Application of quercetin in resisting bombyx mori nuclear polyhedrosis virus

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of quercetin or pharmaceutically acceptable salt thereof in preparation of a bombyx mori nuclear polyhedrosis virus resisting product. The invention firstly discloses that quercetin or pharmaceutically acceptable salt thereof can be used as a medicine for inhibiting proliferation of the bombyx mori nuclear polyhedrosis virus or preventing virus infection, and develops a new direction for treating or preventing diseases related to bombyx mori nuclear polyhedrosis virus infection in future.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of quercetin in resisting Bombyx mori nuclear polyhedrosis virus. Background Art

[0002] Bombyx mori septicemia is the most serious virus disease in current sericulture production. Every year, the occurrence of the disease causes significant economic losses to the sericulture industry. Its pathogen is the nuclear polyhedrosis virus of Bombyx mori, and the main transmission routes are ingestion infection through mulberry leaves and wound infection. This silkworm disease occurs throughout the year, and is most serious during high-temperature periods. The time from infection to onset is about 3 to 6 days. "Prevention first, comprehensive control" is an important principle for controlling Bombyx mori septicemia. However, at present, no specific medicine for treating Bombyx mori septicemia has been found, and there is an urgent need for new drugs against nuclear polyhedrosis virus to be put on the market.

[0003] Quercetin (QR), with the chemical name of 3,3′,4′,5,6-pentahydroxyflavone, also known as quercetagetin; quercetoflavone, etc. Quercetin widely exists in many plants, mainly in the form of glycosides, and can be obtained by acid hydrolysis. The molecular formula of this compound is C 15 H 10 O 7 , with a molecular weight of 302.24, CAS registration number: 117-39-5, and its chemical structure is shown in formula (Ⅰ).

[0004]

[0005] The main pharmacological effects reported in the existing literature are: anti-tumor effect: exerting anti-cancer properties by inhibiting the growth and proliferation of tumor cells, inducing apoptosis of tumor cells, and reversing tumor multi-drug resistance. Antioxidant effect: Quercetin can directly scavenge reactive oxygen free radicals, and can also play an antioxidant role by acting on oxidation-related enzymes; in addition, quercetin can chelate metal ions, thereby inhibiting the catalytic role of metal ions in the process of free radical generation. Anti-inflammatory effect: Quercetin can inhibit the expression of inflammatory factors and inflammation-related enzymes. Research shows that quercetin can reduce the expression of inflammatory factors such as interleukin-1, interleukin-6, and interleukin-10, thereby achieving an anti-inflammatory effect. In addition, quercetin produces an anti-inflammatory effect by inhibiting the expression of inducible nitric oxide synthase, cyclooxygenase 2, and C-reactive protein at the gene transcription level and protein translation level. Antibacterial effect: Quercetin is a plant-derived bactericide and has an inhibitory effect on a variety of bacteria, such as Escherichia coli, Staphylococcus aureus, etc. In addition, quercetin also has cardiovascular protection, blood glucose regulation, and immunosuppressive effects.

[0006] It can be seen that quercetin has various pharmacological effects such as anti-tumor, anti-inflammatory and anti-bacterial infection, etc., but there are few studies and applications in the aspect of anti-virus; due to quercetin can regulate the body's immune response and antioxidant effect, it is often used in combination with other drugs to enhance the drug efficacy and reduce related side effects. At present, there is no study on quercetin as a drug for preparing anti-Bombyx mori nuclear polyhedrosis virus, and there is no report on its use as a broad-spectrum anti-Bombyx mori nuclear polyhedrosis virus drug. Summary of the Invention

[0007] At present, there is no specific drug for treating the septicemia of Bombyx mori in the market. When Bombyx mori is infected with the virus, measures such as "eliminating the source of transmission" are often taken, that is, cleaning the infected diseased silkworms in time to cut off the transmission of the virus; however, there are some drawbacks such as untimely cleaning of diseased silkworms, heavy workload of the breeders and helplessness in dealing with the already infected silkworms. The purpose of the present invention is to propose a new use of quercetin in the application of preparing a drug for inhibiting virus proliferation or preventing virus infection. The anti-Bombyx mori nuclear polyhedrosis virus effect of quercetin was evaluated at the cellular and in vivo levels, laying a good foundation for its further development and application.

[0008] The purpose of the first aspect of the present invention is to provide the application of quercetin or its pharmaceutically acceptable salt in the preparation of a product for anti-Bombyx mori nuclear polyhedrosis virus.

[0009] The purpose of the second aspect of the present invention is to provide the application of quercetin or its pharmaceutically acceptable salt in inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus or in the preparation of a product for inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus.

[0010] The purpose of the third aspect of the present invention is to provide the application of quercetin or its pharmaceutically acceptable salt in the preparation of a product for treating or preventing Bombyx mori nuclear polyhedrosis virus infection.

[0011] The purpose of the fourth aspect of the present invention is to provide a method for inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus for non-therapeutic purposes in vitro.

[0012] In order to achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0013] The first aspect of the present invention provides the application of quercetin or its pharmaceutically acceptable salt in the preparation of a product for anti-Bombyx mori nuclear polyhedrosis virus.

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

[0015] In some embodiments of the present invention, the metal salts include 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 the organic base includes the salt formed with at least one of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine.

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

[0020] In some embodiments of the present invention, the salt formed with the organic acid includes the salt formed with 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, p-toluenesulfonic acid.

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

[0022] In some embodiments of the present invention, the salt formed with the acidic amino acid includes the salt formed with at least one of the following acidic amino acids: aspartic acid, 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 drug includes a pharmaceutically acceptable excipient, and / or any one or more other active ingredients.

[0025] In some embodiments of the present invention, the pharmaceutically acceptable excipient includes at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an antiadhesive, a chelating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickening agent, a clathrant, a humectant, an absorbent, a diluent, a flocculant and an anti-flocculant, a filter aid, a release retardant, a carrier.

[0026] In some embodiments of the present invention, for the convenience of drug administration, the active ingredient quercetin or its pharmaceutically acceptable salts can be processed into specific dosage forms together with any one or several pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hypromellose, povidone, starch paste, and syrup, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and cross-linked povidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and colloidal silicon dioxide, etc.), coloring agents (such as titanium dioxide, sunset yellow, methylene blue, and medicinal ferric oxide, etc.), coating materials (such as acrylic resin, hypromellose, and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol, and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate, etc.), bacteriostatic agents (such as phenol, benzyl alcohol, and thimerosal, etc.), or can also be isotonicity regulators (such as sodium chloride and glucose, etc.).

[0027] In some embodiments of the present invention, the dosage forms of the product include gastrointestinal dosage forms or parenteral dosage forms.

[0028] In some embodiments of the present invention, the gastrointestinal dosage forms include at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[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, scored tablets, sustained-release and controlled-release dosage forms such as sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches, etc.

[0030] In some embodiments of the present invention, the parenteral dosage forms include at least one of injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0031] In some embodiments of the present invention, the injection dosage forms include, but are not limited to, injection solutions, injection solutions for injection, intravenous drip injection solutions, injection suspensions, sterile powders for injection, intravenous injection needles, aqueous injections, injection emulsions, powder injections, injections, sterile powder injections, and freeze-dried 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] The second aspect of the present invention provides the use of quercetin or its pharmaceutically acceptable salt in inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus or in preparing a product for inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus.

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

[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 drug 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 solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, carriers.

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

[0040] In some embodiments of the present invention, the gastrointestinal administration dosage form includes at least one of powders, tablets, granules, capsules, sustained - release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets.

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

[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] The third aspect of the present invention provides the use of quercetin or its pharmaceutically acceptable salt in the preparation of a product for treating or preventing Bombyx mori nucleopolyhedrovirus infection.

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

[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 drug 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 solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickening agents, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, carriers.

[0049] In some embodiments of the present invention, the dosage form of the drug includes an oral dosage form or a parenteral dosage form.

[0050] In some embodiments of the present invention, the oral dosage form includes at least one of powders, tablets, granules, capsules, sustained release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, chewable tablets.

[0051] In some embodiments of the present invention, the parenteral dosage form includes at least one of injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[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] The fourth aspect of the present invention provides a method for inhibiting the proliferation / replication of Bombyx mori nucleopolyhedrovirus for non - therapeutic purposes in vitro, including the step of treating cells with quercetin or its pharmaceutically acceptable salt.

[0055] In some embodiments of the present invention, during the treatment process, 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 treatment process, the effective dose of quercetin or its pharmaceutically acceptable salt is 60 - 150 μg / g; preferably 90 - 110 μg / g.

[0057] The beneficial effects of the present invention are as follows:

[0058] The present invention discloses for the first time that quercetin or its pharmaceutically acceptable salt can be used as a drug to inhibit the proliferation of Bombyx mori nucleopolyhedrovirus or prevent virus infection, opening up a new direction for the treatment or prevention of diseases related to Bombyx mori nucleopolyhedrovirus infection in the future. Specifically as follows:

[0059] After infecting cells with Bombyx mori nucleopolyhedrovirus, the present invention detected the virus level after treating the cells with quercetin at different concentrations and observed the changes in the virus level at different time points after treating the virus - infected cells with quercetin. The results showed that 10 μM quercetin could significantly reduce the replication level after Bombyx mori nucleopolyhedrovirus infection, and quercetin showed good anti - Bombyx mori nucleopolyhedrovirus effects in the early, middle, and late stages (12 - 48 hours) of virus proliferation; in addition, quercetin not only had an inhibitory effect after virus infection, but also played an anti - Bombyx mori nucleopolyhedrovirus role before virus infection and during the adsorption process, indicating that the drug also had a certain preventive effect.

[0060] Quercetin not only inhibits the replication of Bombyx mori nucleopolyhedrovirus at the cellular level, but also has the same effect at the in - vivo level; treating silkworms with nucleopolyhedrovirus and quercetin, and through the survival curve, it can be seen that quercetin has a certain protective effect on virus - infected silkworms. Therefore, quercetin in the present invention has important application value and research significance in anti - Bombyx mori nucleopolyhedrovirus. Brief Description of the Drawings

[0061] Figure 1 For detecting the gene level of BmNPV 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 For detecting the protein level of BmNPV after treatment with different concentrations of quercetin by WB.

[0063] Figure 3 For detecting the gene level of BmNPV at different time points after treatment with quercetin by RT-PCR. In the figure, *** represents p < 0.001.

[0064] Figure 4 For detecting the protein level of BmNPV at different time points after treatment with quercetin by WB.

[0065] Figure 5 For the CC 50 determination results of quercetin.

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

[0067] Figure 7 For detecting the gene level of BmNPV after 1-hour pretreatment with quercetin by RT-PCR. In the figure, *** represents p < 0.001.

[0068] Figure 8 For detecting the protein level of BmNPV after simultaneous treatment with quercetin and virus by WB.

[0069] Figure 9 For detecting the protein level of BmNPV after 1-hour pretreatment with quercetin by WB.

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

[0071] Figure 11 For detecting the protein level of BmNPV at different time points after treatment with quercetin at the in vivo level.

[0072] Figure 12 For the survival curve of silkworms infected with BmNPV under the action of quercetin. Specific embodiments

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

[0074] It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present 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. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

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

[0077] Example 1 Detecting the anti-Bombyx mori nucleopolyhedrovirus effect of quercetin at the cellular level

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

[0079] After the Bombyx mori nucleopolyhedrovirus infects the cells, the virus level is detected after treating the cells with quercetin at different concentrations. The process is as follows:

[0080] Inoculate Bombyx mori ovary (BmN) cells into a 12-well plate, and the medium used is an insect medium (purchased from Thermo Fisher Scientific, USA) containing 10 v / v% fetal bovine serum (purchased from AusGeneX, Australia). When the cell density reaches 70% - 80%, add Bombyx mori nucleopolyhedrovirus (BmNPV) with a green fluorescent label to each well, with a multiplicity of infection (MOI) of 5, incubate in a 28°C incubator for 1 hour, remove the old medium and wash once with phosphate buffer (PBS), add fresh serum-containing medium, and continue to culture the cells for 12 hours. Dissolve and dilute the quercetin powder (purity 95%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) with anhydrous dimethyl sulfoxide (DMSO). Add quercetin solutions at different concentrations to the medium to make their working concentrations 500 pM, 1 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM respectively. The control group is added with a DMSO solution without quercetin, and the cells continue to grow in a 28°C constant temperature incubator for 36 hours. Remove the medium and wash 3 times with PBS, then resuspend the cells with PBS and transfer them to a centrifuge tube, centrifuge at 3000 rpm for 5 minutes, and discard the supernatant.

[0081] Real-time fluorescence quantitative PCR (RT-PCR) is used to detect the virus proliferation level: Extract the genomic DNA of the cells according to the genomic DNA extraction kit instructions, measure and adjust the DNA concentration, mix the template, primers, and quantitative PCR amplification premix evenly, and then place them in a quantitative PCR instrument to detect the replication levels of the virus genes (ie1 and gp64) and the internal reference gene (GAPDH) in the samples according to the set program.

[0082] The virus proliferation level was detected by Western blotting (WB): The cell pellet was resuspended with lysis buffer containing protease inhibitor, lysed on ice for 15 - 30 minutes, centrifuged at 12000 rpm for 15 minutes at 4°C, the supernatant was taken and protein loading buffer was added, boiled at 100°C for 10 minutes, after briefly centrifuging the sample, polyacrylamide gel electrophoresis was carried out, then the proteins in the gel were transferred to a polyvinylidene fluoride membrane, blocked with milk, and then incubated with the corresponding primary antibody and secondary antibody in sequence, and the protein bands were exposed using a hypersensitive luminescence solution.

[0083] The RT-PCR detection results were as Figure 1 shown. There was a concentration-dependent relationship between the inhibitory effect of quercetin on Bombyx mori nucleopolyhedrovirus. Low-concentration quercetin (≤5 μM) had no significant inhibitory effect on the virus. 10 μM quercetin could significantly reduce the DNA level of BmNPV, and almost no BmNPV could be detected after treatment with 20 μM quercetin.

[0084] The WB detection results were as Figure 2 shown. As the concentration of quercetin gradually increased, the virus band (EGFP) weakened. The band of 10 μM quercetin was significantly reduced, and almost no band could be detected with 20 μM quercetin.

[0085] In addition, cells infected with the virus were treated with quercetin, and the changes in virus levels at different time points were observed. The inhibitory effect of quercetin on Bombyx mori nucleopolyhedrovirus was preliminarily analyzed by detecting the virus level, as follows:

[0086] BmN cells were seeded in 12-well plates. When the cell density reached 70% - 80%, BmNPV (MOI = 5) was added to the medium. After incubating at 28°C for 1 hour, the virus was removed, fresh medium was added and the cells were cultured for another 12 hours. A 10 μM quercetin solution was added to the cell medium, and the cells were collected at 12 hours, 24 hours, and 48 hours respectively. The virus proliferation level was detected by RT-PCR and WB (the same RT-PCR and WB detection procedures as above).

[0087] The RT-PCR detection results were as Figure 3 shown. Quercetin showed an obvious inhibitory effect on different replication stages of Bombyx mori nucleopolyhedrovirus. The virus gene levels at each time point after quercetin treatment were significantly lower than those of the control group. The WB detection results were as Figure 4 shown. The virus protein levels at each time point after quercetin treatment were significantly weaker than those of the control group.

[0088] The safety of quercetin was investigated by CCK8 cell experiment. The results showed that the CC 50 of quercetin was 68.36 μM, which was lower than the effective concentration (10 μM) for inhibiting the virus ( Figure 5 ), indicating that quercetin had good safety.

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

[0090] The virus replication cycle includes different stages such as adsorption, penetration, biosynthesis, and release. Quercetin was used to treat different links of virus replication, and the antiviral effects of quercetin on different stages of the virus cycle were analyzed by detecting the virus replication level, as follows:

[0091] BmN cells were inoculated into 12-well plates, and the cells were grown in an incubator at 28 °C until their density reached 70% - 80%. According to the characteristics of the virus replication cycle, the drug treatment methods were divided into two types; the first treatment method: first, add 10 μM quercetin solution to the medium, incubate at 28 °C for 1 hour, then remove the quercetin and wash once with PBS, and then add cell medium containing BmNPV (MOI = 5). After incubating at 28 °C for 1 hour, remove the virus and wash once, and continue to culture for 36 hours before collecting the cells. The second treatment method: mix BmNPV (MOI = 5) and 10 μM quercetin and incubate at 4 °C for 1 hour, then add the mixed solution to the medium, incubate at 28 °C for 1 hour, replace with fresh medium, and continue to culture for 36 hours before collecting the cells. RT-PCR and WB were used to detect the virus proliferation level (the same RT-PCR and WB detection procedures as in (1)).

[0092] The RT-PCR detection results are as Figures 6 - 7 shown. When quercetin and the virus were added to the cells simultaneously, the gene level of the virus decreased significantly ( Figure 6 ); when the cells were pretreated with quercetin for 1 hour and then the virus was added, the gene level of the virus also decreased significantly ( Figure 7 ). WB was used to detect the virus proliferation level, and the results are as Figures 8 - 9 shown. When quercetin and the virus were added simultaneously, the virus band decreased significantly ( Figure 8 ); when quercetin was added first and then the virus was added, the band also decreased significantly ( Figure 9 ). This indicates that quercetin can produce inhibitory effects on different stages of the replication of Bombyx mori nucleopolyhedrovirus, that is, quercetin can not only prevent the adsorption of BmNPV to cells, but also has a certain effect on the prevention of BmNPV.

[0093] Example 2 Analyze the anti-Bombyx mori nucleopolyhedrovirus effect of quercetin at the in vivo level

[0094] (1) The role of quercetin in inhibiting virus proliferation in Bombyx mori

[0095] Bombyx mori was treated with Bombyx mori nucleopolyhedrovirus and quercetin, and the antiviral effect of quercetin in vivo was analyzed by detecting the virus level in the fat body, as follows:

[0096] Fifth-instar day-2 P50 silkworms were randomly divided into 4 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 per silkworm) using a micro syringe, and the other two groups were injected with the same volume of cell culture medium. 24 hours after virus injection, the control + quercetin group and the virus + quercetin group were injected with quercetin solution (6 μg per silkworm), and the other two groups were injected with the same volume of DMSO solution. Silkworm fat body tissues were collected at 24 hours, 48 hours, and 72 hours after virus injection. RT-PCR and WB were used to detect the virus proliferation level (the detection process was the same as in Example 1).

[0097] The RT-PCR detection results are as Figure 10 shown. After quercetin injection, the BmNPV gene level in the silkworm fat body was significantly reduced; the WB detection results are as Figure 11 shown. After quercetin injection, the BmNPV protein level in the fat body was also significantly reduced. The above results indicate that quercetin also has an inhibitory effect on BmNPV proliferation in silkworms.

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

[0099] Virus infection and drug administration were carried out by feeding mulberry leaves, and the death indexes of silkworms were recorded to analyze the protective effect of quercetin on silkworms after infection with Bombyx mori nucleopolyhedrovirus, as follows:

[0100] Fifth-instar day-1 P50 silkworms were randomly divided into 4 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 the occlusion-derived virions (ODV) of Bombyx mori nucleopolyhedrovirus by feeding mulberry leaves, and the virus dose was 5.46×10 9 per silkworm; the same volume of PBS solution was used as a control treatment. Quercetin dissolved in DMSO was diluted with 50% ethanol. On the second day after virus challenge, drug administration was carried out by feeding mulberry leaves, and the drug dose was 100 μg / g of silkworm weight (i.e., 100 micrograms of the drug were fed to each gram of silkworm), once a day until the silkworms died; the same volume of drug-free solution (DMSO) was used as a control treatment, and the death of silkworms in each group was recorded daily starting from drug administration.

[0101] The results are as Figure 12 shown. On the 4th day after drug administration, all the silkworms in the BmNPV + DMSO group died; while some of the silkworms in the BmNPV + QR group still survived and all died until the 8th day after drug administration, significantly prolonging the survival time of the silkworms, indicating 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 in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of quercetin or a pharmaceutically acceptable salt thereof in the preparation of a product resistant to Bombyx mori nuclear polyhedrosis virus.

2. Use of quercetin or a pharmaceutically acceptable salt thereof in inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus or in preparing a product for inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus.

3. Use of quercetin or a pharmaceutically acceptable salt thereof in the preparation of a product for treating or preventing Bombyx mori nuclear polyhedrosis virus infection.

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 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.

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

6. The use according to claim 5, characterized in that: The drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

8. The use according to claim 5, characterized in that: The dosage form of the drug includes a dosage form for gastrointestinal administration and a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

9. An in vitro method for inhibiting the proliferation of Bombyx mori nuclear polyhedrosis virus for non-therapeutic purposes, comprising the step of treating cells with quercetin or a pharmaceutically acceptable salt thereof.

10. The method according to claim 9, characterized in that During the treatment, the effective concentration of quercetin or a pharmaceutically acceptable salt thereof is 10-50 μM.

Images