Use of scopoletin in the preparation of drugs for treating white spot syndrome

Abstract

The present application belongs to the technical field of fishery drugs, and relates to specific therapeutic activity of scopoletin, and particularly to application of scopoletin in preparation of drugs against white spot syndrome. The present application verifies through experiments that scopoletin can up-regulate Ago2 and Dicer2 genes which are key members in an anti-virus mechanism of RNAi of prawns, and can endow prawns and other aquatic animals with certain resistance to white spot syndrome virus.

Description

Technical Field

[0001] This invention belongs to the field of fish medicine technology, and relates to the specific therapeutic activity of scopolamine, specifically the application of scopolamine in the preparation of drugs for vitiligo syndrome. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] White Spot Syndrome (WSS) is one of the most damaging diseases in shrimp farming. The pathogen is White Spot Syndrome Virus (WSSV), an enveloped double-stranded DNA virus that can cause rapid death in shrimp within 3-10 days.

[0004] Based on current research, there is a lack of effective means to prevent and control WSSV. Shrimp rely on their innate immune system to construct an effective antiviral defense network, which provides a molecular basis for developing novel prevention and control strategies. The core mechanism of shrimp's antiviral immunity is RNAi (RNA interference), where the Dicer-2 protein recognizes and cleaves viral long double-stranded RNA, generating virus-specific small interfering RNA (siRNA). Subsequently, these siRNAs are loaded into the RNA-induced silencing complex formed by the Ago2 protein, which targets and cleaves viral messenger RNA through base complementarity, thereby inhibiting viral gene expression and replication at the posttranscriptional level. Previous experimental data have shown that upregulating the expression of shrimp Dicer-2 or Ago2 genes significantly enhances their antiviral ability. Therefore, if a new antiviral drug can be developed that can simultaneously increase the expression levels of key members of the RNAi mechanism to improve the antiviral ability of shrimp, it could effectively inhibit WSSV infection, treat white spot syndrome, and be of great significance for protecting the healthy development of shrimp farming. Summary of the Invention

[0005] Scopolamine, also known as hyoscyamine, has been reported for various medicinal uses, primarily including anti-inflammatory, neuroprotective, and antidiabetic effects, with no documented applications in veterinary medicine. Experiments have demonstrated that scopolamine can induce the expression of key members of the antiviral RNAi mechanism in shrimp, inhibit the replication of WSSV gene mRNA and intact viral particles, and alleviate the damage caused by white spot syndrome. This indicates that scopolamine can confer antiviral capabilities to shrimp by inducing the expression of antiviral genes. This invention discloses for the first time the application of scopolamine as an antiviral drug in shrimp and other aquatic animals, and its use differs from known applications of scopolamine.

[0006] Based on the above research findings, this invention proposes the application of scopolamine lactone in the preparation of drugs for treating vitiligo syndrome. Specifically, the technical solution provided by this invention is as follows:

[0007] The first aspect concerns the application of scopolamine lactone in the preparation of drugs for vitiligo syndrome.

[0008] The scopolamine lactone described in this invention has the CAS number 92-61-5, and its chemical structural formula is shown below:

[0009] .

[0010] In some embodiments, the drug has at least one of the following effects:

[0011] (a) Inducing the expression of Ago2 and Dicer2 genes, core components of the RNAi mechanism in aquatic animals;

[0012] (b) Inhibit the mRNA transcription of the vitiligo syndrome virus structural protein VP28 gene;

[0013] (c) Inhibits the replication of intact viral particles of vitiligo syndrome virus;

[0014] (d) Inhibit shrimp mortality caused by white spot syndrome virus.

[0015] In some embodiments, the active ingredient of the drug is scopolamine lactone or its pharmaceutically acceptable salt, solvate, or hydrate.

[0016] The pharmaceutically acceptable salts described in this invention refer to salts that are pharmaceutically considered safe, effective, and suitable for use in pharmaceutical preparations. These salts can be salts formed with inorganic or organic acids, or salts formed with inorganic or organic bases. Specifically, inorganic acids can be sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, etc.; organic acids can be acetic acid, oxalic acid, citric acid, fumaric acid, p-benzenesulfonic acid, etc.; inorganic bases can be sodium hydroxide, potassium hydroxide, ammonia, etc.; and organic bases can be methylamine, ethylamine, ethanolamine, etc.

[0017] The solvate described in this invention refers to the crystal structure formed by the crystallization of scopolamine lactone and organic solvent.

[0018] The hydrates described in this invention refer to the crystal structure formed by the crystallization of scopolamine lactone and water.

[0019] Since feed additives can also contain appropriate amounts of drugs, in some embodiments, the drugs are used as feed additives for aquatic animals.

[0020] In some implementations, the drug is administered via feed addition or water injection. Specifically, water injection includes, but is not limited to, soaking, bathing, and sprinkling.

[0021] In some embodiments, the dosage of the drug is 10-50 μg / L (i.e., 10-50 μg per liter of water). Specifically, the dosage can be 20-50 μg / L, 30-50 μg / L, 40-50 μg / L, 45-50 μg / L, 48-50 μg / L, etc.

[0022] In some embodiments, the drug is a composition. The composition may take different forms, such as granules, powders, liquid formulations, etc., depending on the recipient's needs.

[0023] Specifically, the composition includes pharmaceutical excipients. These excipients may be palatability enhancers, binders, film-forming agents, diluents, disintegrants, antioxidants, penetration enhancers, preservatives, etc.

[0024] In some implementations, the drug is administered to aquatic animals. Specifically, the aquatic animals are shrimp.

[0025] Secondly, a method for preventing or treating white spot syndrome in shrimp, the method comprising: applying a drug to the shrimp, said drug having an active ingredient of scopolamine lactone or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0026] In some implementations, the drug is administered via feed additives or water. Specifically, water administration includes, but is not limited to, soaking, immersion, and splashing.

[0027] In some implementation schemes, the concentration of the applied drug is 10-50 μg / L (i.e., 10-50 μg per liter of water). Specifically, the concentration can be 20-50 μg / L, 30-50 μg / L, 40-50 μg / L, 45-50 μg / L, 48-50 μg / L, etc.

[0028] The beneficial effects of this invention are as follows:

[0029] 1. This invention reveals for the first time that scopolamine can combat viruses by activating the innate immune pathways of shrimp. Experiments have shown that scopolamine application to shrimp after soaking significantly induces upregulation of the expression of key components of the RNA interference (RNAi) mechanism (Ago2 and Dicer2). This indicates that scopolamine can enhance the shrimp's own key antiviral immune defense capabilities.

[0030] 2. Scopolamine effectively inhibits the replication cycle of White Spot Syndrome Virus (WSSV) in multiple stages. Specifically, it inhibits viral gene transcription: Scopolamine can significantly reduce the mRNA transcription level of the WSSV key structural protein VP28 gene, interfering with viral protein synthesis at the source. (2) Blocks viral particle assembly: Scopolamine can effectively inhibit the production of complete WSSV viral particles during viral replication, cutting off the infection chain. (3) Reduces host mortality: Ultimately, the above effects translate into significant protective effects, effectively inhibiting shrimp mortality caused by WSSV infection and improving the survival rate of farmed shrimp.

[0031] 3. This invention provides novel candidate compounds and a clear research direction for the development of effective anti-WSSV drugs. Scopolamine, as a known active ingredient, has had its preventive and therapeutic effects verified in vivo, and its mechanism of action is clear. This lays a solid foundation for developing a highly effective, safe, and novel targeted antiviral drug for veterinary medicine, and is of great significance for solving the current industrial problem of the lack of effective treatments for white spot syndrome in shrimp farming. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 This is a graph showing the results of confirming the optimal dosage of scopolamine lactone in shrimp according to Example 1 of the present invention.

[0034] Figure 2 This is a graph showing the effect of scopolamine lactone in Example 2 of the present invention on the expression of the core components of the shrimp RNAi mechanism, the Ago2 and Dicer2 genes.

[0035] Figure 3 This is a diagram showing the effect of scopolamine lactone in Example 3 of the present invention on the transcription of the VP28 gene, a structural protein of vitiligo syndrome virus, at the mRNA level.

[0036] Figure 4 This is a graph showing the effect of scopolamine lactone on the replication of intact viral particles of vitiligo syndrome virus in Example 4 of the present invention.

[0037] Figure 5 This is a graph showing the effect of scopolamine lactone in Example 5 of the present invention on shrimp mortality caused by white spot syndrome virus infection. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0039] Example 1

[0040] To determine the optimal dosage of scopolamine for shrimp: Scopolamine (HY-N0342, MedChemExpress) was dissolved in DMSO as a stock solution. 240 shrimp (average 5 mg per shrimp) were randomly divided into four groups of 60 shrimp each. Immersion concentrations of 0, 10, 50, and 250 μg / L were used for each group, and survival rates were recorded every 24 hours.

[0041] The results are as follows Figure 1 As shown, immersion administration of 10 or 50 μg / L did not affect the survival of shrimp.

[0042] Example 2

[0043] Animal treatment: Scopolamine lactone was dissolved in DMSO as a stock solution. The soaking concentrations for each group were 0, 10, and 50 μg / L. After 6 h of culture, total tissue samples were extracted from shrimp. RNA was obtained from the shrimp using RNA extraction buffer after 6 h of culture, and then reverse transcribed into cDNA using a reverse transcription kit (R323-01; Vazyme). Finally, qRT-PCR was used to detect the expression levels of the Ago2 and Dicer2 genes. The primer sequences for detecting the Ago2 and Dicer2 genes are: Dicer2-RTF: 5'-AGGAAATGCAATGTCGTGGTT-3' (as shown in SEQ ID NO.1), Dicer2-RTR: 5'-ACGAGCCCCCCCCCTAGATT-3' (as shown in SEQ ID NO.2); Ago2-RTF: 5'-TAGTGGCAAGAAGAGTAG-3' (as shown in SEQ ID NO.3), Ago2-RTR: 5'-AACGCATGAAAAATGAGTA-3' (as shown in SEQ ID NO.4). β-actin was used as an internal control, with primer sequences of β-actin-RTF: 5'-TGAAGATCCTGACGGAGCGT-3' (as shown in SEQ ID NO.5), β-actin-RTR: 5'-GAACCTCTCGTTGCCGATG-3' (as shown in SEQ ID NO.6). The qRT-PCR program was set to 94 °C for 5 min, 94 °C for 10 s, and 60 °C for 1 min, for a total of 40 cycles. The final data were analyzed using 2... ΔΔCT The method processes the data and performs three independent replicate experiments.

[0044] The results are as follows Figure 2As shown, immersion administration of 50 μg / L scopolamine can induce the expression of Ago2 and Dicer2 genes, the core components of the shrimp RNAi mechanism.

[0045] Example 3

[0046] Animal treatment: Scopolamine was dissolved in DMSO as a stock solution. Group 1: Shrimp were immersed in scopolamine at a concentration of 50 μg / L; Group 2: An equal volume of DMSO was added to the water. Both groups of shrimp were cultured for 6 hours after treatment and fed a diet containing WSSV (10 μg / L virus). 6 Virus particles were ensured to be completely ingested. After 24 h and 48 h of culture, RNA samples were obtained from shrimp tissues using RNA extraction reagents. The RNA was then reverse transcribed into cDNA using a reverse transcription kit. Changes in WSSV mRNA were detected using qRT-PCR technology, with primers VP28-RTF: 5'-AGCTCCAACACCTCCTCCTTCA-3' (as shown in SEQ ID NO.7) and VP28-RTR: 5'-TTACTCGGTCTCAGTGCCAGA-3' (as shown in SEQ ID NO.8).

[0047] The test results are attached. Figure 3 As shown, scopolamine can inhibit the mRNA transcription of the VP28 gene, a structural protein of vitiligo syndrome virus.

[0048] Example 4

[0049] The treatment and detection methods for the experimental animals were the same as in Example 3. Both groups of shrimp were cultured for 6 hours after treatment and fed with feed containing WSSV. The virus content in the feed was 10... 6 Virus particles were ensured to be completely ingested. After 24 h and 48 h of culture, total DNA samples were extracted from the intestinal tissue using the QIAamp DNA Blood Mini Kit (51104; Qiagen), and qRT-PCR was used to detect changes in WSSV DNA.

[0050] The test results are attached. Figure 4 As shown, scopolamine can inhibit the replication of intact viral particles of vitiligo syndrome virus.

[0051] Example 5

[0052] The treatment and detection methods for the experimental animals were the same as in Example 3. Both groups of shrimp were cultured for 6 hours after treatment and fed with feed containing WSSV. The virus content in the feed was 10... 6 Virus particles were collected, ensuring all shrimp were consumed, and the survival rate of each group of shrimp was recorded.

[0053] The test results are attached. Figure 5 As shown, scopolamine can inhibit shrimp mortality caused by white spot syndrome virus.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of scopolamine lactone in the preparation of drugs for vitiligo syndrome.

2. The application as described in claim 1, characterized in that, The drug has at least one of the following effects: (a) Inducing the expression of Ago2 and Dicer2 genes, core components of the RNAi mechanism in aquatic animals; (b) Inhibit the mRNA transcription of the vitiligo syndrome virus structural protein VP28 gene; (c) Inhibits the replication of intact viral particles of vitiligo syndrome virus; (d) Inhibit shrimp mortality caused by white spot syndrome virus.

3. The application as described in claim 1, characterized in that, The active ingredient of the drug is scopolamine lactone or a pharmaceutically acceptable salt thereof.

4. The application as described in claim 1, characterized in that, The drug is used as a feed additive for aquatic animals.

5. The application as described in claim 1, characterized in that, The drug is administered via feed additives or water.

6. The application as described in claim 1, characterized in that, The drug was administered at a concentration of 50 μg / L.

7. The application as described in claim 1, characterized in that, The drug is a composition.

8. The application as described in claim 7, characterized in that, The composition includes pharmaceutical excipients.

9. The application as described in claim 1, characterized in that, The drug is to be administered to aquatic animals.

10. The application as described in claim 9, characterized in that, The aquatic animal in question is a shrimp.

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