CN110711190B - Terpenoid inhibitor for resisting fish diseases and preparation method and application thereof - Google Patents
Terpenoid inhibitor for resisting fish diseases and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a terpenoid inhibitor for resisting fish diseases, which comprises the following components: the composition comprises at least one of curcumenol, curcuminoid and curcumenol; according to the mass ratio, curcumenol: curcuminoids: curcumenol=0-8: 0-1.5:0-1.5. the terpenoid inhibitor for resisting the fish viruses provided by the invention 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 a terpenoid 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 comprising terpenoids, saponins, flavonoids, polysaccharides, alkaloids and the like, and the active chemical components have the characteristics of diversified structures and diversified biological activities, and can play an antiviral role by destroying the structural integrity of viruses, influencing 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 a terpenoid 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 a terpenoid inhibitor against fish diseases: the composition comprises at least one of curcumenol, curcuminoid and curcumenol; according to the mass ratio, curcumenol: curcuminoids: curcumenol=0-8: 0-1.5:0-1.5.
preferably, the components include curcumenol, curdione and curcumenol; according to the mass ratio, curcumenol: curcuminoids: curcumenol=2-8: 0.5-1:0.5-1.
preferably, the mass ratio of curcumenol, curcuminoid and curcumenol in the components is as follows: curcumenol: curcuminoids: curcumenol=2: 1:1.
according to another aspect of the present invention, there is provided a method for preparing the above terpenoid inhibitor against fish virus: is prepared from curcumenol of plant source, curcumodione of plant source and curcumenol of plant source according to the mass ratio.
Preferably, one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong hort is selected as the plant source, and curcumenol is extracted; selecting one of honeysuckle, eucommia bark, malva fruit, dandelion, red sage root, safflower and ligusticum chuanxiong as the plant source, and extracting the curcuma zedoary diketone; and (3) selecting one of honeysuckle, eucommia ulmoides, malva fruits, dandelion, red sage roots, safflower and ligusticum chuanxiong as the plant source, and extracting the curcumenol.
Preferably, honeysuckle is used as the plant source, and the curcumenol, the curcumenol and the curcumenol are extracted respectively.
According to another aspect of the present invention, there is provided a composite formulation: the terpenoid inhibitor with fish virus resistance is used as active ingredient for preventing and/or inhibiting iridovirus.
Preferably, the iridovirus is grouper iridovirus.
Preferably, the concentration of the terpenoid inhibitor is 125-500 μg/mL.
Preferably, the concentration of the terpenoid inhibitor is 500 μg/mL.
Curcumenol, curdione and curcumenol can produce remarkable inhibition effect on garrupa iridovirus infection by influencing the virus replication and infection process. In the terpene inhibitor for resisting fish diseases prepared by compounding curcumenol, curcumenol and curcumenol, the curcumenol and the curcumenol which are taken together as active ingredients are synergistic, so that the terpene inhibitor can effectively inhibit the garrupa iridovirus under the premise of not exceeding the safe use concentration range of each active ingredient in the terpene inhibitor. Compared with the method which adopts curcumenol, curcumenol and curcumenol as single active ingredients respectively, the method adopts curcumenol, curcumenol and curcumenol to prepare the terpenoid inhibitor, can reduce the effective concentration of each active ingredient and the dosage of each active ingredient, 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 terpenoid inhibitor on iridovirus. In addition, the curcumenol diketone and the curcumenol adopted in the invention can be extracted by common medicinal plants, can be widely used as plant sources for extracting the effective components, and can avoid drug efficacy problems such as drug resistance and the like and food safety problems such as drug residues and the like by using the curcumenol, the curcumenol and the curcumenol of the plant sources to replace common antibiotics and other chemical drugs in the existing antiviral veterinary drugs. In conclusion, the terpenoid inhibitor for resisting the fish viruses and the compound formulation preparation using the terpenoid 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.
Drawings
FIG. 1 is the optical lens observation results of the cytotoxic effects of curcumenol, and curcumenol at different concentrations on spleen cells of Epinephelus respectively;
FIG. 2 shows the results of cell activity assays of the effects of curcumenol, and curcumenol at different concentrations on spleen cell viability of Epinephelus respectively;
FIG. 3 shows the inhibition effect of curcumenol, curcumenol alone on Epinephelus iridovirus at different concentrations (not exceeding the safe use concentration range);
FIG. 4 shows the results of cell activity assays of the effect of various concentrations of the compound formulation on spleen cell viability of groupers;
FIG. 5 is the inhibitory effect of a compound formulation of varying concentration (not outside the safe use concentration range) on Epinephelus iridovirus;
figure 6 is the inhibition of replication synthesis of garrupa iridovirus in host cells by the highest safe use concentration of the complex 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。
Curcumenol: chenopodium biosciences limited product (analytical purity > 98%), product number E-017, CAS #4871-97-0. Curcuminoids: chenopodium biosciences limited product (analytical purity > 98%), product number E-009, CAS #13657-68-6. Curcumenol: chengduremia biotechnology Co., ltd (analytical purity > 98%), product number E-003, CAS #19431-84-6.
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 cells/well, cultured for 24 hours at 28 ℃, and then three plant source compound monomers of curcumenol, curcuminoids and curcumenol are respectively diluted into different concentrations in a cell culture medium (curcumenol: 1000, 500, 250 mug/mL; curcuminoids: 1000, 500, 250 mug/mL; curcumenol: 1000, 500, 250 mug/mL); the experimental group is to incubate curcumenol, curcumenol diketone and curcumenol with GS cells in 96-well plates at 28 ℃ for 48h respectively, and observe cell morphology by 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
The observation result of the cytoscope is shown in figure 1, the GS cells of the control group have no obvious change, and when the curcumenol concentration in the experimental group is up to 1000 mug/mL, the morphology of the GS cells has no obvious change, which is the same as that of the cells of the control group; when the concentration of the curcuminoids and the curcumenol is higher than 1000 mug/mL, obvious morphological changes and cytopathy of the GS cells of the experimental group appear, including cell rounding, shrinkage, falling off from the cell culture surface and the like. When the concentration of curcuminoids and curcumenol is lower than 500 mug/mL, the morphology of GS cells is not changed obviously, and the GS cells are the same as those of the 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
The GS cells are transferred into a 96-well plate according to 10000 cells/well, cultured for 24 hours at 28 ℃, and then three plant source compound monomers of curcumenol, curcuminoids and curcumenol are respectively arranged in a cell culture medium to be different in concentration (curcumenol: 1000, 500, 250, 125 mug/mL; curcuminoids: 1000, 500, 250, 125 mug/mL; curcumenol: 1000, 500, 250, 125 mug/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
FIG. 2 shows the results of cell viability assays for each of three different plant-derived compound monomers, with the concentration of curcumenol at 1000, 500, 250, 125 μg/mL, and the cell viability of the experimental group reaching 100%; when the concentration of the curcuminoids and the curcumenol is lower than 500 mug/mL, the cell survival rate of the experimental group is over 95 percent. When the observation result of the light microscope of the example 1 is combined, and the concentration of the plant source compound monomer curcumenol is up to 1000 mug/mL, the cell is still free from obvious cytotoxicity, and the highest safe working concentrations of the curcuminoids and the curcumenol are 500 mug/mL.
Example 3
1. Main instrument and reagent
Real-time fluorescent quantitative PCR technique (qRT-PCR).
2. Experimental method
Culturing curcumenol, curcuminoid and curcumenol in cellThe plant compound monomer solution prepared in the embodiment is tested by RT-qPCR technology under the condition of different concentrations for inhibiting the effect of the garrupa iridovirus infection. 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 and incubated at 28 ℃. 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. 3, 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 monomer curcumenol, curcuminoid and curcumenol in the embodiment independently serve as effective components, and have an inhibiting effect on the infection of the garrupa iridovirus.
Example 4
The weight ratio is as follows: curcumenol: curcuminoids: curcumenol=2: 1:1, weighing curcumenol, curcumenol diketone and curcumenol, uniformly mixing the above 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 curcumenol, curcumenol and curcumenol 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 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. 4, where the cell viability of the experimental group with the compound formulation concentration below 500 μg/mL exceeded 95%, i.e., the highest safe use concentration of the compound formulation was 500 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 (500, 250, 125 mug/mL), and the effect of inhibiting the infection of the garrupa iridovirus under the condition of different concentrations is detected by using the 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 experimental groups and pairsRNA is extracted according to cells and culture mediums in the group, and is reversely transcribed into cDNA, the cDNA is taken as a template, beta-actin gene is taken 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 that the inhibition effect of the compound formulation prepared in the embodiment on the grouper iridovirus is judged. The control group of this experiment was GS cells to which only SGIV-Gx virus was added.
3. Experimental results
As shown in FIG. 5, 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 with the SGIV-Gx and the safe concentration is added is obviously reduced, which indicates that the composite formulation with the concentration of 500 mug/mL, 250 mug/mL and 125 mug/mL can have obvious inhibition effect on the infection of the garrupa iridovirus, wherein the inhibition effect of the composite formulation with the concentration of 500 mug/mL is the most obvious.
In addition, by comparing the test result (fig. 5) of this example with the test result (fig. 3) of example 3, the effective concentration of the compound formulation prepared in this example is significantly lower than that of curcumenol, curcuminoid, and curcumenol alone as the effective ingredients, respectively, whereas when the concentrations of curcumenol, curcuminoid, and curcumenol alone as the effective ingredients are at the same level as that of the compound formulation prepared in this example, the inhibitory effect of the compound formulation prepared in this example on garrupa iridovirus is more significant. Therefore, the composite formula preparation prepared by the embodiment shows that the curcumenol, the curcuminoid and the curcumenol which are taken together as active ingredients can synergistically improve the effect of inhibiting the iridovirus infection of the groupers.
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. 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 having a concentration of 500. 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 this embodiment, the control group treatment method is: 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 cell culture medium was added to the cells (the composition and amount of cell culture medium of the control group were kept consistent with those in the experimental group of this example), and the culture was continued at 28℃for 12 hours. 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. 6, 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 (3)
1. The application of the terpenoid inhibitor in preparing a medicament for preventing or inhibiting the garrupa iridovirus is characterized in that:
the terpene inhibitor contains curcumenol, curcuminoid and curcumenol as active ingredients;
according to the mass ratio, curcumenol: curcuminoids: curcumenol=2: 1:1.
2. The use according to claim 1, wherein: and preparing a compound formulation by using the terpenoid inhibitor, wherein the concentration of the terpenoid inhibitor in the compound formulation is 125-500 mug/mL.
3. The use according to claim 2, wherein: in the compound formulation, the concentration of the terpenoid inhibitor is 500 μg/mL.
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