Use of cinnamomum cassia in preparing a drug for preventing and treating largemouth bass virus

Abstract

The application belongs to the technical field of aquaculture, and discloses application of Cinnamomum cassia in preparation of a large-mouth bass virus prevention and treatment medicine. A Cinnamomum cassia extract is used to treat fathead minnow muscle cell lines (FHM), and the expression amount of the large-mouth bass iridovirus ICP46 gene is significantly reduced, indicating that the Cinnamomum cassia extract has a good anti-large-mouth bass iridovirus effect. The Cinnamomum cassia extract has a clear anti-large-mouth bass iridovirus mechanism, and can play a role in the following ways: destroying the structure of large-mouth bass iridovirus particles, interfering with the binding of large-mouth bass iridovirus and host cell surface receptors, interfering with the invasion of large-mouth bass iridovirus into host cells, and inhibiting the replication of large-mouth bass iridovirus. The Cinnamomum cassia extract is naturally obtained and does not produce chemical residues, has the potential to be further developed into an anti-large-mouth bass iridovirus medicine, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology and relates to the prevention and control of aquatic animal diseases, specifically the application of cinnamon in the preparation of drugs for the prevention and control of largemouth bass virus. Background Technology

[0002] Largemouth bass is an important aquaculture species; however, due to genetic degradation, increased water pollution, and the proliferation of pathogenic microorganisms, diseases frequently occur in largemouth bass. Largemouth bass iridovirus (LMBV), a typical viral pathogen of largemouth bass, can cause significant economic losses and seriously affect the healthy development of the largemouth bass aquaculture industry. Currently, effective drugs for controlling LMBV in largemouth bass are still scarce, and the largemouth bass aquaculture industry is facing a predicament of difficulty in controlling viral diseases. Therefore, there is an urgent need to develop highly effective and safe antiviral drugs for the control of largemouth bass iridovirus.

[0003] In recent years, the extraction of natural active ingredients from medicinal plants has become a research hotspot in aquaculture disease prevention and control, and the search for antiviral drugs for aquatic animals from medicinal plants holds great promise. Medicinal plants contain a variety of natural active ingredients, such as terpenoids, saponins, flavonoids, polysaccharides, and alkaloids, some of which have been proven to have antiviral effects. The antiviral mechanisms of these natural active ingredients mainly include: disrupting viral structure, interfering with viral replication in host cells, or activating the antiviral immune response in fish. Given the safety and lack of environmental pollution associated with medicinal plants, developing natural active plant ingredients that combat aquatic animal viruses could solve food safety issues caused by antibiotics and other chemical drugs, representing an important development direction for achieving environmentally friendly, efficient disease prevention and high-quality ecological aquaculture. Summary of the Invention

[0004] In order to achieve efficient control of largemouth bass iridovirus, reduce the use of chemical drugs, and improve the quality of largemouth bass, this invention provides the application of cinnamon in the preparation of drugs for the prevention and treatment of largemouth bass virus.

[0005] Furthermore, in the above applications, cinnamon extract has been shown to have an antiviral effect against largemouth bass iridovirus.

[0006] Furthermore, in the above applications, at a concentration of 0.0125 mg / mL, the cinnamon extract has the effect of disrupting the particle structure of largemouth bass iridovirus.

[0007] Furthermore, in the above applications, at a concentration of 0.0125 mg / mL, the cinnamon extract has the effect of interfering with the binding of largemouth bass iridovirus to receptors on the surface of host cells.

[0008] Furthermore, in the above applications, at a concentration of 0.0125 mg / mL, the cinnamon extract has the effect of interfering with the invasion of largemouth bass iridovirus into host cells.

[0009] Furthermore, in the above applications, at a concentration of 0.0125 mg / mL, the cinnamon extract has the effect of inhibiting the replication of largemouth bass iridovirus.

[0010] Furthermore, in the above application, the host cell is the fathead flounder muscle cell line.

[0011] Furthermore, in the above application, the preparation method of the cinnamon extract is as follows: cinnamon powder pretreated with petroleum ether is extracted with methanol, the methanol extract is collected and the methanol solvent is removed to obtain dried cinnamon extract.

[0012] Furthermore, the pretreatment method is as follows: cinnamon powder is soaked in petroleum ether for 1 hour, then sonicated three times at 60°C for 5 minutes each time, and the petroleum ether is discarded; then fresh petroleum ether is added and the operation is repeated once. Petroleum ether dosage: 250 mL of petroleum ether is used for every 25 g of cinnamon powder.

[0013] Furthermore, the methanol extraction method is as follows: The pretreated cinnamon powder is soaked in methanol for 1 hour, then sonicated three times at 60°C for 5 minutes each time. The methanol extract is then collected by filtration; fresh methanol is added and the process is repeated once. Methanol dosage: 250 mL of methanol is used for every 25 g of pretreated cinnamon powder.

[0014] Furthermore, the dried cinnamon extract was dissolved in DMSO to prepare a 100 mg / mL stock solution, which was then diluted for use.

[0015] The present invention also provides a drug, wherein the drug uses cinnamon extract as the sole active ingredient, and the drug has an anti-largemouth bass iridovirus effect.

[0016] Compared with the prior art, the present invention "Application of Cinnamon in the Preparation of Drugs for the Prevention and Treatment of Largemouth Bass Virus" has the following beneficial effects: Cinnamon extract has a significant effect against largemouth bass iridovirus. Experiments have shown that at a concentration of 0.0125 mg / mL, cinnamon extract can achieve the effect of fighting largemouth bass iridovirus by disrupting the particle structure of largemouth bass iridovirus, interfering with the binding of largemouth bass iridovirus to receptors on the surface of host cells, interfering with the invasion of largemouth bass iridovirus into host cells, and inhibiting the replication of largemouth bass iridovirus.

[0017] The cinnamon extract provided by this invention can safely and effectively prevent and control largemouth bass iridovirus. It is safe, environmentally friendly, and leaves no chemical residues, meeting the requirements of sustainable development. Based on the research results of this invention, it is hoped that commercially applicable drugs for the prevention and treatment of largemouth bass virus can be further developed, promoting high-quality ecological aquaculture of largemouth bass and reducing economic losses caused by largemouth bass virus diseases. Attached Figure Description

[0018] Figure 1 Cell morphology after treatment with different concentrations of cinnamon extract. Figure 1 In this context, "Control" indicates the control group.

[0019] Figure 2 Cell survival rate after treatment with different concentrations of cinnamon extract. Figure 2 In the text, "Con." indicates the control group.

[0020] Figure 3 The crystal violet staining results are for the control group, LMBV test group, and cinnamon extract test group. Figure 3 A darker color after staining indicates a higher number of live cells.

[0021] Figure 4 The relative expression level of the ICP46 gene in Example 3.

[0022] Figure 5 The relative expression level of the ICP46 gene in Example 4.

[0023] Figure 6 The relative expression level of the ICP46 gene in Example 5.

[0024] Figure 7 The relative expression level of the ICP46 gene in Example 6. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] The quantitative experimental data involved in the embodiments of the present invention are expressed as mean ± standard deviation (±s). SPSS 17.0 statistical software was used to perform statistical processing on the inter-group comparison data using the one-way level ANOVA method.

[0027] The fathead monnow cell line (FHM) was isolated from fathead monnows, and L15 medium was used as the culture medium for FHM cells. Largemouth bass ranavirus (LMBV) was isolated from diseased largemouth bass in Beihai, Guangxi.

[0028] In this embodiment of the invention, LMBV is diluted to 10 in cell culture medium before use. 6 TCID 50 / mL.

[0029] The cinnamon used in this embodiment of the invention was purchased from Yixin Pharmacy in Nanning, Guangxi, and its place of origin is Shiyan, Hubei.

[0030] The cinnamon extract used in the examples was prepared by the following method: Cinnamon powder was soaked in petroleum ether for 1 hour at a ratio of 250 mL per 25 g of cinnamon powder, followed by sonication at 60°C for 5 minutes each time, and the petroleum ether was discarded. This process was repeated once with fresh petroleum ether to obtain pretreated cinnamon powder. The pretreated cinnamon powder was then soaked in methanol for 1 hour at a ratio of 250 mL per 25 g of cinnamon powder, followed by sonication at 60°C for 5 minutes each time, and the methanol extract was collected by filtration. This process was repeated once with fresh methanol. The collected methanol extract was then evaporated using a rotary evaporator to remove the methanol solvent, yielding dried cinnamon extract. For each use, the dried cinnamon extract was dissolved in DMSO to prepare a 100 mg / mL stock solution, which was then diluted with cell culture medium to the required concentration before use.

[0031] The primers for the ICP46 gene and β-actin internal reference gene in this example were synthesized by Sangon Biotech (Shanghai) Co., Ltd. ICP46 gene primers: ICP46-F: 5'-CAACTGCAGACTGGTCCTGA-3'; ICP46-R: 5'-AAAGCCTGTTGAGGAGACGA-3'.

[0032] Primers for the internal reference gene β-actin: β-actin-F: 5'-TCTTCCAGCCATCCTTCCTTGG-3'; β-actin-R: 5'-CTGCATACGGTCAGCAATGCC-3'.

[0033] Example 1 This embodiment provides the method for obtaining a cell-safe concentration of cinnamon extract.

[0034] FHM cells were transduced into 96-well plates, with 1×10⁶ cells per well. 5 Cells were cultured at 28°C for 24 hours. Cinnamon extract was diluted with cell culture medium to different concentrations (0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, 0.00625 mg / mL). These concentrations of cinnamon extract were added to 96-well plates and co-incubated with FHM cells at 28°C for 48 hours. Cell morphology was observed using an optical microscope. After observation, 20 μL of WST-8 solution was added to each well, and the cells were cultured at 28°C for another 4 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Cell viability was calculated as follows: Cell viability = OD of experimental group 450 / Control group OD 450 ×100%.

[0035] Cell morphology observed under an optical microscope at different concentrations of cinnamon extract as follows: Figure 1 As shown. Figure 1 As can be seen, when the concentration of cinnamon extract is ≤0.0125mg / mL, the morphology of FHM cells does not change significantly compared with that of the control group cells.

[0036] Statistical results of cell viability corresponding to different concentrations of cinnamon extract are as follows: Figure 2 As shown. Figure 2 As can be seen, when the concentration of the cinnamon extract was ≤0.0125 mg / mL, the survival rate of FHM cells was greater than 95%, and there was no significant cytotoxic effect compared with the control group. Figure 1 and Figure 2 The results show that the safe concentration of cinnamon extract in FHM cells is 0.0125 mg / mL.

[0037] Example 2 This embodiment demonstrates the effects of different concentrations of cinnamon extract on largemouth bass iridovirus.

[0038] FHM cells were seeded into 24-well culture plates at a concentration of 1 × 10⁶ cells / well. 6 Incubate at 28°C for 18 hours with cells / mL, then discard the culture medium.

[0039] 400 μL of fresh cell culture medium was added to the control group cells; 0.3 μL (10⁻⁶) was added to the LMBV test group cells. 6 TCID 50 LMBV + 400 μL fresh cell culture medium ( / mL).

[0040] The cinnamon extract experimental groups were prepared by adding 400 μL of cinnamon extract at different concentrations (0.0125 mg / mL, 0.00625 mg / mL, 0.003125 mg / mL) + 0.3 μL (10 6 TCID 50 LMBV ( / mL).

[0041] Both the control and experimental groups were replicated in triplicate. After 48 hours of culture, the cell culture medium was removed, and the cells were washed once with PBS buffer. Then, 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 1 hour. After 1 hour, the cells were washed three more times with PBS buffer, and 300 μL of 0.1% crystal violet solution was added for staining at room temperature for 2 hours. After staining, the dye was discarded, and the cells were air-dried upside down. The intensity of cell color was observed to determine cell viability, and photographs were taken for recording.

[0042] Crystal violet staining results as follows Figure 3 As shown, the LMBV experimental group (only LMBV was added) Figure 3 The color of the LMBV in the sample became significantly lighter, indicating a significant decrease in the number of viable cells. The cinnamon extract test group (containing LMBV and different concentrations of cinnamon extract) showed a significant decrease in color. Figure 3 The H, M, and L components in the extract were significantly darker, indicating a significantly higher number of live cells, suggesting that the cinnamon extract has a significant anti-LMBV effect.

[0043] Example 3 This embodiment demonstrates the effect of co-incubation of LMBV virus and cinnamon extract on largemouth bass iridovirus.

[0044] FHM cells were seeded into 12-well culture plates at a concentration of 1 × 10⁶ cells / well. 6 Cells / mL, incubated in a 28°C incubator for 18 hours.

[0045] The experimental group will use LMBV (10 6 TCID 50 The LMBV virus particles were incubated with 0.0125 mg / mL cinnamon extract at 4°C for 2 h, then centrifuged at 25000 × g for 1 h at 4°C, and the supernatant was discarded. The LMBV virus particles obtained by centrifugation were resuspended in 100 μL TN buffer and mixed well.

[0046] The control group will have LMBV (10 6 TCID 50 After incubating the cells ( / mL) with the cell culture medium at 4°C for 2 hours, the above-mentioned centrifugation and resuspending steps were performed.

[0047] Replace the cell culture medium for FHM cells in the 12-well cell culture plate, and then inoculate the FHM cells with 20 μL of the suspension prepared in the experimental group and the control group at 28℃ for 48 h.

[0048] Cell samples were collected from each well in both the experimental and control groups. Total RNA was extracted, and the expression of the ICP46 gene of LMBV was detected by RT-qPCR. Each sample was tested in triplicate.

[0049] The relative expression level of the ICP46 gene was measured by RT-qPCR as follows: Figure 4 As shown, compared with the control group (LMBV-FHM), the relative expression level of the ICP46 gene in the experimental group (cinnamon extract + LMBV-FHM) was significantly decreased. This indicates that after 1 hour of co-incubation with LMBV virus and cinnamon extract, the cinnamon extract disrupted the LMBV virus particle structure, thereby reducing the infectivity of LMBV on FHM cells.

[0050] Example 4 This embodiment demonstrates the effect of directly mixing LMBV with cinnamon extract on largemouth bass iridovirus.

[0051] FHM cells were seeded into 12-well culture plates at a concentration of 1 × 10⁶ cells / well. 6 Cells / mL, incubated in a 28°C incubator for 18 hours.

[0052] The experimental group used 0.3 μL of LMBV (10 6 TCID 50 The mixture of cinnamon extract (0.0125 mg / mL) and cinnamon extract (0.0125 mg / mL) was inoculated into FHM cells in a 12-well cell culture plate and incubated at 4°C for 1 h.

[0053] The control group received only 0.3 μL of LMBV (10 6 TCID 50 ( / mL), incubate at 4℃ for 1 hour.

[0054] After incubation, the supernatant was discarded, and the FHM cells in both the experimental and control groups were washed twice with cell culture medium and cultured at 28°C for another 12 hours. Cell samples from each well in both the experimental and control groups were collected, and the expression of the ICP46 gene was detected using the same method as in Example 3. Each sample was tested in triplicate.

[0055] The relative expression level of the ICP46 gene was measured by RT-qPCR as follows: Figure 5 As shown, compared with the control group (LMBV only), the experimental group (LMBV + cinnamon extract) showed a significantly lower relative expression level of the ICP46 gene. This indicates that during LMBV infection of host cells, cinnamon extract can interfere with the binding of LMBV to receptors on the host cell surface, thereby reducing LMBV infectivity.

[0056] Example 5 This embodiment demonstrates the effect of cinnamon extract against largemouth bass iridovirus during LMBV infection of FHM cells.

[0057] FHM cells were seeded into 12-well culture plates at a concentration of 1 × 10⁶ cells / well. 6 Cells / mL were incubated at 28°C for 18 hours. 800 μL of pre-cooled (4°C) cell culture medium was then mixed with 0.3 μL of LMBV (10⁻⁶ / mL). 6 TCID 50 Mix ( / mL), inoculate into FHM cells, and incubate at 4℃ for 1 h to allow LMBV to adsorb onto the surface of FHM cells, then discard the culture medium.

[0058] The experimental group had cinnamon extract at a concentration of 0.0125 mg / mL added to FHM cells; the control group simply had the cell culture medium replaced with fresh one.

[0059] After culturing at 28°C for 2 hours, the supernatant was discarded, and the FHM cells were washed twice with cell culture medium and cultured at 28°C for another 10 hours. Cells from each well in the experimental and control groups were collected, and the expression of the ICP46 gene was detected using the same method as in Example 3. Each sample was tested in triplicate.

[0060] The relative expression level of the ICP46 gene was measured by RT-qPCR as follows: Figure 6 As shown, 1 hour after LMBV infection of FHM cells, the relative expression level of the ICP46 gene in the experimental group (treated with cinnamon extract) was significantly lower than that in the control group (treated with cell culture medium only). This indicates that cinnamon extract can interfere with LMBV invasion of host cells during the LMBV infection process in FHM cells.

[0061] Example 6 This embodiment demonstrates the effect of cinnamon extract against largemouth bass iridovirus after successful LMBV infection of FHM cells.

[0062] FHM cells were seeded into 12-well cell culture plates at a concentration of 1 × 10⁶ cells / well. 6 Cells / mL were cultured in a 28°C incubator for 18 hours. 800 μL of pre-cooled (4°C) cell culture medium was then mixed with 0.3 μL of LMBV (10⁻⁶ / mL). 6 TCID 50 Mix the medium ( / mL) thoroughly, inoculate into FHM cells, and incubate at 4°C for 1 hour. Remove the cell culture medium containing LMBV, add fresh cell culture medium, and continue culturing at 28°C for 2 hours to allow LMBV to infect FHM cells.

[0063] After the culture was completed, the cell culture medium was discarded, and cinnamon extract at a concentration of 0.0125 mg / mL was added; the control group was replaced with an equal volume of new cell culture medium.

[0064] After culturing cells at 28°C for 8 hours, the cell culture medium was discarded, and the FHM cells were washed twice with cell culture medium and then cultured for another 2 hours at 28°C. Cells from each well in the experimental and control groups were collected, and the expression of the ICP46 gene was detected using the same method as in Example 3. Each sample was tested in triplicate.

[0065] The relative expression level of the ICP46 gene was measured by RT-qPCR as follows: Figure 7 As shown, 3 hours after LMBV infection of FHM cells, compared with the control group (only the cell culture medium was changed), the relative expression level of the ICP46 gene in the experimental group (treated with cinnamon extract) was significantly downregulated, indicating that LMBV replication in FHM cells was significantly reduced. This demonstrates that even after successful infection of host cells, cinnamon extract can still significantly inhibit LMBV replication in host cells.

[0066] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. The application of cinnamon in the preparation of drugs for the prevention and treatment of largemouth bass virus, characterized in that, A cinnamon extract was prepared using cinnamon as a raw material, and the cinnamon extract has an antiviral effect against largemouth bass iridovirus. The safe and effective concentration of the cinnamon extract is 0.003125~0.0125 mg / mL; The method for preparing the cinnamon extract is as follows: Cinnamon powder pretreated with petroleum ether was extracted with methanol, the methanol extract was collected and the methanol solvent was removed to obtain dried cinnamon extract. The pretreatment method is as follows: soak cinnamon powder in petroleum ether for 1 hour, then sonicate it 3 times at 60°C for 5 minutes each time, discard the petroleum ether, and then add new petroleum ether and repeat the operation once. Petroleum ether dosage: Use 250mL of petroleum ether for every 25g of cinnamon powder; The methanol extraction method is as follows: the pretreated cinnamon powder is soaked in methanol for 1 hour, then sonicated 3 times at 60°C for 5 minutes each time, and the methanol extract is collected by filtration. Methanol dosage: Use 250 mL of methanol for every 25 g of pretreated cinnamon powder.

2. The application according to claim 1, characterized in that, At a concentration of 0.0125 mg / mL, the cinnamon extract has the effect of disrupting the particle structure of largemouth bass iridovirus.

3. The application according to claim 1, characterized in that, At a concentration of 0.0125 mg / mL, the cinnamon extract interferes with the binding of largemouth bass iridovirus to receptors on the surface of host cells.

4. The application according to claim 1, characterized in that, At a concentration of 0.0125 mg / mL, the cinnamon extract has the effect of interfering with the invasion of largemouth bass iridovirus into host cells.

5. The application according to claim 1, characterized in that, At a concentration of 0.0125 mg / mL, the cinnamon extract inhibited the replication of largemouth bass iridovirus.

6. The application according to claim 3 or 4, characterized in that, The host cells are the fathead flounder muscle cell line.

7. A drug, characterized in that, The active ingredient of the drug includes cinnamon extract, and the drug has an anti-largemouth bass iridovirus effect; the preparation method of the cinnamon extract is as described in claim 1.

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