Use of ginkolide A in preparation of drug for resisting leukoplakia syndrome

Abstract

The application relates to application of ginkgo biloba lactone A in preparation of drugs against white spot syndrome, aiming at the problem that effective treatment drugs are still lacking for the destructive damage of white spot syndrome virus (WSSV) to aquatic crustaceans, and providing a new application of ginkgo biloba lactone A; through in-vitro experiment of Hpt cells of astacus leptodactylus and in-vivo feeding experiment of astacus leptodactylus, it is verified that the ginkgo biloba lactone A can significantly inhibit WSSV replication at a concentration of 100 muM; the astacus leptodactylus is fed with 40 mg / kg feed for 10 days, the survival rate after being infected with WSSV is increased by about 53.3%, meanwhile, the expression of immune-related genes is up-regulated, the apoptosis rate of blood cells is reduced, and the virus replication is significantly inhibited; a safe and efficient new drug for the prevention and control of WSSV in aquatic breeding is provided, and has important industrial application value.

Description

Technical Field

[0001] This invention relates to the field of disease prevention and control technology in aquaculture, specifically to the application of ginkgolide A in the preparation of drugs for treating vitiligo syndrome. Background Technology

[0002] White spot syndrome virus (WSSV) is one of the most serious viral pathogens threatening aquaculture of crustaceans such as shrimp. In Fujian and Taiwan, the infection rate in farmed shrimp exceeds 75-90%, causing global shrimp farming losses of over ten billion US dollars annually. Currently, there is a lack of safe and effective control strategies for WSSV.

[0003] Ginkgolide A (GA) is a highly active platelet-activating factor (PAF) receptor antagonist molecule isolated from ginkgo leaves. It is known to have anti-inflammatory and immunomodulatory effects and is used in the medical field to treat osteoarthritis, metabolic diseases caused by diabetes, etc. However, there are no reports on its anti-WSSV infection in aquaculture.

[0004] Therefore, this application aims to fill this gap by providing a green and highly effective anti-WSSV drug. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in the related art. Therefore, the object of this invention is to provide the application of ginkgolide A in the preparation of drugs for treating vitiligo syndrome.

[0006] Therefore, in a first aspect of the present invention, the present invention proposes the use of ginkgolide A in the preparation of an anti-leukoderma drug, said drug being used to treat leukoderma virus infection in aquatic crustaceans.

[0007] According to the present invention, a new application of ginkgolide A is provided; through in vitro Hpt cell experiments and in vivo feeding experiments with red claw crayfish, it was verified that ginkgolide A at a concentration of 100 μM can significantly inhibit WSSV replication; feeding red claw crayfish with 40 mg / kg feed for 10 days can increase the survival rate of WSSV infection by about 53.3%, while upregulating the expression of immune-related genes, reducing the apoptosis rate of blood cells, and significantly inhibiting viral replication; this provides a safe and efficient new drug for the prevention and control of WSSV in aquaculture, and has important industrial application value.

[0008] Optionally, the aquatic crustacean is the redclaw crayfish.

[0009] Optionally, the drug is used in the form of a feed additive.

[0010] Furthermore, the dosage of Ginkgolide A added to the feed is 40 mg / kg.

[0011] Optionally, the optimal antiviral activity concentration of the ginkgolide A in in vitro hematopoietic tissue Hpt cells is 100 μM.

[0012] Optionally, the drug exerts its antiviral effect against vitiligo syndrome through at least one of the following mechanisms: (1) Inhibits the expression of VP28 and IE1 genes and viral replication of WSSV; (2) Upregulate the expression of phenol oxidase, anti-lipopolysaccharide factor, and immune-related genes of ns1-a-like binding protein of influenza A virus in the blood cells of aquatic crustaceans; (3) Reduce the apoptosis rate of blood cells in aquatic crustaceans after WSSV infection.

[0013] In a second aspect, the present invention provides a medicament against vitiligo syndrome virus, said medicament containing ginkgolide A and pharmaceutically acceptable excipients.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0015] Figure 1 To utilize crayfish Hpt cells according to embodiments of the present invention, WSSV replication levels were detected by WB / RT-qPCR through GA pre-incubation, co-incubation, and WSSV infection followed by incubation. Figure 2 According to an embodiment of the present invention, ginkgolide A was added to the feed and crayfish were fed for 10 days. The survival rate of crayfish infected with WSSV was statistically analyzed. Figure 3 The expression levels of immune-related genes in blood cells were measured by feeding crayfish with 40 mg / kg GA feed for 10 days according to an embodiment of the present invention. Figure 4 The apoptosis rate of blood cells after crayfish were infected with WSSV were determined by feeding them 40 mg / kg GA feed for 10 days according to an embodiment of the present invention. Figure 5 The viral replication level in blood cells after crayfish were infected with WSSV were determined by feeding them 40 mg / kg GA feed for 10 days according to an embodiment of the present invention. Detailed Implementation

[0016] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0017] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0018] The test materials used in this invention are all commercially available products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0019] Modified L15 complete culture medium: 50 μg / mL streptomycin, 60 μg / mL penicillin, 50 μg / mL gentamicin, 2 mM L-glutamine, 1 μM phenylthiourea and 5 μM β-mercaptoethanol were dissolved in L15 medium and stored at 4 °C.

[0020] Ginkgolide A was purchased from Shanghai Yuanye Biotechnology. The structural formula of Ginkgolide A is as follows:

[0021] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0022] Example 1: Validation of the anti-WSSV activity of Ginkgolide A (GA) (1) Select male red claw crayfish, cut open the head and thorax of the crayfish, remove the upper hematopoietic tissue (Hpt) of the stomach and place it in an EP tube containing 600 μL collagenase solution, and digest at room temperature for 45 min.

[0023] (2) Centrifuge at 500 g at room temperature for 3 min, discard the supernatant, add 1 mL of sterile CPBS to wash the Hpt precipitate, centrifuge at 500 g for 3 min, and discard the supernatant.

[0024] (3) Add 1 mL of sterile CPBS and gently blow up the precipitate. Then, gently blow up the precipitate about 15 times. Centrifuge at 500 g for 3 min and aspirate the supernatant. The precipitate at this time is Hpt cells.

[0025] (4) Add 1 mL of modified L15 complete culture medium, gently pipette the suspended cells, and filter out cell debris.

[0026] (5) Cell count.

[0027] (6) Seeding the prepared Hpt cells: 96-well plate 1.0×10 5 Cells / well, cultured at 20℃ for 30 min, and after the cells adhered, crayfish Plasma was added to each well of a 96-well plate at 0.2 μL and incubated at 20℃.

[0028] (7) Hpt cells were seeded in 96-well cell culture plates. After the cells were stable, ginkgolide A (100 μM) was added as follows: Figure 1 Cells were added as shown in the schematic diagram. After 12 h of WSSV infection (MOI=1), cell samples were collected using SDS loading buffer for Western blot detection of VP28 protein expression levels. Simultaneously, total RNA was extracted from Hpt cells and reverse transcribed to synthesize cDNA as a template for gene amplification. RT-qPCR was used to detect WSSV-VP28 and IE1 gene expression. RT-qPCR detection: The SYBR Green method was used for quantification of the target genes, with 16S as an internal control gene. The reaction system is shown in the table below:

[0029] The RT-qPCR experiment was performed using an ABI 7500 instrument, and the reaction procedure is as follows: 50 ℃ for 2 min 95 ℃ for 10 min 95 ℃ for 15 s, 60 ℃ for 1 min; 40 cycles; After the above reaction is completed, a melting curve quantitative PCR reaction is performed. The reaction procedure is as follows: 95 ℃ 15 s 60 ℃ for 1 min, 95 ℃ for 15 s; 40 cycles; The obtained data is used in 2 -ΔΔCt The calculation method is used to calculate the RQ (Relative quantification) value.

[0030] Primer list

[0031] The results are as follows Figure 1As shown, the expression levels of WSSV-VP28 and WSSV-IE1 genes were significantly reduced under the three GA treatments, indicating that GA has anti-WSSV activity.

[0032] Example 2: Survival rate statistics of red swamp crayfish after adding ginkgolide A (GA) to feed. (1) Healthy red claw crayfish (male, 11±0.5 cm, 17±0.5 g) were divided into 5 groups of 15 crayfish each and kept at room temperature for one week.

[0033] (2) Add 0 mg / kg, 20 mg / kg, 40 mg / kg and 80 mg / kg GA to commercial crayfish feed respectively, feed them with GA-containing feed for 10 days and count the survival rate.

[0034] (3) Remove the shell from the tail of the WSSV-infected shrimp and grind it into a paste. Add 8-10 mL of CFS (crayfish physiological saline: 5.4 mM KCl, 0.2 M NaCl, 2.6 mM MgCl2•6H2O, 10 mM CaCl2•2H2O and 2 mM NaHCO3 dissolved in 1 L of double-distilled water, pH=6.8). After mixing well, proceed as follows: Figure 2 The schematic diagram shows that each crayfish was injected with 100 μL of the solution, and the temperature in the rearing room was raised to 26°C within 24 hours.

[0035] (4) Statistics on crayfish survival rate.

[0036] The results are as follows Figure 2 As shown, crayfish fed a diet of 40 mg / kg GA had a survival rate approximately 53.3% higher after WSSV infection compared to crayfish fed a diet of 0 mg / kg GA. This indicates that ginkgolide A has good potential for production and application.

[0037] Example 3: Addition of Ginkgolide A (GA) to feed increased the expression level of immune-related genes in red swamp crayfish. (1) Healthy red claw crayfish (male, 11±0.5 cm, 17±0.5 g) were divided into two groups of 15 crayfish each and kept at room temperature for one week.

[0038] (2) Add 0 mg / kg and 40 mg / kg GA to commercial crayfish feed respectively, feed them with GA-containing feed for 10 days, extract blood lymphocytes, centrifuge at 800 g for 5 min at 4℃, extract RNA by Trizol method, reverse reverse to cDNA, and detect by real-time PCR. Cq Expression levels of immune genes such as HSP70, ALF, proPO, JAK, STAT, C-type-lectin, LYZ, Caspase, myosin, and NS1abp.

[0039] The results are as follows Figure 3 As shown, feeding redclaw crayfish with a diet containing 40 mg / kg ginkgolide A significantly increased the expression of immune-related genes, including the phenol oxidase gene (…). Cq PO), anti-lipopolysaccharide factor ( Cq ALF), influenza A virus non-structural protein ns1-a-like binding protein (ALF), Cq The expression levels of antiviral genes such as ns1abp were significantly upregulated.

[0040] Example 4: Analysis of apoptosis rate of red swamp crayfish blood cells after feed supplementation with ginkgolide A (GA) (1) Healthy red claw crayfish (male, 11±0.5 cm, 17±0.5 g) were divided into 5 groups of 15 crayfish each and kept at room temperature for one week.

[0041] (2) Add 0 mg / kg and 40 mg / kg GA to commercial crayfish feed respectively, feed them with GA-containing feed for 10 days, and count the survival rate.

[0042] (3) Remove the shell from the tail of the WSSV-infected shrimp and grind it into a paste. Add 8-10 mL of CFS (crayfish physiological saline: 5.4 mM KCl, 0.2 M NaCl, 2.6 mM MgCl2•6H2O, 10 mM CaCl2•2H2O and 2 mM NaHCO3 dissolved in 1 L of double-distilled water, pH=6.8). After mixing well, proceed as follows: Figure 2 The schematic diagram shows that each crayfish was injected with 100 μL of the solution, and the temperature in the rearing room was raised to 26°C within 24 hours. Blood lymphocytes were collected 24 hours later.

[0043] (4) Centrifuge at 500 g at room temperature for 3 min, discard the supernatant, add 1 mL of sterile citrate buffer (CPBS) to wash the Hpt precipitate, centrifuge at 500 g for 3 min, and discard the supernatant.

[0044] (5) Cell counting, collect 1-5 × 10⁻⁵ cells. 5 cell.

[0045] (6) Discard CPBS and add 100 μL of 1×Binding Buffer to resuspend the cells.

[0046] (7) Add 5 μL Annexin V-FITC and 10 μL PI Staining Solution according to the cell apoptosis detection kit and mix gently.

[0047] (8) React in the dark at room temperature for 10-15 min.

[0048] (9) Add 400 μL of 1×Binding Buffer, mix well and place on ice. Detect the sample with flow cytometry or fluorescence microscopy within 1 hour.

[0049] The results are as follows Figure 4 As shown, crayfish fed with 40 mg / kg GA showed a significantly lower rate of hematopoietic apoptosis after WSSV infection compared to crayfish fed with 0 mg / kg GA.

[0050] Example 5: Analysis of Virus Replication Levels in Red Claw Crawfish After Addition of Ginkgolide A (GA) to Feed (1) Healthy red claw crayfish (male, 11±0.5 cm, 17±0.5 g) were divided into 5 groups of 15 crayfish each and kept at room temperature for one week.

[0051] (2) Add 0 mg / kg and 40 mg / kg GA to commercial crayfish feed respectively, and feed crayfish with GA-containing feed for 10 days.

[0052] (3) Remove the shell from the tail of the WSSV-infected shrimp and grind it into a paste. Add 8-10 mL of CFS (crayfish physiological saline: 5.4 mM KCl, 0.2 M NaCl, 2.6 mM MgCl2•6H2O, 10 mM CaCl2•2H2O and 2 mM NaHCO3 dissolved in 1 L of double-distilled water, pH=6.8). After mixing well, proceed as follows: Figure 2 The schematic diagram shows that each crayfish was injected with 100 μL of the solution, and the temperature in the rearing room was raised to 26°C within 24 hours.

[0053] (4) Every 24 h, crayfish blood lymphocytes were extracted, centrifuged at 800 g for 5 min at 4℃, RNA was extracted by Trizol method, reversed to cDNA, and the expression level of WSSV-VP28 gene was detected by real-time PCR.

[0054] The results are as follows Figure 5 As shown, crayfish fed a diet of 40 mg / kg GA showed significantly lower viral replication levels in hematopoietic cells after WSSV infection compared to crayfish fed a diet of 0 mg / kg GA.

[0055] In summary, according to embodiments of the present invention, the antiviral activity of ginkgolide A was verified in vitro using a redclaw crayfish Hpt cell model, and its function in WSSV infection was also verified: feeding redclaw crayfish with 40 mg / kg ginkgolide A significantly increased the expression of immune-related genes in redclaw crayfish, including the phenol oxidase gene (…). Cq PO), anti-lipopolysaccharide factor ( Cq ALF), influenza A virus non-structural protein ns1-a-like binding protein (ALF), CqThe expression levels of antiviral genes such as ns1abp were significantly upregulated. Simultaneously, the viral replication level in crayfish was significantly reduced, improving the survival rate of crayfish infected with the virus. Therefore, the preparation of a new drug against WSSV infection using ginkgolide A has promising application prospects.

[0056] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0057] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of ginkgolide A in the preparation of drugs for vitiligo syndrome, characterized in that, The drug is used to treat white spot syndrome virus infection in aquatic crustaceans.

2. The application according to claim 1, characterized in that, The aquatic crustacean mentioned is the redclaw crayfish.

3. The application according to claim 1, characterized in that, The drug is used in the form of a feed additive.

4. The application according to claim 3, characterized in that, The dosage of Ginkgolide A added to the feed is 40 mg / kg.

5. The application according to claim 1, characterized in that, The optimal antiviral activity concentration of Ginkgolide A in in vitro hematopoietic tissue Hpt cells is 100 μM.

6. The application according to claim 1, characterized in that, The drug exerts its antiviral effect against vitiligo syndrome through at least one of the following mechanisms: (1) Inhibits the expression of VP28 and IE1 genes and viral replication of WSSV; (2) Upregulate the expression of phenol oxidase, anti-lipopolysaccharide factor, and immune-related genes of ns1-a-like binding protein of influenza A virus in the blood cells of aquatic crustaceans; (3) Reduce the apoptosis rate of blood cells in aquatic crustaceans after WSSV infection.

7. A drug for treating vitiligo syndrome virus, characterized in that, The drug contains ginkgolide A and pharmaceutically acceptable excipients.

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