Application of dihydromyricetin in preparation of medicine for preventing and / or treating poxvirus infection

Abstract

The invention provides application of dihydromyricetin in preparation of a medicine for preventing and / or treating poxvirus infection. The dihydromyricetin is effectively combined with the bispecific phosphatase coded by the poxvirus and inhibits the in-vitro enzyme activity of the bispecific phosphatase, has an obvious inhibiting effect on the poxvirus, and can be used for preparing the medicine for preventing and / or treating poxvirus infection. Furthermore, a pharmaceutical composition taking dihydromyricetin as an active ingredient can also be used for preparing a medicine for preventing and / or treating poxvirus infection.

Description

Technical Field

[0001] The present invention relates to the fields of veterinary medicine and medical technology, and in particular to the use of dihydromyricetin in preparing medicines for preventing and / or treating infections of multiple poxvirus family members. Background Art

[0002] Poxvirus is an ancient virus family with a large number of members, including smallpox virus, vaccinia virus, cowpox virus, monkeypox virus, contagious pustular stomatitis virus, bovine papular stomatitis virus, sheeppox virus, bovine nodular dermatitis virus, swinepox virus, fowlpox virus and other poxviruses related to humans and animals. Many members are zoonotic. After infection, humans and animals often develop local or systemic purulent skin lesions. It is an important pathogen that seriously affects public health and safety, and has attracted people's high attention.

[0003] Ovine infectious pustular virus (ORFV) is a representative species of the genus Parapoxvirus in the family Poxviridae. It mainly infects small and medium-sized ruminants such as sheep and goats, and occasionally infects wild even-toed ungulate mammals such as musk oxen, moose, reindeer and camels. Infected animals are characterized by the formation of proliferative lesions such as pustules, ulcers and warty thick scabs on the skin / mucous membranes of the lips, gums, eyelids, nose, ears, hoof crowns, tail roots and udders. Clinically, infected animals have feeding disorders, resulting in a decline in their growth / reproductive performance or even death, causing economic losses to the sheep industry; people can also be infected through direct or indirect contact with sick sheep, which poses a potential occupational risk. Among them, herders, veterinarians, butchers, breeders, fur processors and other practitioners are prone to infection. ORFV is widely distributed in sheep flocks in sheep-raising countries and regions around the world, and frequent outbreaks have seriously hindered the development of the sheep industry and related industries.

[0004] Protein phosphorylation mediated by protein kinases and phosphatases is the most common, basic, and important mechanism for regulating and controlling the activity and function of proteins and enzymes. Protein phosphorylation and dephosphorylation modifications play an important role in the viral replication cycle by regulating the function of viral proteins. The poxvirus life cycle is also regulated by its own protein phosphorylation and dephosphorylation network. Dual-specificity phosphatases (vDUSPs) are widely present in members of the Poxviridae family. They regulate viral transcription, affect the morphogenesis of viral particles, and interfere with the host's immune response by phosphorylating tyrosine and serine of specific viral and cellular substrates, and dephosphorylating threonine residues. Given the key role of dual-specificity phosphatases in the poxvirus life cycle, they are considered ideal targets for new anti-poxvirus agents.

[0005] Studies have shown that dihydromyricetin has antioxidant, anti-inflammatory, anti-tumor, alcohol-detoxifying and liver-protecting effects, but other pharmacological effects of dihydromyricetin are still unclear. Summary of the invention

[0006] The purpose of the present invention is to provide a new application of dihydromyricetin in the field of preparing drugs for the prevention and / or treatment of poxviruses. In the research of the present invention, it was found that dihydromyricetin can effectively inhibit the activity of poxvirus dual-specificity phosphatase, so that it can be used to prevent and / or treat poxvirus infection, and further used to prepare drugs for the prevention and / or treatment of poxvirus infection.

[0007] In one aspect, the present invention provides the use of dihydromyricetin in the preparation of a drug for preventing and / or treating poxvirus infection. Preferably, the poxvirus is a common poxvirus for humans, livestock and poultry, including at least one of ovine contagious pustular virus (ORFV), monkeypox virus (MPXV), sheeppox virus (SPPV), bovine nodular skin disease virus (LSDV), fowlpox virus (FWPV), goatpox virus (GTPV), cowpox virus (VACV) or swinepox virus.

[0008] On the other hand, the present application provides a pharmaceutical composition for preventing and / or treating poxvirus infection, which comprises dihydromyricetin as an active ingredient. Preferably, the poxvirus is a common poxvirus in humans, livestock and poultry, and the poxvirus includes at least one of ovine contagious pustular virus (ORFV), monkeypox virus (MPXV), sheeppox virus (SPPV), bovine nodular skin disease virus (LSDV), fowlpox virus (FWPV), goatpox virus (GTPV), cowpox virus (VACV) or swinepox virus.

[0009] Dihydromyricetin can effectively inhibit the activity of poxvirus dual-specific phosphatase, so it can be used to prevent and / or treat poxvirus infection, and further used to prepare drugs for preventing and / or treating poxvirus infection. Furthermore, a pharmaceutical composition containing dihydromyricetin can also be used to prepare drugs for preventing and / or treating poxvirus infection.

[0010] In order to achieve the above object, the present invention provides the use of dihydromyricetin in the preparation of a drug for preventing and / or treating poxvirus infection.

[0011] Preferably, the poxvirus comprises at least one of ovine pox virus ORFV, monkeypox virus MPXV, sheeppox virus SPPV, bovine lumpy skin disease virus LSDV, fowlpox virus FWPV, goatpox virus GTPV, cowpox virus VACV or swinepox virus.

[0012] Preferably, dihydromyricetin inhibits the activity of the poxvirus dual-specificity phosphatase. Dihydromyricetin prevents and / or treats poxvirus infection by inhibiting the activity of the poxvirus dual-specificity phosphatase.

[0013] In any of the above, the effective concentration of dihydromyricetin is preferably greater than 0.1 μM, more preferably 0.1 μM to 100 μM, and more preferably 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM and ranges therebetween.

[0014] Furthermore, the inhibitory concentration of dihydromyricetin on the dual-specific phosphatase of the contagious pustular virus of sheep is preferably 0.1 μM to 100 μM, and more preferably 0.1, 1, 10, 100 μM and the range therebetween. Furthermore, the half effective concentration (IC 50 ) was 8.29±1.06μM. Furthermore, the median effective concentration (IC 50 ) was 11.59±1.79μM.

[0015] Furthermore, the inhibitory concentration of dihydromyricetin on monkeypox virus dual specific phosphatase is 0.1 μM to 100 μM, and more preferably 0.1, 1, 10, 100 μM and the range therebetween. Furthermore, the half effective concentration (IC 50 ) was 11.09±0.19μM.

[0016] Furthermore, the inhibitory concentration of dihydromyricetin on vaccinia virus dual specificity phosphatase is preferably 0.1 μM to 100 μM, and more preferably 0.1, 1, 10, 100 μM and the range therebetween. Furthermore, the half effective concentration (IC 50 ) was 7.31±0.30μM.

[0017] Furthermore, the inhibitory concentration of dihydromyricetin on the dual specific phosphatase of capripoxvirus is preferably 0.1 μM to 100 μM, and more preferably 0.1, 1, 10, 100 μM and the range therebetween. Furthermore, the half effective concentration (IC 50 ) was 28.37±2.42μM.

[0018] Furthermore, the concentration of dihydromyricetin for bovine papular stomatitis virus dual specificity phosphatase is preferably 0.1 μM to 100 μM, and more preferably 0.1, 1, 10, 100 μM and the range therebetween. Furthermore, the half effective concentration (IC 50 ) was 14.67±2.40μM.

[0019] The present invention also provides a pharmaceutical composition for preventing and / or treating poxvirus infection, wherein the pharmaceutical composition comprises dihydromyricetin.

[0020] Preferably, the pharmaceutical composition contains the pharmaceutically active ingredient dihydromyricetin in the form of a monomer, or in the form of a plant extract containing dihydromyricetin.

[0021] Preferably, in any of the above items, the plant extract is from at least one of a Camellia sinensis extract and a Ziziphus jujuba extract.

[0022] Preferably, any of the above items is that the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0023] Preferably, the pharmaceutically acceptable carrier and / or excipient is selected from at least one of a solvent, a diluent, a disintegrant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a release agent, a coating agent, or an antioxidant.

[0024] Preferably, any of the above items is that the solvent includes distilled water, glycerol, dimethyl sulfoxide, ethanol, propylene glycol, polyethylene glycol, fatty oil, etc.

[0025] Preferably, any of the above items is that the diluent comprises starch, sucrose, dextrin, pregelatinized starch, mannitol or the like.

[0026] Preferably, in any of the above items, the disintegrant comprises microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone or the like.

[0027] Preferably, in any of the above items, the glidant comprises micro powder silica gel, talcum powder and the like.

[0028] Preferably, any of the above items is that the binder comprises carboxymethyl cellulose, gelatin, polyvinyl pyrrolidone, povidone, starch slurry or the like.

[0029] Preferably, any of the above items is that the lubricant includes sodium stearate fumarate, magnesium stearate, talc, hexagonal boron nitride, sodium benzoate, etc.

[0030] Preferably, any of the above items is that the dispersant includes sodium pyrophosphate, sodium tripolyphosphate, polyoxyethylene alkylphenol ether, and the like.

[0031] Preferably, any of the above items is that the suspending agent includes glycerol, syrup, sorbitol, gum arabic, gum tragacanth, sodium alginate, methylcellulose, etc.

[0032] Preferably, any of the above items is that the isotonic agent comprises sodium chloride, glucose, glycerol, boric acid, borax and the like.

[0033] Preferably, in any of the above items, the wetting agent comprises polysorbate, polyoxyethylene castor oil, poloxamer or the like.

[0034] Preferably, any of the above items is that the emulsifier includes fatty acids, sorbitan fatty acids, monoglycerides, lanolin, sodium lauryl sulfate, and the like.

[0035] Preferably, in any of the above items, the buffer comprises acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, lactic acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc.

[0036] Preferably, in any of the above items, the antioxidant comprises sodium sulfite, sodium bisulfite, butylated p-hydroxyanisole, and the like.

[0037] Preferably, the pharmaceutical composition is formulated into any one of the dosage forms of powder, tablet, pill, emulsion, pellet, capsule, suspension or tincture. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the inhibition rate curve of different concentrations of dihydromyricetin on the dual specific phosphatase of contagious pustular virus in the preferred embodiment 2 of the present invention.

[0039] Figure 2 This is the inhibition rate curve of different concentrations of dihydromyricetin on monkeypox virus dual specificity phosphatase in preferred embodiment 2 of the present invention.

[0040] Figure 3 This is the inhibition rate curve of dihydromyricetin at different concentrations on vaccinia virus dual specificity phosphatase in preferred embodiment 2 of the present invention.

[0041] Figure 4 This is the inhibition rate curve of dihydromyricetin at different concentrations on capripox virus dual specificity phosphatase in preferred embodiment 2 of the present invention.

[0042] Figure 5 This is the inhibition rate curve of different concentrations of dihydromyricetin on bovine papular stomatitis virus dual specificity phosphatase in preferred embodiment 2 of the present invention.

[0043] Figure 6 This is the toxicity determination of different concentrations of dihydromyricetin on OFTu cells in the preferred embodiment 3 of the present invention.

[0044] Figure 7 The results are as follows: The inhibitory effects of different concentrations of dihydromyricetin on sheep contagious pustular virus in the preferred embodiment 3 of the present invention. DETAILED DESCRIPTION

[0045] The present invention is described more clearly and completely through the following examples, but the described examples are only part of the examples of the present application, rather than all the examples. The examples are provided to help understand the present invention and should not be used to limit the scope of protection of the present invention.

[0046] The present invention has no particular restrictions on the extraction methods of the rattan tea extract and the jujube extract. The technicians can extract by existing techniques, which are all applicable to the present invention. Exemplary extraction methods include but are not limited to: refluxing the rattan tea and / or the jujube with n-butanol or ethanol; or, extracting the rattan tea and / or the jujube with hot water to obtain the rattan tea extract and / or the jujube extract containing dihydromyricetin.

[0047] The pharmaceutical composition of the present invention is a pharmaceutically acceptable dose of dihydromyricetin, i.e., an applied dose, which can be changed according to the age / monthly age, sex and weight of the subject to be treated, the specific disease to be treated or the pathological state, the severity of the disease or pathological state, the route of administration and the judgment of the diagnosis. Taking these factors into consideration, it is determined that the applied dose can generally be 0.01-1000mg / kg / day, specifically 1-100mg / kg / day. However, the scope of the present disclosure is not limited to the applied dose in any way.

[0048] The present application is described in detail below with reference to specific embodiments.

[0049] Dihydromyricetin (CAS No.: 27200-12-0) was purchased from Chengdu Ruifensidan Biotechnology Co., Ltd. The structural formula is as follows:

[0050]

[0051] Sources of materials used in the examples:

[0052] Viruses and cells: ORFV-SY17 (GenBank: MG712417.1) strain, which is a strain that has been disclosed in the prior art and can be obtained by the public through literature resource sharing; OFTu immortalized cells (sheep turbinate immortalized cell line OFTu, CCTCC NO: C2022191) were prepared, constructed, identified and preserved in this laboratory at the China Center for Type Culture Collection (Wuhan, China). The culture was received and registered by the collection on June 23, 2022; the collection completed the viability test of the culture on June 27, 2022, and the result was viable; storage address: Wuhan University, Wuhan, China; Tel: 027-68752319.

[0053] Reagents: CCK-8 kit (Cell Counting Kit-8) was purchased from Meilun Biotechnology Co., Ltd., and fetal bovine serum was purchased from Biological Industries, Israel. Other reagents not mentioned in the present invention are all commercially available products.

[0054] Example 1

[0055] Example 1 provides the inhibitory effect of dihydromyricetin on poxvirus dual specificity phosphatase.

[0056] Dissolve dihydromyricetin in dimethyl sulfoxide (DMSO) solvent and prepare different concentrations. Add 80 μL of dihydromyricetin with different concentrations, purified dual-specific phosphatase and reaction buffer to a 96-well ELISA plate, set up a control group (DMSO only) and a blank group (no substrate), and incubate at 37°C for 30 minutes. Subsequently, add 20 μL of substrate pNPP (final concentration of 1 mM), continue incubation at 37°C for 30 minutes, and finally add 100 μL of 1M NaOH stop solution to terminate the reaction and detect the absorbance value at 405 nm.

[0057] According to the absorbance value, the formula: relative enzyme activity (%) = 100% × [(OD 实验 -OD 空白 ) / (OD 对照 -OD 空白 )]; inhibition rate (%) = 100% - relative enzyme activity (%), respectively calculate the inhibition rate of dihydromyricetin on the dual specific phosphatase of sheep infectious pustular virus, monkeypox virus, cowpox virus, sheeppox virus and bovine papular stomatitis virus; the inhibition rate curve is drawn with dihydromyricetin concentration (μM) as the horizontal axis and inhibition rate as the vertical axis, such as Figure 1-Figure 5 The half inhibitory concentration (IC 50 ), IC values ​​of dihydromyricetin against dual specificity phosphatases of venom, monkeypox, cowpox, capripox and bovine papular stomatitis virus 50 The values ​​were 8.29±1.06μM, 11.09±0.19μM, 7.31±0.30μM, 28.37±2.42μM and 14.67±2.40μM, respectively. The above results show that dihydromyricetin can effectively inhibit multiple poxvirus dual-specific phosphatases.

[0058] Example 2

[0059] Example 2 provides a molecular docking method to predict the interaction between dihydromyricetin and different poxvirus dual-specificity phosphatases.

[0060] Preparation of small molecule ligand dihydromyricetin: The structure of dihydromyricetin was downloaded from https: / / www.rcsb.org / ; it was opened with ChemBioDraw Ultra 12.0 software, converted into a three-dimensional structure with ChemBio3D Ultra 12.0 software, and energy optimized using the MMFF94 force field. Then, non-polar hydrogens were merged and rotatable bonds were defined using AutodockTools 1.5.6, and finally converted into PDBQT format and saved.

[0061] Macromolecular receptor preparation: Download the resolved structure of poxvirus dual-specific phosphatase from https: / / www.rcsb.org / or predict the structural model of poxvirus dual-specific phosphatase that has not been resolved on https: / / swissmodel.expasy.org / , preferably, the dual-specific phosphatase of sheep contagious pustulosis virus (PDB ID: 5NCR), monkeypox virus dual-specific phosphatase (PDB ID: 8GZ4), and cowpox virus dual-specific phosphatase (PDB ID: 3CM3); use SWISS-MODEL to model the structures of sheeppox virus and bovine epidemic stomatitis virus dual-specific phosphatase. Use AutodockTools1.5.6 to add polar hydrogen and charge, and finally convert to PDBQT format and save.

[0062] Select appropriate active pocket coordinates, size_x = 15, size_y = 15, size_z = 15. The parameter exhaustiveness is set to 16 to increase the accuracy of the calculation. Unless otherwise specified, other parameters use the default values. Finally, select the conformation with the highest score and use PyMoL1.7.6 for result analysis.

[0063] The results are shown in Table 1:

[0064]

[0065] Example 3

[0066] Example 3 provides the inhibitory effect of dihydromyricetin on sheep contagious pustular virus.

[0067] Dihydromyricetin was dissolved in dimethyl sulfoxide (DMSO) solvent and prepared at different concentrations. Different concentrations of dihydromyricetin were added to a dense monolayer of OFTu cells, incubated for 72 hours, and then CCK-8 solution (10 μL / well) was added. The cells were exposed to the solution at 37°C for 3 hours, and the absorbance of the cells at 450 nm was measured to evaluate the toxic effect of dihydromyricetin on OFTu cells. The results are shown in Table 1. Figure 6The toxicity of different concentrations of dihydromyricetin on OFTu cells was determined by CCK-8 method, and the half toxic concentration (CC 50 ) was 423.9±22.4μM. To further clarify the effect of different concentrations of dihydromyricetin on ORFV infection, ORFV was inoculated into OFTu cells at MOI=1. After adsorption for 1 hour, the cells were replaced with maintenance medium containing different concentrations of dihydromyricetin and continued to be cultured. Samples were collected after 72 hours and the TCID 50 The virus titer was determined by the method. Figure 7 As shown in Figure 2, as the concentration of dihydromyricetin increased, the virus titer decreased significantly. The IC value of dihydromyricetin for ORFV was calculated by software fitting. 50 It is 11.59±1.79μM.

[0068] In summary, dihydromyricetin can inhibit the infection of ovine contagious pustular virus by inhibiting the activity of multiple poxvirus dual-specific phosphatases, which shows that dihydromyricetin can be used to prevent and / or treat poxvirus infection, and further used to prepare drugs for preventing and / or treating poxvirus infection.

[0069] Example 4

[0070] Embodiment 4 provides a pharmaceutical composition, which comprises dihydromyricetin. The dihydromyricetin is provided in monomeric form.

[0071] Example 5

[0072] Embodiment 5 provides a pharmaceutical composition, which contains dihydromyricetin. The dihydromyricetin component is provided in the form of a plant extract.

[0073] Preferably, the plant extract is from at least one of a Camellia oleifera extract and a Ziziphus jujuba extract.

[0074] Example 6

[0075] Example 6 provides a pharmaceutical composition comprising dihydromyricetin and a pharmaceutically acceptable carrier and / or excipient.

[0076] The pharmaceutically acceptable carrier and / or excipient is selected from at least one of a solvent, a diluent, a disintegrant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a release agent, a coating agent, or an antioxidant.

[0077] The pharmaceutical composition is prepared into any dosage form of powder, tablet, pill, emulsion, drop pill, capsule, suspension or tincture.

[0078] The pharmaceutically acceptable carrier and / or excipient is selected from at least one of a solvent, a diluent, a disintegrant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a release agent, a coating agent, or an antioxidant.

[0079] The solvent includes at least one of distilled water, glycerol, dimethyl sulfoxide, ethanol, propylene glycol, polyethylene glycol, fatty oil, etc.

[0080] The diluent includes at least one of starch, sucrose, dextrin, pregelatinized starch, mannitol and the like.

[0081] The disintegrant includes at least one of microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and the like.

[0082] The glidant includes at least one of micro-powdered silica gel, talcum powder and the like.

[0083] The binder includes at least one of carboxymethyl cellulose, gelatin, polyvinyl pyrrolidone, povidone, starch slurry and the like.

[0084] The lubricant includes at least one of sodium stearate fumarate, magnesium stearate, talc, hexagonal boron nitride, sodium benzoate and the like.

[0085] The dispersant includes at least one of sodium pyrophosphate, sodium tripolyphosphate, polyoxyethylene alkylphenol ether, and the like.

[0086] The suspending agent includes at least one of glycerin, syrup, sorbitol, gum arabic, gum tragacanth, sodium alginate, methylcellulose and the like.

[0087] The isotonic agent includes at least one of sodium chloride, glucose, glycerol, boric acid, borax and the like.

[0088] The wetting agent includes at least one of polysorbate, polyoxyethylene castor oil, poloxamer and the like.

[0089] The emulsifier includes at least one of fatty acids, sorbitan fatty acids, monoglycerides, lanolin, sodium lauryl sulfate and the like.

[0090] The buffer includes at least one of acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, lactic acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, and the like.

[0091] The antioxidant includes at least one of sodium sulfite, sodium bisulfite, butylated p-hydroxyanisole, and the like.

[0092] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of dihydromyricetin in the preparation of drugs for preventing and / or treating poxvirus infection.

2. The use according to claim 1, characterized in that The poxvirus includes at least one of ovine pox virus ORFV, monkeypox virus MPXV, sheeppox virus SPPV, bovine lumpy skin disease virus LSDV, fowlpox virus FWPV, goatpox virus GTPV, cowpox virus VACV or swinepox virus.

3. The use according to claim 1, characterized in that Dihydromyricetin inhibits the activity of the poxvirus dual-specificity phosphatase.

4. A pharmaceutical composition for preventing and / or treating poxvirus infection, characterized in that: The pharmaceutical composition comprises dihydromyricetin.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition contains the pharmaceutically active ingredient dihydromyricetin in the form of a monomer, or in the form of a plant extract containing the dihydromyricetin component.

6. The pharmaceutical composition according to claim 5, characterized in that The plant extract is selected from at least one of a rattan tea extract and a jujube extract.

7. The pharmaceutical composition according to any one of claims 4 to 6, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutically acceptable carrier and / or excipient is selected from at least one of a solvent, a diluent, a disintegrant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a release agent, a coating agent, or an antioxidant.

9. The pharmaceutical composition according to any one of claims 4 to 6, characterized in that The pharmaceutical composition is prepared into any dosage form of powder, tablet, pill, emulsion, drop pill, capsule, suspension or tincture.

Images