Use of acacetin in the preparation of drugs for preventing and treating vitiligo syndrome

Abstract

The application belongs to the technical field of disease prevention and treatment and aquaculture, and relates to application of acacetin in preparation of a drug for preventing and treating white spot syndrome. The application discloses for the first time that acacetin has the biological activity of resisting white spot syndrome virus, verifies that the acacetin can significantly inhibit the replication of the white spot syndrome virus in the body of a procambarus clarkii, and simultaneously improve the survival rate of the procambarus clarkii infected by the white spot syndrome virus. The acacetin has good antiviral effect on the white spot syndrome virus in crustacean aquaculture, and can be applied to prevention and treatment of water animal diseases caused by the white spot syndrome virus.

Description

Technical Field

[0001] This invention belongs to the field of disease prevention and aquaculture technology, and relates to the application of farnesin in the preparation of drugs for the prevention and treatment of vitiligo syndrome. Background Technology

[0002] Acacetin is a natural flavonoid widely found in various legumes and asteraceae plants, possessing a good foundation for chemical synthesis and extraction. Its chemical structure is 5,7-dihydroxy-4'-methoxyflavonoid. Recent studies have shown that acacetin exhibits significant activities in anti-inflammatory, antiviral, antioxidant, and antitumor effects, including anti-inflammatory effects through regulation of the NF-κB signaling pathway, antitumor effects through inhibition of topoisomerases, and antioxidant responses through activation of the Nrf2 pathway. It has been widely applied in medicine, food, and agriculture. Particularly in studies of viral infection mechanisms, acacetin demonstrates good targeted regulatory capabilities, showing potential as a novel antiviral lead compound. Despite increasingly in-depth pharmacological research on acacetin, systematic research on its application in the prevention and control of viruses in aquatic animals remains lacking.

[0003] White Spot Syndrome (WSS) is a common disease in aquatic animals caused by White Spot Syndrome Virus (WSSV). Currently, there are no effective prevention and control methods for WSS. As a member of the Baculoviridae family, the interaction between WSSV's envelope protein VP28 and other structural proteins and host cell membrane receptors is a key step in the infection process. Current prevention and control strategies face three major technical bottlenecks: the lack of an adaptive immune system in crustaceans hinders vaccine development; chemically synthesized drugs are prone to causing ecotoxicity accumulation; and the high-frequency recombination characteristics of the viral genome lead to rapid mutation of antiviral targets. Existing antiviral agent research has significant limitations: chemically synthesized drugs, such as amantadine, easily induce drug-resistant viral strains; the complex composition of plant crude extracts makes quality control difficult; and interfering RNA technology suffers from low in vivo delivery efficiency. Therefore, the development of an antiviral drug with a well-defined structure that can prevent and treat WSSV infection and WSS would be of great significance for protecting the healthy development of the aquaculture industry. Summary of the Invention

[0004] This invention discloses for the first time the bioactivity of farnesin against white spot syndrome virus (WSSV), verifying that farnesin can significantly inhibit the replication of WSSV in *Procambarus clarkii* and simultaneously improve the survival rate of WSSV-infected *Procambarus clarkii*. Farnesin exhibits good antiviral activity against WSSV in crustacean aquaculture and can be applied to the prevention and control of aquatic animal diseases caused by WSSV.

[0005] On the one hand, this invention relates to the application of farnesin in the preparation of drugs for the prevention and treatment of vitiligo syndrome.

[0006] The chemical structure of farnesin described in this invention is shown below:

[0007] .

[0008] In some embodiments, the drug is a composition. The composition may take different forms, such as powder, liquid formulation, etc., depending on the needs of the recipient.

[0009] Furthermore, in the application provided by the present invention, the pathogen of the vitiligo syndrome is the White Spot Syndrome Virus.

[0010] Furthermore, in the application provided by the present invention, the active ingredient of the drug is farnesin or a pharmaceutically acceptable salt thereof.

[0011] The pharmaceutically acceptable salts described in this invention refer to salts that are pharmaceutically considered safe, effective and suitable for use in pharmaceutical preparations, such as hydrochlorides, sulfates, nitrates, acetates, oxalates, citrates, fumarates, p-benzenesulfonates, etc.

[0012] In addition, the water-soluble prodrugs or pharmaceutically acceptable derivatives of farnesin formed after modification can be directly converted into farnesin in animals to exert pharmacological effects. These include farnesin salts, esters, ethers, stereoisomers, or prodrug molecules.

[0013] Furthermore, in the applications provided by this invention, the drug includes pharmaceutical excipients. These pharmaceutical excipients may be binders, diluents, fillers, preservatives, absorption enhancers, etc.

[0014] Furthermore, in the application provided by the present invention, the drug is applied to aquatic animals or the habitat of aquatic animals.

[0015] Furthermore, in the application provided by this invention, the aquatic animal is a shrimp.

[0016] Furthermore, in the application provided by the present invention, the drug is administered by injection.

[0017] Furthermore, in the application provided by the present invention, the concentration of the drug is not less than 30 mg / kg.

[0018] Furthermore, in the application provided by the present invention, the concentration of the drug administered is not higher than 110 mg / kg.

[0019] Furthermore, in the applications provided by this invention, the drug has one or more of the following effects:

[0020] Inhibits the replication of white spot syndrome virus in aquatic animals;

[0021] Reduce mortality in aquatic animals caused by white spot syndrome virus;

[0022] Preventing infection of aquatic animals with white spot syndrome virus.

[0023] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0024] This invention reveals for the first time that the natural flavonoid compound farnesin possesses strong inhibitory activity against White Spot Syndrome Virus (WSSV), filling a gap in its application in the field of aquatic virus prevention and control. Rigorous in vivo experiments demonstrated that farnesin effectively inhibits WSSV replication in *Procambarus clarkii*, achieving inhibition rates of 71.18% and 83.09% at concentrations of 40 mg / kg and 80 mg / kg, respectively. Simultaneously, it significantly improved the survival rate of infected individuals, reaching 21% and 35%, respectively, far exceeding the 2% of the virus control group. As a structurally defined single compound, farnesin overcomes the shortcomings of traditional plant crude extracts, such as complex composition and difficulty in quality control. Furthermore, its natural source avoids the problems of drug resistance and ecotoxicity associated with chemically synthesized drugs. Safety evaluation clarified the safe usage concentration range of 30–90 mg / kg, providing a reliable basis for practical application. This invention provides a novel, highly efficient, safe, and quality-controllable antiviral drug for aquaculture, which is of great significance for promoting green and healthy aquaculture. It also provides important technical reference and development prospects for the application of farnesin in the prevention and control of viruses in other aquatic animals. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is an absolute quantitative standard curve.

[0027] Figure 2 The inhibition rate of WSSV replication copy number in gill tissue after injection of 40 mg / kg or 80 mg / kg concentrations for 24 hours.

[0028] Figure 3The cumulative survival rate of *Procambarus clarkii* under different treatments was statistically analyzed using the Kaplan-Meier method. TM buffer represents the treatment group injected with TM buffer only; WSSV represents the treatment group injected with WSSV virus; WSSV-farnesin (80 mg / kg) represents the treatment group injected with both WSSV virus and 80 mg / kg farnesin; WSSV-farnesin (40 mg / kg) represents the treatment group injected with both WSSV virus and 40 mg / kg farnesin. Detailed Implementation

[0029] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0030] Example 1

[0031] This embodiment provides an assay for the antiviral activity of acacia extract against vitiligo syndrome.

[0032] (1) Test materials

[0033] Viral material: White spot syndrome virus was provided by the Aquatic Diseases Laboratory of Northwest A&F University.

[0034] Preparation of the test solution: Accurately weigh the acacia oleracea standard and prepare a standard solution with a concentration of 100 mg / mL using chromatographic grade DMSO. Then dilute it with DMSO to different concentrations of 10 mg / mL, 15 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 60 mg / mL, and 80 mg / mL. Filter the solution using a 0.22 μm organic syringe filter and transfer it to a brown chromatographic bottle for use as a standard dilution.

[0035] Experimental animals: The red swamp crayfish used in the experiment were purchased from the Xianyang Aquatic Products Market in Shaanxi Province.

[0036] (2) Safety evaluation

[0037] Before conducting antiviral activity assays, a safety evaluation of farnesin in Procambarus clarkii was carried out.

[0038] Healthy red swamp crayfish were randomly selected and divided into groups of 12, placed in plastic tanks at a water temperature of 24–27°C for experimental treatment. A blank control group and drug-treated groups were included. The blank control group received only TM buffer (100 mM Tris-HCl, 10 mM MgCl2, pH=7.5), while the drug-treated groups received injections of different concentrations of farnesin. The tested concentrations of farnesin were: 30 mg / kg, 50 mg / kg, 70 mg / kg, 90 mg / kg, 110 mg / kg, 130 mg / kg, and 150 mg / kg. The injection volume was 100 μL per crayfish. After injection, the survival status of the crayfish was observed and recorded for 72 hours.

[0039] Safety evaluation results showed that within the tested time (72 h) and concentration range (30–90 mg / kg), farnesin was safe for *Procambarus clarkii*, and the crayfish did not exhibit any adverse symptoms. Within the tested time (72 h) and concentration range (110–150 mg / kg), 1–6 crayfish died in each group. Therefore, the safe concentration for the use of farnesin was determined to be higher than 90 mg / kg but not higher than 110 mg / kg. Subsequent experiments will use farnesin concentrations lower than 90 mg / kg on *Procambarus clarkii*.

[0040] (3) Antiviral activity detection

[0041] Healthy red swamp crayfish were randomly selected and divided into three groups (WSSV treatment group, a combination of farnesin and WSSV treatment group, and a blank control group), with 25 crayfish in each group and three parallel experiments. The experiment was conducted in culture water at 25℃. The tested concentrations of farnesin were 40 mg / kg and 80 mg / kg. Each red swamp crayfish was injected with 100 μL of the treatment solution. The drug treatment method was immediate abdominal injection after premixing WSSV virus and the monomeric compound (6.9 × 10⁻⁶ virus doses per crayfish). 7 (Copies, premixed: the virus solution and the drug solution were mixed at 25℃ and injected immediately). After 24 hours, 5 red swamp crayfish were randomly selected from each treatment group, and gill tissue was collected and stored at -80℃ for later use.

[0042] The collected samples were used for absolute quantification of WSSV genomic DNA copy number. The viral genome copy number in the gill tissue of the treated samples was detected using real-time PCR. DNA was extracted from the collected tissue samples using a marine animal tissue genomic DNA extraction kit (Beijing, Tiangen). The concentration and purity of the extracted DNA were detected using a micro-spectrophotometer, and the DNA concentration was then uniformly adjusted to 50 ng / μL. RT-qPCR was then used to construct... Figure 1 The absolute quantitative standard curve shown is used to quantify the viral copy number in tissues.

[0043] DNA was extracted from the gill tissue of *Procambarus clarkii* according to the instructions of the extraction kit, and real-time PCR quantification was performed using a CFX-96 PCR machine. The quantification primer used was VP28-141, and the primer sequences are shown in Table 1.

[0044] Table 1: Sequence of primer VP28-141

[0045]

[0046] The quantitative reagent kit uses Vazyme's AceQ ® qPCR SYBR ® The quantitative reaction system of Green Master Mix is ​​shown in Table 2.

[0047] Table 2: RT-qPCR reaction system

[0048]

[0049] The reaction procedure is shown in Table 3.

[0050] Table 3: RT-qPCR reaction procedure

[0051]

[0052] (4) Survival rate detection

[0053] After determining the safe concentration, the protective effect of farnesin on red swamp crayfish was tested in vivo. Healthy red swamp crayfish were randomly divided into three groups of 100 each, and injected with TM buffer (control group), TM-WSSV mixture (positive control group), and drug-WSSV mixture (treatment groups, 40 mg / kg and 80 mg / kg), respectively. Red swamp crayfish in each group were observed for 10 days, and mortality was recorded. The survival rate was calculated using the formula: Survival rate (%) = (Number of surviving crayfish in the treatment group / Total number in each group) × 100%.

[0054] Depend on Figure 2 It was found that different concentrations of farnesin (40 mg / kg and 80 mg / kg) significantly reduced the copy number of WSSV in Procambarus clarkii and had good anti-WSSV biological activity, with inhibition rates of 71.18% and 83.09%, respectively.

[0055] Depend on Figure 3It was found that farnesin significantly reduced the mortality rate of *Procambarus clarkii* infected with WSSV. During the experiment, no deaths or only a small number of deaths were observed in *Procambarus clarkii* treated with TM buffer. *Procambarus clarkii* treated with WSSV showed a large mortality rate, with a survival rate of 2% at 9 days. Treatment with different concentrations of farnesin (40 and 80 mg / kg) improved the survival rate of diseased *Procambarus clarkii*, reaching 21% and 35%, respectively.

[0056] Depend on Figure 2 and Figure 3 It can be seen that farnesin can significantly inhibit the replication of WSSV in Procambarus clarkii and at the same time improve the survival rate of WSSV-infected Procambarus clarkii.

[0057] In summary, the results of the embodiments of this invention fully demonstrate that farnesin can significantly reduce the expression of WSSV replication-related genes, thereby inhibiting WSSV replication in *Procambarus clarkii* and improving the survival rate of virus-infected hosts. These embodiments provide a theoretical basis for developing farnesin as a highly effective and practical anti-WSSV drug, and also provide an important reference for research on the antiviral effects of farnesin on other aquatic animal viruses.

[0058] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. Application of farnesin as the sole active ingredient in the preparation of drugs for the prevention and treatment of vitiligo syndrome.

2. The application according to claim 1, characterized in that, The pathogen of the vitiligo syndrome is the White Spot Syndrome Virus.

3. The application according to claim 1, characterized in that, The active ingredient of the drug is farnesin or a pharmaceutically acceptable salt thereof.

4. The application according to claim 1, characterized in that, The drug includes pharmaceutical excipients.

5. The application according to claim 1, characterized in that, The drug is applied to aquatic animals or their habitats.

6. The application according to claim 5, characterized in that, The aquatic animal in question is a shrimp.

7. The application according to claim 1, characterized in that, The drug is administered by injection.

8. The application according to claim 7, characterized in that, The concentration of the drug administered shall not be less than 30 mg / kg.

9. The application according to claim 7, characterized in that, The concentration of the drug administered shall not exceed 110 mg / kg.

10. The application according to claim 1, characterized in that, The drug has one or more of the following effects: Inhibits the replication of white spot syndrome virus in aquatic animals; Reduce mortality in aquatic animals caused by white spot syndrome virus; Preventing infection of aquatic animals with white spot syndrome virus.

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