and cells infected only with FV3 virus served as the positive control (FV3). Each treatment group was performed in triplicate, and the results are expressed as the mean ± SD of the three groups.

[0037] 3. Experimental Results RT-qPCR test results showed that, Figure 3 As shown, the relative expression level of the FV3 ORF82 gene in EPC cells of the epicatechin + FV3 group was significantly lower than that in the FV3 group, indicating that epicatechin inhibited the replication of FV3 in EPC cells.

[0038] Example 4 1. Main instruments and reagents Optical microscope.

[0039] 2. Experimental Methods EPC cells (1×10) 5 Cells were inoculated into 12-well plates (number cells / well) and cultured at 28°C for 18 h. Then, epicatechin was prepared to a concentration of 12.5 µg / mL using L15 medium. 800 µL of this concentration of epicatechin was added to EPC cells and incubated at 28°C for 6 h. The medium containing epicatechin was discarded, and the cells were washed twice with PBS and then added to 800 µL of L15 medium containing 20 µL of FV3. Cells treated only with L15 medium but not infected with FV3 served as the negative control (Control), while EPC cells first treated with 800 µL of L15 medium for 6 h and then infected with FV3 (20 µL of FV3 added to 800 µL of L15 medium) served as the positive control (FV3). Cells in each group were further infected with the virus at 28°C for 48 h, and the cytopathic effects were observed using an optical microscope.

[0040] 3. Experimental Results Optical microscopy observations show that, for example Figure 4As shown, cells in the FV3 group exhibited significant cytopathic effects 48 h after viral infection, while the cytopathic effects of EPC cells in the epicatechin + FV3 group were lower than those in the FV3 group, indicating that epicatechin pretreatment of EPC cells can also reduce the cytopathic effects induced by FV3 infection.

[0041] Example 5 1. Main instruments and reagents Real-time quantitative PCR (qRT-PCR) technology.

[0042] 2. Experimental Methods EPC cells (1×10) 5 Cells were inoculated into 12-well plates (number cells / well) and cultured at 28°C for 18 h. Then, epicatechin was prepared to a concentration of 12.5 µg / mL using L15 medium. 800 µL of this concentration of epicatechin was added to EPC cells and incubated at 28°C for 6 h. After discarding the epicatechin-containing medium, the cells were washed twice with PBS and then added to 800 µL of L15 medium containing 20 µL of FV3. Cells treated only with L15 medium but not infected with FV3 served as the negative control (Control), while EPC cells first treated with 800 µL of L15 medium for 6 h and then infected with FV3 (20 µL of FV3 added to 800 µL of L15 medium) served as the positive control (FV3). Cells in each group were cultured at 28°C for 48 h, and RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, the relative expression level of the FV3 ORF82 gene in each group was detected by RT-qPCR. Each treatment group was set up in triplicate, and the results were expressed as the mean ± SD of the three groups.

[0043] 3. Experimental Results RT-qPCR test results showed that, Figure 5 As shown, the relative expression level of the FV3 ORF82 gene in EPC cells with epicatechin + FV3 was significantly lower than that in cells in the FV3 group. This suggests that epicatechin pretreatment of cells for 6 h may have activated the host's immune response, enabling the cells to establish an antiviral state and thus inhibiting subsequent FV3 infection.

[0044] 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. Use of epicatechin in the preparation of a drug for resisting frog iridovirus.

2. The use of epicatechin in the preparation of a drug for resisting frog iridovirus according to claim 1, wherein the frog iridovirus is Rana virus type 3.

3. The use of epicatechin in the preparation of a drug for resisting frog iridovirus according to claim 1, wherein the frog includes bullfrog.

4. The use of epicatechin in the preparation of a drug for resisting frog iridovirus according to claim 1, wherein a working solution prepared from the epicatechin is used to prevent and / or treat frog iridovirus, and the concentration of the epicatechin in the working solution is 3.125 μg / mL-12.5 μg / mL.

5. The use of epicatechin in the preparation of a drug for resisting frog iridovirus according to claim 4, wherein the concentration of the epicatechin in the working solution is 6.25 μg / mL-12.5 μg / mL.

6. The use of epicatechin in the preparation of a drug for resisting frog iridovirus according to claim 1, wherein the working solution is prepared according to the following procedure: the epicatechin is dissolved in a solvent to a concentration of 40-60 mg / mL, and then diluted with L15 culture solution to a cell safety concentration to obtain the working solution, and the concentration of the epicatechin in the working solution is 3.125 μg / mL-12.5 μg / mL.

7. Use of epicatechin in the preparation of frog feed for resisting frog iridovirus.

8. The use of epicatechin in the preparation of frog feed for resisting frog iridovirus according to claim 7, wherein the frog iridovirus is Rana virus type 3.

9. The use of epicatechin in the preparation of frog feed for resisting frog iridovirus according to claim 7, wherein the frog includes bullfrog.

10. The use of epicatechin in the preparation of frog feed for resisting frog iridovirus according to claim 7, wherein the mass fraction of the epicatechin in the frog feed is 0.01-0.05%. ​ ​ ​ ​ ​ ​ ​ ​

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