CN110548022B - Organic acid inhibitor for resisting fish diseases and preparation method and application thereof - Google Patents
Organic acid inhibitor for resisting fish diseases and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an organic acid inhibitor for resisting fish diseases, which comprises the following components: the component comprises at least one of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid; isochlorogenic acid A is calculated according to the mass ratio: isochlorogenic acid B: isochlorogenic acid C: caffeic acid=0-4: 0-4:0-4:0-1. the organic acid inhibitor for resisting the fish viruses can safely and efficiently prevent and inhibit the garrupa iridovirus, meets the requirements of green pollution-free sustainable development, can promote the high-quality ecological culture of aquaculture and brings non-negligible economic benefits.
Description
Technical Field
The invention belongs to the field of disease control of aquatic animals, and particularly relates to an organic acid inhibitor for resisting fish diseases, and a preparation method and application thereof.
Background
In recent years, the ocean economy of China rapidly develops, and the data of the Chinese ocean economy statistical publication in 2017 show that the total national ocean production value in 2017 reaches 7.76 trillion yuan, wherein the ocean fishery economy accounts for more than 17.8% of the ocean economy. The grouper has fine and smooth quality and rich nutrition, is one of the biggest sea water farmed fishes in south China, and has extremely high economic value in the living seafood market. However, under the conditions of high-density and intensive cultivation, frequent outbreaks of various cultivation diseases cause huge economic losses. The garrupa iridovirus is called as ocean foot-and-mouth disease, and the mortality rate of the garrupa iridovirus caused by the garrupa iridovirus is extremely high, and the garrupa iridovirus is a main viral pathogen causing garrupa morbidity. However, no effective antiviral drug is available in the market aiming at the garrupa iridovirus disease at present, and the garrupa breeding industry is in the dilemma of no drug availability. Therefore, development of a new thought for developing efficient antiviral drugs for prevention, control and treatment of iridovirus disease in grouper culture is urgently needed.
In recent years, the search of natural active ingredients with antiviral action from medicinal plants has become a research hotspot for developing novel plant-derived medicines at home and abroad. China has rich medicinal plant resources, the medicinal plants have long use history, and the application prospect of screening antiviral drugs from the medicinal plants is extremely wide. The medicinal plant contains a plurality of natural active chemical components, mainly contains organic acids, saponins, flavonoids, polysaccharides, alkaloids and the like, has the characteristics of diversified structures and diversified biological activities, and can play an antiviral role by destroying the structural integrity of viruses, affecting the synthesis of proteins and genetic materials of the viruses or activating the immune system of fish bodies. The medical plants for aquatic products can be studied systematically, so that the problems of drug resistance and drug residue caused by chemical drugs such as antibiotics can be effectively solved, and the medical plants for aquatic products are an important development direction for realizing green pollution-free, efficient disease prevention and high-quality ecological cultivation of aquatic products.
Disclosure of Invention
The invention aims to provide an organic acid inhibitor for resisting fish diseases, and a preparation method and application thereof, so as to effectively prevent and treat garrupa iridovirus.
According to one aspect of the present invention, there is provided an organic acid inhibitor against fish diseases: the component comprises at least one of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid; isochlorogenic acid A is calculated according to the mass ratio: isochlorogenic acid B: isochlorogenic acid C: caffeic acid=0-4: 0-4:0-4:0-1.
preferably, the components include isochlorogenic acid a, isochlorogenic acid B, isochlorogenic acid C and caffeic acid; isochlorogenic acid A is calculated according to the mass ratio: isochlorogenic acid B: isochlorogenic acid C: caffeic acid=0.5-4: 0.5-4:1-4:0.25-1.
preferably, the mass ratio of the isochlorogenic acid A, the isochlorogenic acid B, the isochlorogenic acid C and the caffeic acid in the components is as follows: isochlorogenic acid a: isochlorogenic acid B: isochlorogenic acid C: caffeic acid=2: 2:4:1.
according to another aspect of the present invention, there is provided a method for preparing the above organic acid inhibitor against fish virus: is prepared from plant source isochlorogenic acid A, plant source isochlorogenic acid B, plant source isochlorogenic acid C and plant source caffeic acid according to a mass ratio.
Preferably, one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong hort is selected as a plant source, and isochlorogenic acid A is extracted; selecting one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong hort as a plant source, and extracting isochlorogenic acid B; selecting one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong hort as a plant source, and extracting isochlorogenic acid C; one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong hort is selected as a plant source, and caffeic acid is extracted.
Preferably, honeysuckle is used as a plant source, and isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid are respectively extracted.
According to another aspect of the present invention, there is provided a composite formulation: the organic acid inhibitor for resisting fish viruses is used as an active ingredient for preventing and/or inhibiting iridovirus.
Preferably, the iridovirus is grouper iridovirus.
Preferably, the concentration of the organic acid inhibitor is 62.5-300 μg/mL.
Preferably, the concentration of the organic acid inhibitor is 250 μg/mL.
The isochlorogenic acid A, the isochlorogenic acid B, the isochlorogenic acid C and the caffeic acid can generate remarkable inhibition effect on the infection of the grouper iridovirus by destroying the structure of the grouper iridovirus and influencing the viral replication and infection process. In the organic acid inhibitor for resisting fish diseases, which is prepared by compounding isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid, the isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid which are taken together as active ingredients are synergistic, so that the organic acid inhibitor can effectively inhibit the grouper iridovirus under the premise of not exceeding the safe use concentration range of each active ingredient. Compared with the method which adopts isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid as single active ingredients respectively, the method adopts isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid to prepare the organic acid inhibitor together, so that the effective action concentration of each active ingredient can be reduced, and the dosage of each active ingredient is reduced, thereby reducing the raw material cost of the active ingredient, improving the production benefit, reducing the side effect influence generated by the active ingredient, reducing the drug residue, improving the safety of the drug, and simultaneously not reducing the inhibiting effect of the organic acid inhibitor on iridovirus. In addition, the isochlorogenic acid A, the isochlorogenic acid B, the isochlorogenic acid C and the caffeic acid adopted by the invention can be extracted by common medicinal plants, can be widely used for extracting the effective components, and can avoid the drug effect problems such as drug resistance and the like and the food safety problems such as drug residues by replacing common antibiotics and other chemical drugs in the existing antiviral veterinary drugs with the isochlorogenic acid A, the isochlorogenic acid B, the isochlorogenic acid C and the caffeic acid of the plant sources. In conclusion, the organic acid inhibitor for resisting the fish viruses and the compound formula preparation using the organic acid inhibitor can safely and efficiently prevent and inhibit the garrupa iridovirus, meet the requirements of green pollution-free sustainable development, promote the high-quality ecological cultivation of aquaculture and bring non-negligible economic benefits.
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FIG. 1 is a photo-microscopic observation of the cytotoxic effects of different concentrations of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid on spleen cells of Epinephelus respectively;
FIG. 2 shows the results of cell activity assays of the effect of different concentrations of isochlorogenic acid A on spleen cell viability of groupers;
FIG. 3 shows the results of cell activity assays of the effect of different concentrations of isochlorogenic acid B on spleen cell viability of groupers;
FIG. 4 shows the results of cell activity assays of the effect of different concentrations of isochlorogenic acid C on spleen cell viability of groupers;
FIG. 5 shows the results of cell activity assays of the effect of varying concentrations of caffeic acid on spleen cell viability of groupers;
FIG. 6 shows the inhibitory effect of different concentrations (not exceeding the safe use concentration range) of the compound monomers isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid on the grouper iridovirus alone;
FIG. 7 shows the results of cell activity assays of the effect of various concentrations of the compound formulation on spleen cell viability of groupers;
FIG. 8 is the inhibitory effect of a compound formulation of varying concentration (not outside the safe use concentration range) on Epinephelus iridovirus;
FIG. 9 is the destructive effect of a compound formulation of different concentrations (not outside the safe use concentration range) on the structure of Epinephelus iridovirus;
figure 10 is the inhibition of replication and synthesis of garrupa iridovirus in host cells by a safe use concentration of the compound formulation.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments.
Quantitative experimental data referred to in the examples below are expressed as mean ± standard deviation (±s) and the comparison data between groups were statistically processed using the single factor horizontal anova method using SPSS 17.0 statistical software.
The garrupa spleen cells (Grouper Spleen cell, GS) were kept in the laboratory and publicly available from the applicant for use only in repeated experiments of the invention.
The grouper iridovirus (Guangxi strain, grouper iridovirus Guangxi strain, SGIV-Gx) was isolated from the diseased pearl gentian grouper (Epineplus yellow crofttattus) in North Guangxi sea, stored in applicant laboratory, publicly available from applicant, and used only for repeated experiments of the invention. The SGIV-Gx virus was diluted to 10 in cell culture medium prior to use in the examples described below 5 TCID 50 /mL。
Isochlorogenic acid a: chengduremia biotechnology Co., ltd (analytical purity > 98%), product number Y-068, CAS #2450-53-5. Isochlorogenic acid B: chengdoremia biotechnology Co., ltd (analytical purity > 98%), product number Y-069, CAS#14534-61-3. Isochlorogenic acid C: chengduremia biotechnology Co., ltd (analytical purity > 98%) product number Y-070, CAS #32451-88-0. Caffeic acid: chengduremia biotechnology Co., ltd (analytical purity > 98%), product number K-003, CAS #331-39-5.
Gene primer of main capsid protein (Major capsid protein, MCP) of garrupa iridovirus: forward primer (qMCP-F) 5'-GCACGCTTCTCTCACCTTCA-3' and reverse primer (qMCP-R) 5'-AACGGCAACGGGAGCACTA-3'. The reference gene beta-actin primer: forward primer (. Beta. -actin-F) 5'-TACGAGCTGCCTGACGGACA-3' and reverse primer (. Beta. -actin-R) 5'-GGCTGTGATCTCCTTCTGCA-3'. The primer is synthesized by Shanghai.
Example 1
1. Main instrument and reagent
An optical microscope.
2. Experimental method
The GS cells are transferred into a 96-well plate according to 10000 per well, cultured for 24 hours at 28 ℃, and then the four plant source compound monomers of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid are respectively diluted into different concentrations in a cell culture medium (isochlorogenic acid A:1000, 500, 250 mug/mL; isochlorogenic acid B:1000, 500, 250 mug/mL; isochlorogenic acid C:2000, 1000, 500 mug/mL; caffeic acid: 500, 250, 125 mug/mL); the experimental group is to incubate and culture the isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid with GS cells in 96-well plates for 48h at 28 ℃ respectively, and observe the cell morphology by using a light microscope. The control group of the experiment was GS cells without the above-mentioned monomer of plant-derived compound added to the medium.
3. Experimental results
As shown in FIG. 1, the observation results of the cytoscope are shown in the graph, the control group GS cells have no obvious change, and when the concentration of the isochlorogenic acid A in the experimental group is higher than 500 mug/mL, the concentration of the isochlorogenic acid B in the experimental group is higher than 500 mug/mL, the concentration of the isochlorogenic acid C in the experimental group is higher than 1000 mug/mL, and the concentration of the caffeic acid in the experimental group is higher than 250 mug/mL, the experimental group GS cells have obvious morphological change and cytopathy, including cell rounding, shrinkage, falling off from a cell culture surface and the like. When the concentration of isochlorogenic acid A is lower than 250 mug/mL, the concentration of isochlorogenic acid B is lower than 250 mug/mL, the concentration of isochlorogenic acid C is lower than 500 mug/mL, and the concentration of caffeic acid is lower than 125 mug/mL, the morphology of the GS cells is not obviously changed, and is approximately the same as that of the GS cells of the control group.
Example 2
1. Main instrument and reagent
The enzyme-labeled instrument, 5- (2, 4-disulfophenyl) -3- (2-methoxy-4-nitrophenyl) -2H-tetrazolium sodium inner salt solution (WST-8 solution).
2. Experimental method
GS cells were transferred into 96-well plates at 10000 per well and cultured at 28 ℃ for 24 hours, and then four plant-derived compound monomers of isochlorogenic acid a, isochlorogenic acid B, isochlorogenic acid C and caffeic acid were respectively set to different concentrations in the cell culture medium (isochlorogenic acid a:2000, 1000, 500, 250, 125 μg/mL; isochlorogenic acid B:2000, 1000, 500, 250, 125 μg/mL; isochlorogenic acid C:2000, 1000, 500, 25, 125 μg/mL; caffeic acid: 2000, 1000, 500, 250, 125, 62.5 μg/mL); different concentrations of plant-derived compound monomers were incubated with GS cells in 96-well plates at 28 ℃ for 48h, respectively. To test cell activity, cells in each well were incubated at 28℃for 4 hours with 20. Mu.L of WST-8 solution, and absorbance at 450nm was measured with a microplate reader. The cell viability was calculated as:
cell viability = experimental group OD 450 Control group OD 450 ×100％。
Experiments were repeated three times each. And determining the maximum non-toxic concentration of each plant source compound monomer, namely the maximum safe use concentration according to the cell viability measurement result. The control group of this experiment was GS cells without the addition of plant-derived compound monomers to the medium.
3. Experimental results
FIGS. 2-5 show cell viability assays corresponding to four different plant-derived compound monomers, respectively, with cell viability exceeding 95% for the experimental groups with isochlorogenic acid A concentrations below 250 μg/mL, isochlorogenic acid B concentrations below 250 μg/mL, isochlorogenic acid C concentrations below 500 μg/mL, and caffeic acid concentrations below 125 μg/mL. The highest safe use concentrations of the individual plant-derived compounds, monomer A, B, C and caffeic acid, were determined to be 250 μg/mL, 500 μg/mL, 125 μg/mL, respectively, in combination with the optical observation of example 1.
Example 3
1. Main instrument and reagent
Real-time fluorescent quantitative PCR technique (qRT-PCR).
2. Experimental method
The plant compound monomer solutions prepared in this example were each diluted in a cell culture medium at a multiple ratio and set to different concentrations (isochlorogenic acid A:250, 125, 62.5. Mu.g/mL; isochlorogenic acid B:250, 125, 62.5. Mu.g/mL; isochlorogenic acid C:500, 250, 125. Mu.g/mL; caffeic acid: 125, 62.5, 31.25. Mu.g/mL), and the effect of inhibiting the iridovirus infection of garrupa under different concentration conditions was examined by RT-qPCR technique. GS cells were transferred to 24-well plates at 40000 cells/well and incubated at 28 ℃ for 24 hours. The test plant compound monomer solution was separated from SGIV-Gx virus (10 5 TCID 50 Per mL) were added together to cells in a 24-well plate at 28 ℃Culturing. Culturing for 48 hours, collecting cells and culture mediums in the experimental group and the control group, extracting RNA, reversely transcribing the RNA into cDNA, taking the cDNA as a template, taking a beta-actin gene as an internal reference gene, and detecting the expression condition of a main capsid protein MCP gene of the grouper iridovirus by using RT-qPCR to judge the inhibition effect of the tested plant compound monomer solution on the grouper iridovirus. The control group of this experiment was GS cells to which only SGIV-Gx virus was added.
3. Experimental results
The results of detecting the expression of the MCP gene by the RT-qPCR technology are shown in FIG. 6, and compared with the expression of the MCP gene in the cells of the control group, the expression of the MCP gene in the cells of the experimental group is obviously reduced, which shows that the compound monomers isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid in the embodiment are independently used as effective components, and the compound has an inhibiting effect on the infection of the grouper iridovirus.
Example 4
The weight ratio is as follows: isochlorogenic acid a: isochlorogenic acid B: isochlorogenic acid C: caffeic acid=2: 2:4:1, weighing isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid, uniformly mixing the raw materials according to the mass ratio to obtain a mixed solution, dissolving the mixed solution by using a cell culture medium to prepare a compound formula mother solution with the concentration (total concentration of isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid in the solution) of 1000 mug/mL, filtering and sterilizing by using a 0.22 mu m filter column, and preserving at-20 ℃ for later use.
Example 5
1. Main instrument and reagent
Enzyme-labeled instrument, WST-8 solution.
2. Experimental method
GS cells were transferred into 96-well plates at 10000 cells/well and incubated at 28 ℃ for 24 hours. The mother solution of the compound formulation prepared in example 4 was diluted in a cell culture medium in a multiple ratio gradient and set to compound formulations of different concentrations (1000, 500, 250, 125, 62.5 μg/mL); the composite formulations of different concentrations were added to GS cells in 96-well plates, respectively, and incubated at 28℃for 48h. To test cell activity, cells in each well were incubated at 28℃for 4 hours with 20. Mu.L of WST-8 solution, and absorbance at 450nm was measured with a microplate reader. The cell viability was calculated as:
cell viability = experimental group OD 450 Control group OD 450 ×100％。
Experiments were repeated three times each. The maximum non-toxic concentration of the composite formulation, i.e., the maximum safe use concentration, is determined based on the cell viability assay. The control group of this experiment was GS cells without the addition of the complex formulation to the medium.
3. Experimental results
The results of the cell viability assay of this example are shown in FIG. 7, where the cell viability of the experimental group with the compound formulation concentration below 250 μg/mL exceeded 95%, i.e., the highest safe use concentration of the compound formulation was 250 μg/mL.
Example 6
1. Main instrument and reagent
Real-time fluorescent quantitative PCR technique (qRT-PCR).
2. Experimental method
The compound formulation mother liquor prepared in example 4 is diluted in a cell culture medium in a multiple ratio gradient manner, and is set into compound formulation preparations with different concentrations (250, 125, 62.5 mug/mL), and the effect of inhibiting the garrupa iridovirus infection under the different concentration conditions of the compound formulation prepared in the example is detected by using RT-qPCR technology. GS cells were transferred to 24-well plates at 40000 cells/well and incubated at 28 ℃ for 24 hours. Three different concentrations of the complex formulation were combined with SGIV-Gx virus (10 5 TCID 50 Per mL) were added together to cells in a 24-well plate and incubated at 28 ℃. Culturing for 48 hours, collecting cells and culture mediums in an experimental group and a control group, extracting RNA, reversely transcribing the RNA into cDNA, taking the cDNA as a template, taking a beta-actin gene as an internal reference gene, and detecting the expression condition of a main capsid protein MCP gene of the grouper iridovirus by using RT-qPCR to judge the inhibition effect of the compound formulation prepared in the embodiment on the grouper iridovirus. The control group of this experiment was GS cells to which only SGIV-Gx virus was added.
3. Experimental results
As shown in FIG. 8, the result of detecting the expression of the MCP gene by the RT-qPCR technology is that compared with the expression of the MCP gene in the GS cells of the control group to which the SGIV-Gx virus is only added, the expression of the MCP gene in the cells of the experimental group to which the composite formulation prepared by the SGIV-Gx and the embodiment is added is obviously reduced, which indicates that the composite formulation with the concentration of 250 mug/mL, 125 mug/mL and 62.5 mug/mL has obvious inhibition effect on the iridovirus infection of the garrupa.
In addition, by comparing the test results of this example (fig. 8) with the test results of example 3 (fig. 6), the effective concentration of the compound formulation prepared in this example was significantly lower than that of the isochlorogenic acid a, isochlorogenic acid B, isochlorogenic acid C, and caffeic acid alone as the effective ingredients, respectively, whereas the compound formulation prepared in this example had a significantly higher inhibitory effect on the garrupa iridovirus when the concentrations of the isochlorogenic acid a, isochlorogenic acid B, isochlorogenic acid C, and caffeic acid alone as the effective ingredients were at the same level as that of the compound formulation prepared in this example. Therefore, the compound formulation prepared in the embodiment shows that the isochlorogenic acid A, the isochlorogenic acid B, the isochlorogenic acid C and the caffeic acid which are taken together as active ingredients can be synergistic, and a better inhibition effect on the garrupa iridovirus infection is achieved.
Example 7
1. Main instrument and reagent
Real-time fluorescent quantitative PCR (qRT-PCR), frozen high-speed centrifuge, PBS buffer.
2. Experimental method
GS cells were transferred to 24-well plates at 40000 cells/well and incubated at 28 ℃ for 24 hours. The stock solution of the complex formulation prepared in example 4 was then diluted in cell culture medium to a complex formulation with a concentration of 250. Mu.g/mL (highest safe use concentration), and the complex formulation was combined with SGIV-Gx virus (10 5 TCID 50 Per mL) were added to cells of a 24-well plate together, incubated at 4 ℃ for 30 minutes, centrifuged at 4 ℃ for 30 minutes using a refrigerated high-speed centrifuge 35000g, the complex formulation in the supernatant was removed, the virus precipitated by centrifugation was rinsed with PBS buffer, resuspended in 400 μl of cell culture medium and added to GS fines of a 24-well plateCells, cells were cultured at 28℃for a further 12 hours. In this embodiment, the control group treatment method is: SGIV-Gx Virus (10 5 TCID 50 Per mL) with cell culture medium (composition and amount of cell culture medium of control group was kept consistent with the setting in experimental group of this example) at 4 ℃ for 30 min, then centrifuged at 35000g with a refrigerated high-speed centrifuge at 4 ℃ for 30 min, after removal of supernatant the centrifugally precipitated virus was rinsed with PBS buffer, resuspended in 400 μl of cell culture medium and added to 24-well GS cells, and culture was continued at 28 ℃. After 12 hours, the cells and culture medium in the experimental group and the control group are collected to extract RNA, and the RNA is reversely transcribed into cDNA; then, cDNA is used as a template, beta-actin gene is used as an internal reference gene, and RT-qPCR is used for detecting the expression condition of a main capsid protein MCP gene of the grouper iridovirus, so as to judge the destructive effect of the compound formulation on the grouper iridovirus structure.
3. Experimental results
The result of detecting the expression of the MCP gene by the RT-qPCR technology is shown in FIG. 9, and compared with the expression of the MCP gene in the cells of the control group, the expression of the MCP gene in the cells of the experimental group is obviously reduced, which proves that the compound formulation prepared by the embodiment can play the effect of resisting the infection of the garrupa iridovirus by destroying the structure of the garrupa iridovirus.
Example 8
1. Main instrument and reagent
Real-time fluorescent quantitative PCR (qRT-PCR), frozen high-speed centrifuge, PBS buffer.
2. Experimental method
GS cells were transferred to 24-well plates at 40000 cells/well and incubated at 28 ℃ for 24 hours. SGIV-Gx virus (10 5 TCID 50 /mL) was added to the cells, allowed to stand at 4℃for 30 minutes, and then the cells were transferred to 28℃for 2 hours. The medium in the cells of the 24-well plate was removed, the cells were washed twice with fresh medium, and then the mother liquor of the composite formulation prepared in example 4 was diluted in the cell culture medium to a composite formulation with a concentration of 250. Mu.g/mL (highest safe use concentration), and the composite formulation prepared in this example was added to the cells of the 24-well plate, and the culture was continued at 28℃for 12 hours. In the present embodimentThe control group treatment method is as follows: SGIV-Gx Virus (10 5 TCID 50 /mL) was added to the cells, allowed to stand at 4℃for 30 minutes, and then the cells were transferred to 28℃for 2 hours. The medium in the 24-well plate cells was removed, the cells were washed twice with fresh medium, and then a cell medium control group was added to the cells, and the culture was continued for 12 hours at 28 ℃. And then collecting cells and culture mediums in the experimental group and the control group, extracting total RNA, carrying out reverse transcription on the total RNA to obtain cDNA, taking the cDNA as a template, taking a beta-actin gene as an internal reference gene, and detecting the expression condition of a main capsid protein MCP gene of the grouper iridovirus by using an RT-qPCR technology to judge the inhibition of the compound formulation preparation on the grouper iridovirus in the replication and synthesis process in host cells.
3. Experimental results
The result of detecting the expression of the MCP gene by the RT-qPCR technology is shown in FIG. 10, and compared with the expression of the MCP gene in cells of a control group, the expression of the MCP gene in cells of an experimental group is obviously reduced, which indicates that the compound formulation prepared by the embodiment has the effect of inhibiting the replication and synthesis of the garrupa iridovirus in host cells.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention.
Claims (5)
1. The application of the organic acid inhibitor in preparing a medicament for preventing and/or inhibiting the garrupa iridovirus is characterized in that: the medicine takes the organic acid inhibitor as an active ingredient, and the concentration of the organic acid inhibitor in the medicine is 62.5-300 mug/mL;
the organic acid inhibitor comprises isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C and caffeic acid; the isochlorogenic acid A is calculated according to the mass ratio: the isochlorogenic acid B: the isochlorogenic acid C: caffeic acid=0.5-4: 0.5-4:1-4:0.25-1.
2. the use according to claim 1, wherein: the isochlorogenic acid A is calculated according to the mass ratio: the isochlorogenic acid B: the isochlorogenic acid C: caffeic acid=2: 2:4:1.
3. the use according to claim 1, wherein:
selecting one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong hort as the plant source, and extracting the isochlorogenic acid A;
selecting one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong hort as the plant source, and extracting the isochlorogenic acid B;
selecting one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong hort as the plant source, and extracting the isochlorogenic acid C;
and extracting the caffeic acid by taking one of honeysuckle, eucommia ulmoides, malva fruits, dandelion, red sage roots, safflower and ligusticum chuanxiong hort as the plant source.
4. A use according to claim 3, wherein:
and respectively extracting the isochlorogenic acid A, the isochlorogenic acid B, the isochlorogenic acid C and the caffeic acid by taking honeysuckle as the plant source.
5. The use according to claim 1, wherein: in the medicament, the concentration of the organic acid inhibitor is 250 mug/mL.
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Citations (2)
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|---|---|---|---|---|
| CN101380318A (en) * | 2008-10-16 | 2009-03-11 | 伍义行 | Use of isochlorogenic acid compound and different combinations in hepatitis treatment |
| CN106562100A (en) * | 2016-09-13 | 2017-04-19 | 河北京阳生物医药科技有限公司 | Feed additive for preventing and treating diseases of aquatic organisms and feed for aquatic organisms, and preparation methods thereof |
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| CN101380318A (en) * | 2008-10-16 | 2009-03-11 | 伍义行 | Use of isochlorogenic acid compound and different combinations in hepatitis treatment |
| CN106562100A (en) * | 2016-09-13 | 2017-04-19 | 河北京阳生物医药科技有限公司 | Feed additive for preventing and treating diseases of aquatic organisms and feed for aquatic organisms, and preparation methods thereof |
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