Application of quercetin in the treatment of silkworm nucleopolyhedrovirus

Abstract

This invention belongs to the field of biomedical technology and discloses the application of quercetin or its pharmaceutically acceptable salts in the preparation of products against silkworm nucleopolyhedrovirus. This invention is the first to disclose that quercetin or its pharmaceutically acceptable salts can be used as drugs to inhibit the proliferation of silkworm nucleopolyhedrovirus or prevent viral infection, opening up new directions for the treatment or prevention of diseases related to silkworm nucleopolyhedrovirus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of quercetin in combating silkworm nucleopolyhedrovirus. Background Technology

[0002] Silkworm hemorrhagic septicemia is currently the most serious viral disease affecting silkworm production, causing significant economic losses to the sericulture industry annually. The pathogen is a nucleopolyhedrovirus (NPV) of the silkworm, primarily transmitted through ingestion of mulberry leaves and wound infection. This disease occurs year-round, but is most severe during periods of high temperatures, with the time from infection to onset of symptoms approximately 3-6 days. "Prevention first, comprehensive control" is a crucial principle for controlling silkworm hemorrhagic septicemia; however, no specific antiviral drug has yet been discovered, and there is an urgent need for new drugs targeting NPV.

[0003] Quercetin (QR), chemically known as 3,3′,4′,5,6-pentahydroxyflavone, is also called quercetin or quercetin flavonoid. It is widely found in many plants, primarily in the form of a glycoside, and can be obtained through acid hydrolysis. The molecular formula of this compound is C1. 15 H 10 O7, molecular weight 302.24, CAS Registry Number: 117-39-5, its chemical structure is shown in formula (Ⅰ).

[0004]

[0005] The main pharmacological effects reported in existing literature include: Antitumor effects: Quercetin exerts its anticancer properties by inhibiting tumor cell growth and proliferation, inducing tumor cell apoptosis, and reversing multidrug resistance in tumors. Antioxidant effects: Quercetin can directly scavenge reactive oxygen species (ROS) and also exert its antioxidant effects by acting on oxidation-related enzymes; in addition, quercetin can chelate metal ions, thereby inhibiting the catalytic role of metal ions in the generation of free radicals. Anti-inflammatory effects: Quercetin can inhibit the expression of inflammatory factors and inflammation-related enzymes. Studies have shown that quercetin can reduce the expression of inflammatory factors such as interleukin-1, interleukin-6, and interleukin-10, thereby achieving an anti-inflammatory effect. Furthermore, quercetin produces anti-inflammatory effects by inhibiting the expression of inducible nitric oxide synthase, cyclooxygenase 2, and C-reactive protein at the gene transcription and protein translation levels. Antibacterial effects: Quercetin is a plant-derived bactericide with inhibitory effects on various bacteria, such as Escherichia coli and Staphylococcus aureus. In addition, quercetin also has cardiovascular protective, blood sugar regulating, and immunosuppressive effects.

[0006] It is evident that quercetin possesses various pharmacological effects, including antitumor, anti-inflammatory, and antibacterial activity; however, its research and application in antiviral applications are limited. Because quercetin can regulate the body's immune response and has antioxidant effects, it is often used in combination with other drugs to enhance efficacy and reduce related side effects. Currently, there are no studies on quercetin as a drug for preparing anti-bombyxovirus agents, and even fewer reports on its use as a broad-spectrum anti-bombyxovirus agent. Summary of the Invention

[0007] Currently, there is no specific drug on the market for treating hemorrhagic septicemia in silkworms. When silkworms are infected with the virus, the common approach is to "eliminate the source of transmission," that is, to promptly remove infected silkworms to cut off the spread of the virus. However, this approach has some drawbacks, such as the untimely removal of infected silkworms, the heavy workload of the caretakers, and the lack of effective treatment for already infected silkworms. This invention aims to propose a new use for quercetin in the preparation of drugs that inhibit viral proliferation or prevent viral infection. The antiviral activity of quercetin against silkworm nucleopolyhedrovirus was evaluated at both cellular and in vivo levels, laying a good foundation for its further development and application.

[0008] The first aspect of this invention aims to provide the use of quercetin or a pharmaceutically acceptable salt thereof in the preparation of products against silkworm nucleopolyhedrovirus.

[0009] A second aspect of the present invention aims to provide the use of quercetin or a pharmaceutically acceptable salt thereof in inhibiting the proliferation of silkworm nucleopolyhedrovirus or in the preparation of products that inhibit the proliferation of silkworm nucleopolyhedrovirus.

[0010] A third aspect of the present invention aims to provide the use of quercetin or a pharmaceutically acceptable salt thereof in the preparation of products for the treatment or prevention of silkworm nucleopolyhedrovirus infection.

[0011] The fourth aspect of this invention aims to provide a method for inhibiting the proliferation of silkworm nucleopolyhedrovirus in vitro for non-therapeutic purposes.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] A first aspect of the invention provides the use of quercetin or a pharmaceutically acceptable salt thereof in the preparation of a product for treating silkworm nucleopolyhedrovirus.

[0014] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0015] In some embodiments of the present invention, the metal salt includes alkali metal salts and alkaline earth metal salts.

[0016] In some embodiments of the present invention, the alkali metal salt includes at least one of sodium salt and potassium salt.

[0017] In some embodiments of the present invention, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt, and aluminum salt.

[0018] In some embodiments of the present invention, the salt formed with an organic base includes at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0019] In some embodiments of the present invention, the salt formed with the inorganic acid includes at least one of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0020] In some embodiments of the present invention, the salt formed with the organic acid includes at least one of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0021] In some embodiments of the present invention, the salt formed with the basic amino acid includes at least one of the following basic amino acids: arginine, lysine, and ornithine.

[0022] In some embodiments of the present invention, the salt formed with the acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid and glutamic acid.

[0023] In some embodiments of the present invention, the product includes a reagent or a drug.

[0024] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients and / or any one or more other active ingredients.

[0025] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0026] In some embodiments of the present invention, for ease of administration, the active ingredient quercetin or a pharmaceutically acceptable salt thereof may be processed with one or more pharmaceutically acceptable excipients into a specific dosage form. These excipients may be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base adjusters (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic adjusters (such as sodium chloride and glucose).

[0027] In some embodiments of the present invention, the dosage form of the product includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0028] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0029] In some embodiments of the present invention, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0030] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0031] In some embodiments of the present invention, the injectable dosage forms include, but are not limited to, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0032] In some embodiments of the present invention, the effective concentration of quercetin or its pharmaceutically acceptable salt in the product is 10–50 μM; preferably 10–20 μM.

[0033] In some embodiments of the present invention, the effective dose of quercetin or its pharmaceutically acceptable salt in the product is 60–150 μg / g; preferably 90–110 μg / g.

[0034] A second aspect of the invention provides the use of quercetin or a pharmaceutically acceptable salt thereof in inhibiting the proliferation of silkworm nucleopolyhedrovirus or in the preparation of products that inhibit the proliferation of silkworm nucleopolyhedrovirus.

[0035] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0036] In some embodiments of the present invention, the product includes a reagent or a drug.

[0037] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients and / or any one or more other active ingredients.

[0038] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0039] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0040] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0041] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0042] In some embodiments of the present invention, the effective concentration of quercetin or its pharmaceutically acceptable salt in the product is 10–50 μM; preferably 10–20 μM.

[0043] In some embodiments of the present invention, the effective dose of quercetin or its pharmaceutically acceptable salt in the product is 60–150 μg / g; preferably 90–110 μg / g.

[0044] A third aspect of the invention provides the use of quercetin or a pharmaceutically acceptable salt thereof in the preparation of products for the treatment or prevention of silkworm nucleopolyhedrovirus infection.

[0045] In some embodiments of the present invention, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0046] In some embodiments of the present invention, the product includes a reagent or a drug.

[0047] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients and / or any one or more other active ingredients.

[0048] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0049] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0050] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0051] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0052] In some embodiments of the present invention, the effective concentration of quercetin or its pharmaceutically acceptable salt in the product is 10–50 μM; preferably 10–20 μM.

[0053] In some embodiments of the present invention, the effective dose of quercetin or its pharmaceutically acceptable salt in the product is 60–150 μg / g; preferably 90–110 μg / g.

[0054] A fourth aspect of the present invention provides a method for inhibiting the proliferation / replication of silkworm nucleopolyhedrovirus in vitro for non-therapeutic purposes, comprising the step of treating cells with quercetin or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments of the present invention, during the processing, the effective concentration of quercetin or its pharmaceutically acceptable salt is 10–50 μM; preferably 10–20 μM.

[0056] In some embodiments of the present invention, during the processing, the effective dose of quercetin or a pharmaceutically acceptable salt thereof is 60–150 μg / g; preferably 90–110 μg / g.

[0057] The beneficial effects of this invention are:

[0058] This invention discloses for the first time that quercetin or its pharmaceutically acceptable salts can be used as drugs to inhibit the proliferation of silkworm nucleopolyhedrovirus or prevent viral infection, opening up new directions for the treatment or prevention of silkworm nucleopolyhedrovirus-related diseases. Specifically:

[0059] This invention investigates the effects of quercetin on the viral load after infecting silkworm nucleopolyhedrovirus (BNPV) cells with different concentrations of quercetin. It also observes changes in viral load at different time points after treating infected cells with quercetin. The results show that 10 μM quercetin significantly reduces BNPV replication after infection, and that quercetin exhibits good anti-BNPV effects throughout the early, middle, and late stages of viral proliferation (12–48 hours). Furthermore, quercetin not only inhibits viral infection but also exerts anti-BNPV effects before infection and during viral adsorption, indicating that the drug also has a certain preventative effect.

[0060] Quercetin inhibits the replication of silkworm nucleopolyhedrovirus (MPV) not only at the cellular level but also at the in vivo level. Treatment of silkworms with MPV and quercetin yielded survival curves, demonstrating that quercetin provides some protection against MPV infection. Therefore, the quercetin described in this invention has significant application value and research significance in combating silkworm MPV. Attached Figure Description

[0061] Figure 1 To detect the BmNPV gene level after treatment with different concentrations of quercetin by RT-PCR, in the figure, * represents p < 0.05, and *** represents p < 0.001.

[0062] Figure 2 The protein level of BmNPV was measured by Western blotting after treatment with different concentrations of quercetin.

[0063] Figure 3 To detect the BmNPV gene level at different time points after quercetin treatment by RT-PCR, *** in the figure represents p < 0.001.

[0064] Figure 4 Western blot analysis was performed to detect BmNPV protein levels at different time points after quercetin treatment.

[0065] Figure 5 CC of quercetin 50 Measurement results.

[0066] Figure 6 To detect the BmNPV gene level after simultaneous treatment with quercetin and virus by RT-PCR, ** in the figure represents p < 0.01.

[0067] Figure 7 The BmNPV gene level was detected by RT-PCR 1 hour after quercetin pretreatment. In the figure, *** represents p < 0.001.

[0068] Figure 8 Western blot analysis was performed to detect BmNPV protein levels after simultaneous treatment with quercetin and the virus.

[0069] Figure 9 BmNPV protein levels were detected by Western blotting 1 hour after quercetin pretreatment.

[0070] Figure 10 To detect the BmNPV gene level at different time points after quercetin treatment in vivo, * in the figure represents p < 0.05, and ** represents p < 0.01.

[0071] Figure 11 To detect BmNPV protein levels at different time points after quercetin treatment in vivo.

[0072] Figure 12 Survival curves of silkworms infected with BmNPV under the action of quercetin. Detailed Implementation

[0073] The present invention will be further described in detail below through specific embodiments.

[0074] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0075] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0076] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0077] Example 1: Detection of the antiviral effect of quercetin against silkworm nucleopolyhedrovirus at the cellular level

[0078] (1) Inhibitory effect of different concentrations or durations of quercetin on the virus

[0079] After silkworm nucleopolyhedrovirus (MPV) infected cells, the virus levels were detected after treating the cells with different concentrations of quercetin. The procedure is as follows:

[0080] Silkworm ovary (BmN) cells were seeded into 12-well plates using insect medium (Thermo Fisher Scientific, USA) containing 10 v / v% fetal bovine serum (purchased from AusGeneX, Australia). When the cell density reached 70%–80%, silkworm nucleopolyhedrovirus (BmNPV) with a green fluorescent tag was added to each well at a multiplicity of infection (MOI) of 5. The cells were incubated at 28°C for 1 hour. The old medium was removed, and the cells were washed once with phosphate-buffered saline (PBS). Fresh serum-containing medium was added, and the cells were cultured for another 12 hours. Quercetin powder (95% purity, purchased from Aladdin Biochemical Technology Co., Ltd., Shanghai) was dissolved and diluted with anhydrous dimethyl sulfoxide (DMSO). Different concentrations of quercetin solution were added to the culture medium to achieve working concentrations of 500 pM, 1 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. The control group was treated with DMSO solution without quercetin. Cells were incubated at 28°C for 36 hours. The culture medium was removed, and the cells were washed three times with PBS. The cells were then resuspended in PBS and transferred to centrifuge tubes. The cells were centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded.

[0081] Viral replication levels were detected using real-time quantitative PCR (RT-PCR): The genome of the cells was extracted according to the instructions of the genomic DNA extraction kit, the DNA concentration was measured and adjusted, and the template, primers and quantitative PCR amplification premix were mixed evenly and placed in the quantitative PCR instrument. The replication levels of viral genes (ie1 and gp64) and internal reference gene (GAPDH) in the sample were detected according to the set program.

[0082] The viral proliferation level was detected by Western blotting (WB): the cell pellet was resuspended in lysis buffer containing protease inhibitors, lysed on ice for 15-30 minutes, centrifuged at 12,000 rpm for 15 minutes at 4°C, the supernatant was collected and protein loading buffer was added, boiled at 100°C for 10 minutes, and after a brief separation of the sample, polyacrylamide gel electrophoresis was performed. The proteins in the gel were then transferred to a polyvinylidene fluoride membrane, blocked with milk, and incubated with the corresponding primary and secondary antibodies in sequence. The protein bands were then exposed using a high-sensitivity luminescent solution.

[0083] RT-PCR test results as follows Figure 1 As shown, the inhibitory effect of quercetin on silkworm nucleopolyhedrovirus (BmNPV) is concentration-dependent. Low concentrations of quercetin (≤5 μM) have no significant inhibitory effect on the virus, while 10 μM quercetin can significantly reduce the DNA level of BmNPV, and BmNPV is almost undetectable after treatment with 20 μM quercetin.

[0084] WB test results are as follows Figure 2 As shown, the viral band (EGFP) weakens as the quercetin concentration gradually increases, the band of 10 μM quercetin is significantly reduced, and the band of 20 μM quercetin is almost undetectable.

[0085] In addition, quercetin was used to treat virus-infected cells, and changes in viral levels at different time points were observed. The inhibitory effect of quercetin on silkworm nucleopolyhedrovirus was preliminarily analyzed by detecting viral levels, as detailed below:

[0086] BmN cells were seeded into 12-well plates. When the cell density reached 70%–80%, BmNPV (MOI = 5) was added to the culture medium. After incubation at 28°C for 1 hour, the virus was removed, fresh culture medium was added, and the cells were cultured for another 12 hours. 10 μM quercetin solution was added to the cell culture medium, and cells were collected at 12, 24, and 48 hours. Viral proliferation levels were detected by RT-PCR and Western blotting (using the same procedures as described above).

[0087] RT-PCR test results as follows Figure 3 As shown, quercetin exhibited significant inhibitory effects on the replication of silkworm nucleopolyhedrovirus at different stages, and the viral gene levels at all time points after quercetin treatment were significantly lower than those in the control group. Western blot (WB) results are shown below. Figure 4 As shown, the viral protein levels at each time point after quercetin treatment were significantly lower than those in the control group.

[0088] The safety of quercetin was investigated using CCK8 cell assays. Results showed that quercetin's CCK8 activity was low. 50 = 68.36 μM, lower than the effective concentration for inhibiting the virus (10 μM) ( Figure 5 This indicates that quercetin has a good safety profile.

[0089] (2) Inhibitory effects of quercetin at different stages of the replication cycle

[0090] The viral replication cycle includes different stages such as adsorption, invasion, biosynthesis, and release. Quercetin treatment was applied to different stages of viral replication, and the antiviral effect of quercetin on different stages of the viral cycle was analyzed by detecting the viral replication level, as detailed below:

[0091] BmN cells were seeded into 12-well plates and grown in an incubator at 28°C until the cell density reached 70%–80%. Based on the characteristics of the viral replication cycle, the drug treatment methods were divided into two types: The first treatment method: 10 μM quercetin solution was added to the culture medium, incubated at 28°C for 1 hour, then the quercetin was removed and the cells were washed once with PBS. Then, cell culture medium containing BmNPV (MOI=5) was added, incubated at 28°C for 1 hour, then the virus was removed and the cells were washed once. After culturing for 36 hours, the cells were collected. The second treatment method: BmNPV (MOI=5) and 10 μM quercetin were mixed and incubated at 4°C for 1 hour. Then, the mixed solution was added to the culture medium, incubated at 28°C for 1 hour, then the culture medium was replaced with fresh medium. After culturing for 36 hours, the cells were collected. The viral proliferation level was detected by RT-PCR and WB (same as the RT-PCR and WB detection process in (1)).

[0092] RT-PCR test results as follows Figures 6-7 As shown, when quercetin and virus are added to cells simultaneously, the viral gene level is significantly reduced. Figure 6 ); Adding virus to cells after quercetin pretreatment for 1 hour also significantly reduced the viral gene level. Figure 7 WB detection of viral proliferation level, results as follows Figures 8-9 As shown, when quercetin and the virus were added simultaneously, the viral band was significantly reduced. Figure 8 Adding quercetin first and then the virus resulted in a significant reduction in the band count. Figure 9 This indicates that quercetin can inhibit different stages of BmNPV replication, meaning that quercetin not only prevents BmNPV from adsorbing to cells, but also plays a role in the prevention of BmNPV.

[0093] Example 2: Analysis of the effect of quercetin against silkworm nucleopolyhedrovirus at the in vivo level

[0094] (1) The inhibitory effect of quercetin on viral proliferation in silkworms

[0095] Silkworms were treated with silkworm nucleopolyhedrovirus and quercetin. The antiviral effect of quercetin in vivo was analyzed by detecting the virus level in the fat body, as detailed below:

[0096] Fifth instar, 2-day-old P50 silkworms were randomly divided into four groups (control + DMSO group, control + quercetin group, virus + DMSO group, and virus + quercetin group), with 15 silkworms in each group. First, the virus + DMSO group and the virus + quercetin group were injected with BmNPV (7.5 μL / silkworm) using a microsyringe, while the other two groups were injected with the same volume of cell culture medium. Twenty-four hours after virus injection, the control + quercetin group and the virus + quercetin group were injected with quercetin solution (6 μg / silkworm), while the other two groups were injected with the same volume of DMSO solution. Fat body tissue was harvested from the silkworms at 24, 48, and 72 hours after virus injection. Virus proliferation levels were detected by RT-PCR and Western blotting (the detection procedure was the same as in Example 1).

[0097] RT-PCR test results as follows Figure 10 As shown, injection of quercetin significantly reduced the BmNPV gene level in the fat body of silkworms; Western blot results are as follows. Figure 11 As shown, the BmNPV protein level in the fat body was also significantly reduced after quercetin injection. These results indicate that quercetin also inhibits BmNPV proliferation in silkworms.

[0098] (2) Protective effect of quercetin on silkworms

[0099] By infecting silkworms with mulberry leaves and administering medication, and recording mortality indicators, the protective effect of quercetin on silkworms infected with silkworm nucleopolyhedrovirus was analyzed, as follows:

[0100] Fifth instar, 1-day-old P50 silkworms were randomly divided into four groups (PBS+DMSO group, PBS+QR group, BmNPV+DMSO group, and BmNPV+QR group), with 45 silkworms in each group. On the first day, the silkworms were infected with occlusion-derived viruses (ODV) by ingesting mulberry leaves, with a viral dose of 5.46 × 10⁻⁶. 9 / head; the same volume of PBS solution was used as a control. Quercetin dissolved in DMSO was diluted with 50% ethanol. On the second day after challenge, the drug was administered by feeding mulberry leaves at a dose of 100 μg / g of silkworm weight (i.e., 100 micrograms of drug per gram of silkworm), once a day until the silkworms died; the same volume of drug-free solution (DMSO) was used as a control. The mortality of silkworms in each group was recorded daily from the start of drug administration.

[0101] The results are as follows Figure 12 As shown, on the 4th day after administration, all silkworms in the BmNPV+DMSO group died; while some silkworms in the BmNPV+QR group survived until the 8th day after administration, which significantly prolonged the survival time of silkworms, suggesting that quercetin has a certain protective effect on silkworms infected with BmNPV.

[0102] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of quercetin or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a product against Bombyx mori nuclear polyhedrosis virus; the effective concentration of the quercetin or the pharmaceutically acceptable salt thereof is 10-50 μM.

2. Use of quercetin or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a product for inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus; the effective concentration of the quercetin or the pharmaceutically acceptable salt thereof is 10-50 μM.

3. Use of quercetin or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a product for treating or preventing Bombyx mori nuclear polyhedrosis virus infection; the effective concentration of the quercetin or the pharmaceutically acceptable salt thereof is 10-50 μM.

4. The use according to any one of claims 1 to 3, characterized in that, The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt with an inorganic acid, a salt with an organic base, and a salt with an organic acid.

5. The use according to any one of claims 1 to 3, characterized in that, The product includes a reagent or a drug.

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