Disclosure of Invention
Paeonol is a micromolecular phenolic compound with a special structure, has wide biological activity, stable chemical property, easily modified structure, low price and easy obtaining, and has a plurality of excellent conditions as a lead structure for drug development. The paeonol and the derivatives thereof have wide prospect in developing new medicaments. The invention aims to explore the feasibility of paeonol and derivatives thereof in the aspect of aquaculture antivirus, and develops a new way for innovative research of antiviral green fishery drugs.
The invention provides application of paeonol in preparing a medicament for resisting crustacean aquatic animal viruses. Based on the experimental animal verification of the invention, paeonol has inhibitory activity on white spot syndrome virus WSSV, and can be used for preventing and treating white spot syndrome of aquatic animals.
In addition, the invention provides the application of the paeonol derivative in preparing the medicament for resisting the virus of the crustacean aquatic animals. Based on the same technical effect, the paeonol derivative can be used for preventing and treating the white spot syndrome of the aquatic animals.
The paeonol derivative is selected from any one or more of the following compounds 1-61.
2, 5-dimethoxyphenol (compound 1), 2-hydroxy-4-methoxybenzonitrile (compound 2), 4-methoxy-1, 2-benzenediol (compound 3), 2-hydroxy-4-methoxybenzoic acid (compound 4), 5-methoxy-2-methylphenol (compound 5), 2-hydroxy-4-methoxybenzaldehyde (compound 6), 2-hydroxy-4-methoxypropiophenone (compound 7), 2-hydroxy-4-methoxyphenylbutanone (compound 8), 2-hydroxy-4-methoxybenzophenone (compound 9), 1- (2-hydroxy-4-methoxyphenyl) -2-acetophenone (compound 10), 2-ethyl-5-methoxyphenol (compound 11), 1- (2-hydroxy-4-methoxybenzene) ethanol (compound 12), paeonol bromide (compound 13), paeonol thiosemicarbazone (compound 14), and potassium paeonol iminoacetate (compound 15);
2, 4-dimethoxyacetophenone (compound 16), 2-allyloxy-4-methoxyacetophenone (compound 17), 2-fluoro-4-methoxyacetophenone (compound 18), 2-acetyl 5-methoxyphenoxyacetic acid (compound 19), p-methoxyacetophenone (compound 20), paeonol acetic acid-2-bromo-ethyl ester (compound 21), paeonol acetic acid-2- (nitrooxy) ethyl ester (compound 22), 2-ethoxy-4-methoxyacetophenone (compound 23), 2-butoxy-4-methoxyacetophenone (compound 24), 2-hexyloxy-4-methoxyacetophenone (compound 25), 2-octyloxy-4-methoxyacetophenone (compound 26), 2-decyloxy-4-methoxyacetophenone (compound 27), 2-dodecyloxy-4-methoxyacetophenone (compound 28), 2-tetradecyloxy-4-methoxyacetophenone (compound 29), 2-hexadecyloxy-4-methoxyacetophenone (compound 30);
2, 4-dihydroxyacetophenone (compound 31), 4-bromo-2-hydroxyacetophenone (compound 32), 4-fluoro-2-hydroxyacetophenone (compound 33), 4-benzyloxy-2-hydroxyacetophenone (compound 34), 4-ethoxy-2-hydroxyacetophenone (compound 35), 2-hydroxy-4-methylacetophenone (compound 36), 4-chloro-2-hydroxyacetophenone (compound 37), 2-hydroxyacetophenone (compound 38), 4-allyloxy-2-hydroxyacetophenone (compound 39);
2-hydroxy-3, 4-dimethoxyacetophenone (compound 40), 2-hydroxy-4, 5-dimethoxyacetophenone (compound 41), 2-hydroxy-4, 6-dimethoxyacetophenone (compound 42), sodium paeonol sulfonate (compound 43), 5-bromo-2-hydroxy-4-methoxyacetophenone (compound 44);
2, 4-dimethoxybenzoic acid (compound 45), 2-4-dihydroxybenzoic acid (compound 46), 2-hydroxy-4-n-octoxybenzophenone (compound 47), 4-hydroxy-2-methylacetophenone (compound 48), 2, 4-dimethylacetophenone (compound 49), p-hydroxyacetophenone (compound 50), 5-bromo-2-hydroxy-4-methoxybenzoic acid (compound 51), 2-hydroxy-4-methoxybenzophenone-5-sodium sulfonate (compound 52), 5-bromo-2-ethoxy-4-methoxyacetophenone (compound 53), 5-bromo-2-butoxy-4-methoxyacetophenone (compound 54), 5-bromo-2-hexyloxy-4-methoxyacetophenone (compound 55), 5-bromo-2-octyloxy-4-methoxyacetophenone (compound 56), 5-bromo-2-decyloxy-4-methoxyacetophenone (compound 57), 5-bromo-2-dodecyloxy-4-methoxyacetophenone (compound 58), 5-bromo-2-tetradecyloxy-4-methoxyacetophenone (compound 59), 5-bromo-2-hexadecyloxy-4-methoxyacetophenone (compound 60), 3-allyl-2, 4-dihydroxyacetophenone (compound 61).
Based on the results of animal experiments, the invention provides a medicament for resisting crustacean aquatic animal white spot syndrome virus, and the active ingredient of the medicament comprises paeonol. The technical personnel in the field can easily know that on the basis that paeonol can be used for preventing and treating white spot syndrome of aquatic animals, the paeonol is combined with a pharmaceutically optional carrier or auxiliary agent to prepare the medicament into a preparation or vaccine convenient for aquaculture. The invention is not limited to the pharmaceutically acceptable carriers or adjuvants.
Based on the results of animal experiments, the invention provides a medicament for resisting crustacean aquatic animal white spot syndrome virus, and the active ingredients of the medicament comprise paeonol derivatives. The paeonol derivative is selected from any one or more of compounds 1-61. The technical personnel in the field can easily know that on the basis that the paeonol derivative can be used for preventing and treating the white spot syndrome of aquatic animals, the paeonol derivative is combined with a pharmaceutically optional carrier or auxiliary agent to prepare the medicament into a preparation or vaccine convenient for aquaculture. The invention is not limited to the pharmaceutically acceptable carriers or adjuvants.
Based on the results of animal experiments, the invention provides a medicament for resisting crustacean aquatic animal white spot syndrome virus, and the active ingredients of the medicament comprise paeonol and paeonol derivatives. The paeonol derivative is selected from any one or more of compounds 1-61. The technical personnel in the field can easily know that the paeonol and the paeonol derivative can be used for preventing and treating the white spot syndrome of aquatic animals, and the medicaments are prepared into a preparation or a vaccine convenient for aquaculture by being matched with a pharmaceutically optional carrier or auxiliary agent. The invention is not limited to the pharmaceutically acceptable carriers or adjuvants.
It is also particularly desirable in view of the wide variety of paeonol derivatives. The invention only verifies the biological activity of the paeonol derivative (compounds 1-61) on the white spot syndrome virus. In light of the teaching or technology of the present invention, those skilled in the art can also continuously verify the white spot syndrome virus inhibiting activity of other paeonol derivatives than the compounds 1-61, the combination of at least two of the paeonol derivatives of the compounds 1-61, the combination of paeonol and at least one of the paeonol derivatives of the compounds 1-61, and the like.
Compared with the prior art, the invention has the following beneficial effects or advantages:
the invention firstly verifies and reveals the biological activity of paeonol and derivatives thereof for resisting white spot syndrome virus. Animal experiment researches show that paeonol and derivatives thereof can obviously inhibit the replication of white spot syndrome virus in bodies of the penaeus vannamei boone and improve the survival rate of the penaeus vannamei boone infected by the white spot syndrome virus. Paeonol and its derivatives have good antiviral effect on white spot syndrome virus in shellfish aquaculture, and can be used for preventing and treating aquatic animal diseases caused by white spot syndrome virus.
The paeonol and the derivatives thereof have the characteristics of low toxicity, low residue, low cost, greenness and safety, and the biological activity of the paeonol and the derivatives thereof can be utilized to prevent and treat white spot syndrome virus which harms crustacean aquaculture, thereby providing a new path for developing and researching green biological agents for aquaculture and expanding new application of the paeonol and the derivatives thereof.
Detailed Description
The present invention will be further described with reference to the following examples and accompanying drawings, but the present invention is not limited to these examples. The embodiment of the invention aims to illustrate the white spot syndrome virus resisting activity of paeonol and derivatives thereof.
Example 1
This example illustrates the determination of the anti-white spot syndrome virus activity of paeonol and its first-position derivative.
(1) Test materials
Virus material: the white spot syndrome virus is provided by the third ocean institute of the national ocean agency and is stored in an aquatic disease laboratory of the northwest agriculture and forestry science and technology university.
Preparing a liquid medicine to be detected: accurately weighing 1-15 standard substances of the compounds respectively, preparing standard solution with concentration of 100 mg/mL by using chromatographic grade methanol, and diluting the standard solution with methanol into different concentrations of 10, 15, 20, 30, 40, 60 and 80 mg/mL; the resulting mixture was filtered through a 0.22 μm organic syringe filter and transferred to a brown chromatographic bottle for use as a standard dilution.
Experimental animals: the test is carried out by adopting penaeus vannamei boone purchased from Shaanxi Yanyang aquatic animals market.
(2) Evaluation of safety
Before the determination of the antiviral activity, the safety evaluation of the paeonol and the derivatives thereof on the penaeus vannamei boone is carried out. Healthy penaeus vannamei boone is randomly selected and grouped into 5 penaeus vannamei boone groups, and the penaeus vannamei boone groups are placed in a plastic box with the water temperature of 25 ℃ for test treatment. The test was set up with a blank control group and a drug treated group. The blank control group was injected with only TM buffer, and the drug-treated group was injected with different concentrations of compound. The tested concentrations of the compounds were: 50. 150 and 200 mg/kg. The injection volume of the drug was 100. mu.L/vial. After the drug injection, the survival status of the shrimps was observed and recorded for 72 hours continuously.
The safety evaluation result shows that the paeonol and the derivatives thereof are safe for the penaeus vannamei boone within the tested time (72 h) and concentration (50-200 mg/kg), and meanwhile, the penaeus vannamei boone does not show uncomfortable symptoms.
(3) Antiviral Activity assay
Healthy penaeus vannamei boone was randomly selected, each was divided into 3 groups (WSSV treatment group, compound and WSSV co-treatment group, blank control group), and 30 each group was subjected to 3 parallel experiments. The test was carried out in a 25 ℃ aquaculture water. The tested concentrations of the compounds were all 50 mg/kg. The volume of the treatment solution for each penaeus vannamei injection is 100. mu.L. The drug treatment method is that the WSSV virus and the monomer compound are premixed and then immediately injected into the abdomen (the virus dosage of each shrimp is 6.9 multiplied by 10)7copies, pre-mix: mixing the virus solution and the medicinal solution at 25 deg.C, and injecting immediately), respectively, after 24 hr, randomly selecting 5 Penaeus vannamei Boone from each treatment group, collecting the branchial tissue of the prawn, and storing at-80 deg.C for use.
The samples collected were used for absolute quantification of the WSSV genomic DNA copy number, respectively. The viral genome copy number in gill tissues of the treated samples was detected using absolute fluorescent quantitative PCR. And extracting the DNA of the collected tissue sample by using a marine animal tissue genome DNA extraction kit. The extracted DNA concentration and purity were measured using a ultramicrospectrophotometer, and then the DNA concentration was adjusted to 50 ng/. mu.L in a lump. The number of viral copies in tissues was quantified by RT-qPCR and an absolute quantitative standard curve constructed (figure 1).
Extracting the DNA of the gill tissue of the penaeus vannamei:
1) weighing about 10 mg of tissue material, fully grinding, putting into a centrifuge tube filled with 200 mu L of GA buffer solution, and carrying out vortex oscillation for 15 sec;
2) adding 20 μ L of protease K (20 mg/mL), mixing by vortex, centrifuging briefly to remove water droplets on the inner wall of the tube cover, standing at 56 deg.C until the tissue is completely dissolved, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;
3) adding 200 μ L buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10 min, cleaning the solution, and centrifuging briefly to remove water droplets on the inner wall of the tube cover;
4) adding 200 μ L of anhydrous ethanol, mixing thoroughly, wherein flocculent precipitate may appear, and centrifuging briefly to remove water drops on the inner wall of the tube cover;
5) adding the solution and flocculent precipitate obtained in the previous step into adsorption column CB3 (placing the adsorption column into a collecting pipe), centrifuging at 12000 rpm for 30 sec, pouring off waste liquid, and placing adsorption column CB3 back into the collecting pipe;
6) adding 500 μ L buffer GD into adsorption column CB3, centrifuging at 12000 rpm for 30 sec, pouring off waste liquid, and placing adsorption column CB3 into a collection tube;
7) adding 600 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12000 rpm for 30 sec, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube;
8) repeating the operation step 7);
9) putting the adsorption column CB3 back into the collecting pipe, centrifuging at 12000 rpm for 2 min, pouring off waste liquid, and placing the adsorption column CB3 at room temperature for a plurality of minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
10) transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 mu L of elution buffer solution ddH into the middle part of the adsorption film2O, standing at room temperature for 2-5 min, centrifuging at 12000 rpm for 2 min, and collecting the solution into a centrifuge tube;
11) adding the solution obtained by centrifugation into an adsorption column CB3, standing at room temperature for 2 min, centrifuging at 12000 rpm for 2 min to obtain a DNA product, and storing the DNA product at-20 ℃ for later use.
The PCR quantitative detection was performed in real time using CFX-96. The quantitative primer used was VP28141The primer sequences are shown in Table 1. The quantitative kit uses AceQ qPCR SYBR Green Master Mix of Vazyme. The quantitative reaction system and the reaction procedure are shown in Table 2 and Table 3, respectively.
TABLE 1 primer VP28141Of (2) a
TABLE 2 RT-qPCR reaction System
TABLE 3 RT-qPCR reaction procedure
(4) Survival detection
After the safe concentration is determined, the protective effect of paeonol and the derivatives thereof on the penaeus vannamei boone is tested in vivo. Healthy penaeus vannamei boone was randomly divided into 3 groups of 100 each, and TM buffer (control group), TM-WSSV mixture (positive control group), and drug-WSSV mixture (treatment group, 50 mg/kg) were injected, respectively. Each group of penaeus vannamei was observed for 7 days and the mortality was recorded. Wherein, the survival rate calculation formula is as follows: survival (%) = number of survivors treated group/total number per group x 100%.
TABLE 4 determination of the anti-white spot syndrome Virus Activity of the Paeonol I-position derivative
As can be seen from Table 4, paeonol and the first-position derivative thereof significantly reduce the copy number of WSSV in Penaeus vannamei Boone, and have good biological activity against WSSV, wherein the inhibition rates of 2-hydroxy-4-methoxybenzophenone and 1- (2-hydroxy-4-methoxyphenyl) -2-acetophenone on viruses are the highest and reach 88%.
As can be seen from table 4, paeonol and its derivatives can significantly reduce the mortality rate of WSSV litopenaeus vannamei. During the test, no or only a small amount of the TM buffer treated penaeus vannamei boone died. WSSV treated Penaeus vannamei Boone suffered from massive death, and the survival rate at 8 d was 0%. The survival rate of the sick Penaeus vannamei Boone treated by the paeonol and the first-position derivative thereof is improved, wherein the survival rate of the 2-hydroxy-4-methoxybenzophenone group is the highest and reaches 57%.
As can be seen from table 4, paeonol and its first-order derivatives can significantly inhibit the in vivo replication of WSSV penaeus vannamei, and at the same time, increase the survival rate of WSSV infected penaeus vannamei.
Example 2
This example shows the determination of the anti-white spot syndrome virus activity of paeonol and its derivatives at position two.
The test materials, antiviral activity assay, and absolute quantification of WSSV genomic DNA copy number used in this example were the same as in example 1. The test results are shown in Table 5.
TABLE 5 determination of the anti-white Spot syndrome Virus Activity of the Paeonol No. two derivatives
As can be seen from Table 5, the paeonol and the second-position derivative thereof remarkably reduce the copy number of the WSSV in the bodies of the penaeus vannamei boone, and have good biological activity of resisting the WSSV, wherein the inhibition rate of the 2-allyloxy-4-methoxyacetophenone on viruses is the highest and reaches 89.36 percent.
As can be seen from Table 5, paeonol and its derivatives at the second position can significantly reduce the mortality rate of WSSV-infected Penaeus vannamei Boone. During the test, no or only a small amount of the TM buffer treated penaeus vannamei boone died. WSSV treated Penaeus vannamei Boone suffered from massive death, and the survival rate at 8 d was 0%. The survival rate of the sick Penaeus vannamei Boone treated by paeonol and its derivatives is improved, wherein the survival rate of 2-octyloxy-4-methoxyacetophenone and 2-decyloxy-4-methoxyacetophenone groups is up to 56%.
As can be seen from table 5, paeonol and its derivatives can significantly inhibit the in vivo replication of WSSV penaeus vannamei, and at the same time, increase the survival rate of WSSV infected penaeus vannamei.
Example 3
This example shows the determination of the anti-white spot syndrome virus activity of paeonol and its four-position derivatives.
The test materials, antiviral activity assay, and absolute quantification of WSSV genomic DNA copy number used in this example were the same as in example 1. The test results are shown in Table 6.
TABLE 6 determination of the anti-white spot syndrome Virus Activity of the Paeonol four-position derivative
As can be seen from Table 6, the paeonol and the four-position derivative thereof remarkably reduce the copy number of the WSSV in the bodies of the penaeus vannamei boone, have good biological activity of resisting the WSSV, and the inhibition rate of the 4-benzyloxy-2-hydroxyacetophenone on viruses is the highest and reaches 92 percent.
As can be seen from table 6, paeonol and its four-position derivatives can significantly reduce the mortality rate of WSSV penaeus vannamei. During the test, no or only a small amount of the TM buffer treated penaeus vannamei boone died. WSSV treated Penaeus vannamei Boone suffered from massive death, and the survival rate at 8 d was 0%. The survival rate of the sick Penaeus vannamei Boone treated by paeonol and the derivatives thereof is improved, wherein the survival rate of the 4-benzyloxy-2-hydroxyacetophenone group is the highest and reaches 58%.
As can be seen from table 6, paeonol and its derivatives can significantly inhibit the in vivo replication of WSSV penaeus vannamei, and at the same time, increase the survival rate of WSSV infected penaeus vannamei.
Example 4
This example shows the determination of the anti-white spot syndrome virus activity of paeonol and other site derivatives.
The test materials, antiviral activity assay, and absolute quantification of WSSV genomic DNA copy number used in this example were the same as in example 1. The test results are shown in Table 7.
TABLE 7 determination of the anti-white spot syndrome Virus Activity of the Paeonol four-position derivative
As can be seen from Table 7, the copy number of WSSV in Penaeus vannamei Boone is significantly reduced by paeonol and other derivatives, and the results prove that the paeonol and other derivatives have good biological activity against WSSV, wherein the inhibition rate of 5-bromo-2-hydroxy-4-methoxyacetophenone on viruses is the highest and reaches 85%.
To further demonstrate the anti-WSSV activity of paeonol and other derivatives, as shown in table 7, paeonol and other derivatives were able to significantly reduce the mortality rate of WSSV infected penaeus vannamei. During the test, no or only a small amount of the TM buffer treated penaeus vannamei boone died. WSSV treated Penaeus vannamei Boone suffered from massive death, and the survival rate at 8 d was 0%. The survival rate of the sick Penaeus vannamei Boone treated by paeonol and its derivatives is improved, wherein the survival rate of the sodium paeonol sulfonate group is the highest, and reaches 53%.
In addition, the paeonol and the derivatives thereof can obviously inhibit the in vivo replication of WSSV penaeus vannamei boone and simultaneously improve the survival rate of the WSSV infected penaeus vannamei boone.
Example 5
This example shows the determination of the anti-white spot syndrome virus activity of paeonol and its multi-position derivatives.
The test materials, antiviral activity assay, and absolute quantification of WSSV genomic DNA copy number used in this example were the same as in example 1. The test results are shown in Table 8.
TABLE 8 determination of the anti-white spot syndrome Virus Activity of the Paeonol four-position derivative
As can be seen from Table 8, the paeonol and the multi-position derivatives thereof significantly reduce the copy number of WSSV in Penaeus vannamei Boone, and the results prove that the paeonol and the multi-position derivatives thereof have good biological activity against WSSV, wherein the inhibition rate of 5-bromo-2-hydroxy-4-methoxybenzoic acid on viruses is the highest and reaches 85%.
To further demonstrate the anti-WSSV activity of paeonol and its multi-position derivatives, as shown in table 8, paeonol and its multi-position derivatives were able to significantly reduce the mortality rate of WSSV infected penaeus vannamei. During the test, no or only a small amount of the TM buffer treated penaeus vannamei boone died. WSSV treated Penaeus vannamei Boone suffered from massive death, and the survival rate at 8 d was 0%. The survival rate of the sick Penaeus vannamei Boone treated by paeonol and the derivatives thereof is improved, wherein the survival rate of the 2-hydroxy-4-methoxybenzophenone-5-sodium sulfonate group is the highest and reaches 54 percent.
In addition, the paeonol and the derivatives thereof can obviously inhibit the in vivo replication of WSSV penaeus vannamei boone and simultaneously improve the survival rate of the WSSV infected penaeus vannamei boone.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.