Application of 18 beta glycyrrhetinic acid in preparation of medicine for resisting porcine pseudorabies virus infection

Abstract

The invention belongs to the technical field of antiviral drugs, and particularly relates to application of 18 beta glycyrrhetinic acid in preparation of drugs for resisting porcine pseudorabies virus infection. The invention provides application of 18 beta glycyrrhetinic acid in preparation of a medicine for resisting porcine pseudorabies virus infection in order to solve the problem that an existing PRV (porcine reproductive virus) new prevalent strain II type lacks vaccines and medicines capable of effectively protecting the existing PRV new prevalent strain II type. Meanwhile, the invention also provides a medicament containing 18 beta glycyrrhetinic acid for resisting porcine pseudorabies virus infection. It is found for the first time that the 18 beta glycyrrhetinic acid can inhibit adsorption and replication of PRV on PK-15 cells, and the 18 beta glycyrrhetinic acid has a very remarkable effect on resisting PRV, so that the 18 beta glycyrrhetinic acid can be used for preparing the medicine for resisting porcine pseudorabies virus infection, is safe, non-toxic and harmless, and provides an effective and reliable natural medicine for prevention, control and adjuvant treatment of porcine pseudorabies virus infection.

Description

Technical Field

[0001] The invention belongs to the technical field of antiviral drugs, and particularly relates to use of 18β-glycyrrhetinic acid in preparing drugs for resisting porcine pseudorabies virus infection. Background Art

[0002] The pathogen of pseudorabies (Pseudorabies, PR) is Pseudorabies Virus (PRV). PRV is an acute infectious pathogen that causes a series of clinical symptoms such as fever, itching, encephalomyelitis, respiratory and nervous system disorders. Neurological symptoms include: movement disorders, muscle tremors, paddling of hind limbs, rotation on the spot, etc. Pigs are the natural hosts of PRV. PRV has a wide range of infection. In addition to infecting pigs, it can also infect many other mammals, mainly ruminants, carnivores and rodents. The World Organization for Animal Health lists PR as one of the diseases that cause huge economic losses to the pig industry.

[0003] PRV, also known as porcine herpesvirus type 1, is one of the main members of the herpesviridae family and belongs to the alphaherpesvirus subfamily. PRV virus particles are spherical with a diameter of 120-130nm. Its genome is double-stranded DNA with a length of about 145Kb. It has a viral envelope, capsid, and membrane on the periphery. It has an icosahedral stereoscopic symmetry and a GC% content of more than 70%.

[0004] Vaccination is the most common way to prevent PR.

[0005] 18β-Glycyrrhetinic acid (18βGA) is a natural oleanane-type triterpenoid compound. Glycyrrhizic acid from the dry roots and rhizomes of Glycyrrhiza uralensis, Glycyrrhiza inflata and Glycyrrhiza glabra is metabolized in plants and the human intestine and degraded into a pentacyclic triterpenoid compound, glycyrrhetinic acid. Glycyrrhetinic acid mainly includes two stereoisomers: 18βGA and 18αGA. 18βGA and its derivatives have multiple pharmacological effects, including anti-cancer, anti-tumor, anti-diabetic, antibacterial, anti-viral, anti-asthmatic, anti-ulcer, liver protection and anti-inflammatory effects. 18β-Glycyrrhetinic acid Alias: (3β,18β)-3-hydroxy-11-oxo-olean-12-ene-29-acid, chemical formula: C30H46O4; molecular weight: 470.68, appearance and shape: white to off-white (pure color); purity (LCMS): 99.88%; melting point: 292-295℃; boiling point: 588.3℃; melts in DMSO and is stable under normal conditions. In recent years, new progress has been made in the study of the antibacterial and antiviral activity of 18βGA at home and abroad. 18βGA can inhibit the expression of the main virulence proteins (saeR, mecA and sbi) of methicillin-resistant Staphylococcus aureus (MRSA) to achieve bactericidal effect. At sublethal doses, it reduces the expression of virulence proteins in Staphylococcus aureus both in vitro and in vivo. Studies have also found that 18βGA has significant inhibitory activity against viruses of multiple families, including Reoviridae and Hepaciviridae. Its mechanisms of viral inhibition include: (1) Effects on specific stages of the replication cycle. For example, 18βGA continuously activates NF-kB, which can override the viral coding mechanism that downregulates its activity, thereby reducing rotavirus replication. 18βGA reduces the titer of rotavirus in cells by regulating the PI3 / Akt pathway. (2) Regulates inflammatory factors and indirectly affects viral replication. For example, 18βGA inhibits the proliferation of viral hepatitis by inhibiting the release of HMGB1 and antagonizing the cytokine activity of HMGB.

[0006] At present, the genetic diversity of PRV is increasing, and traditional PRV vaccine strains lack effective protection against new epidemic strains (PRV type II), which makes it extremely difficult to achieve disease prevention and control and purification by vaccine immunization alone. Therefore, the development of new drugs with high efficiency and low toxicity for the treatment of PRV meets the actual needs of current veterinary clinics and is also an important direction for future research. Summary of the invention

[0007] The technical problem to be solved by the present invention is that there is a lack of effective vaccines and therapeutic drugs for the existing PRV new epidemic strain type II.

[0008] The technical solution of the present invention to solve the above technical problem is: to provide a use of 18β-glycyrrhetinic acid in preparing a drug for resisting pseudorabies virus infection in pigs.

[0009] Wherein, in the above use, the concentration of 18β-glycyrrhetinic acid is 0.391-50 μM.

[0010] Preferably, in the above use, the concentration of 18β-glycyrrhetinic acid is 25 μM.

[0011] Wherein, in the above use, the 18β-glycyrrhetinic acid has a CAS number of 471-53-4.

[0012] Among them, in the above use, the 18β-glycyrrhetinic acid is used to inhibit the infection of pseudorabies virus.

[0013] Furthermore, the 18β-glycyrrhetinic acid inhibits the infection of porcine pseudorabies virus by inhibiting the adsorption or replication of porcine pseudorabies virus.

[0014] Wherein, in the above use, the pseudorabies virus is pseudorabies virus type II. Further, it is strain PRVSC15-01.

[0015] Among them, in the above-mentioned use, the drug is a drug for preventing or treating pseudorabies virus infection in pigs.

[0016] The invention also provides a pharmaceutical preparation for resisting pseudorabies virus infection in pigs, and the pharmaceutical preparation comprises 18β-glycyrrhetinic acid.

[0017] Wherein, the above-mentioned pharmaceutical preparation also includes pharmaceutically acceptable excipients.

[0018] The beneficial effects of the present invention are:

[0019] The present invention provides the use of 18β-glycyrrhetinic acid in preparing a drug for resisting porcine pseudorabies virus infection, and further provides a pharmaceutical preparation. The present invention first discovered through experiments that 18β-glycyrrhetinic acid can inhibit the adsorption and replication of PRV on PK-15 cells, and has a very significant effect in resisting PRV. Therefore, it can be used to prepare a drug for resisting porcine pseudorabies virus infection, which is safe, non-toxic and harmless, and provides a new way to resist PRV. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure shows the cytotoxicity of 18β-glycyrrhetinic acid to PK15 cells.

[0021] Figure 2 This is a diagram showing the inhibitory effect of 18β-glycyrrhetinic acid on PRV in PK15 cells.

[0022] Figure 3The activity of 18β-glycyrrhetinic acid in inhibiting PRV infection in PK15 cells; A shows the effect of 18β-glycyrrhetinic acid on PK15 cell pathological changes caused by PRV infection observed under an inverted microscope; B shows the effect of 18β-glycyrrhetinic acid on the virus TCID50 in PRV-infected PK15 cells; C shows the effect of 18β-glycyrrhetinic acid on the expression of PRV gB protein determined by Western blot.

[0023] Figure 4 The effect of 18β-glycyrrhetinic acid on viral proliferation in PK15 cells after 12h, 24h, 36h, and 48h of PRV infection. A is the effect of 18β-glycyrrhetinic acid on viral copy number in PK15 cells 12h after PRV infection determined by fluorescence quantitative PCR; B is the effect of 18β-glycyrrhetinic acid on viral copy number in PK15 cells 24h after PRV infection determined by fluorescence quantitative PCR; C is the effect of 18β-glycyrrhetinic acid on viral copy number in PK15 cells 36h after PRV infection determined by fluorescence quantitative PCR; D is the effect of 18β-glycyrrhetinic acid on viral copy number in PK15 cells 48h after PRV infection determined by fluorescence quantitative PCR.

[0024] Figure 5 The effects of different modes of action of 18β on the anti-PRV effect of glycyrrhetinic acid, where A is the protective effect of 18β glycyrrhetinic acid against PRV infection, and B is the evaluation of the direct viral killing effect of 18β glycyrrhetinic acid.

[0025] Figure 6 The inhibitory effects of 18β-glycyrrhetinic acid on different stages of the PRV life cycle. A is the effect of 18β-glycyrrhetinic acid on PRV adsorption detected by fluorescence quantitative PCR; B is the effect of 18β-glycyrrhetinic acid on PRV penetration detected by fluorescence quantitative PCR; C is the effect of 18β-glycyrrhetinic acid on PRV synthesis and assembly (replication) detected by fluorescence quantitative PCR; D is the effect of 18β-glycyrrhetinic acid on PRV release detected by fluorescence quantitative PCR. DETAILED DESCRIPTION

[0026] 18β-glycyrrhizic acid is the main biologically active ingredient in licorice, and has multiple pharmacological activities such as anti-inflammatory, antiviral, anticancer, immunomodulatory and neuroprotective. Existing research on it mostly focuses on its antibacterial effect, and there is no report on its effect against pseudorabies virus in pigs.

[0027] The inventors of the present application conducted a large number of experiments in the early stage, screened numerous natural small molecules such as flavonoids, terpenes, alkaloids and phenolic acids, and accidentally discovered that 18β-glycyrrhizic acid has a relatively significant inhibitory effect on PRV. Based on this, they systematically evaluated its antiviral activity, mode of action and its impact on the viral life cycle.

[0028] The present invention discovered for the first time the anti-PRV virus effect of 18β-glycyrrhizic acid. As a natural monomer small molecule with a clear active ingredient and chemical structure, it provides a basis and convenience for subsequent drug development.

[0029] The present invention has discovered for the first time through experiments that 18β-glycyrrhetinic acid has antiviral activity against PRV, and it is expected to become an anti-PRV drug, which is of great significance for the prevention, control and adjuvant treatment of PRV. At the same time, as a natural drug, 18β-glycyrrhetinic acid has pharmacological activity that is unmatched by a single chemotherapy drug, and its range of therapeutic diseases is wider.

[0030] The specific implementation modes of the present invention will be further explained below through examples, but it is not intended to limit the protection scope of the present invention to the scope described in the examples.

[0031] The sources of the test materials used in the examples are as follows:

[0032] 18β-glycyrrhetinic acid was purchased from MedChemexprpress Biotechnology (MCE) with a purity of 99.88%;

[0033] The porcine pseudorabies virus SC15-01 strain was isolated by the Animal Medicine Laboratory of Southwest University for Nationalities and stored in the laboratory.

[0034] PK-15 (porcine kidney epithelial cells) were purchased from the American Cell Line Resource Center (ATCC) and subcultured and preserved by the Animal Medicine Laboratory of Southwest University for Nationalities;

[0035] MTT detection kit was purchased from Solebow Biotechnology Co., Ltd.;

[0036] The cell culture fluid used is composed of 91% DMEM culture medium, 8% fetal bovine serum and 1% penicillin-streptomycin prepared in a sterile environment and stored in a refrigerator at 4°C; the maintenance fluid used in the inoculation process is composed of 99% DMEM culture medium and 1% penicillin-streptomycin prepared in a sterile environment and stored in a refrigerator at 4°C;

[0037] The main instruments and equipment used in the embodiments of the present invention are: a carbon dioxide incubator (Suzhou Jiemei Electronics Co., Ltd., CIB-191CX), a biological safety cabinet (Qingdao Haier Biomedical Co., Ltd., HR900-ⅡA2), an ELISA reader (American Berton Instrument Co., Ltd., ELX800), and a fluorescence quantitative PCR instrument (American Baile Co., Ltd., 778BR07568).

[0038] Other instruments and materials not specified are common commercial products.

[0039] Example 1 Determination of toxicity of 18β-glycyrrhetinic acid on PK-15 cells

[0040] Cytotoxicity test was determined by calculating cell survival rate using MTT method. 18β-glycyrrhetinic acid was dissolved in 1% DMSO and filtered through a 0.22μm microporous filter; starting at 200μM, the drug was diluted to 0.781μM in cell maintenance medium. PK-15 cells were diluted to 2.5×10 4 / mL, transfer to 96-well plate at 200μL per well, culture in 37℃, 5% CO2 incubator for 24h, after PK-15 cells grow into monolayer on 96-well culture plate, add diluted drugs of different concentrations, 150μL per well, make 5 duplicate wells for each concentration, set up blank control group and DMSO control group at the same time, add 150μL cell maintenance solution, place in 37℃, 5% CO2 cell incubator, culture for 48h, discard culture solution, wash each cell well twice with sterile 1mL PBS, add 100μL MTT solution to each well, place in 37℃, 5% CO2 incubator for incubation, remove liquid in wells after 4h, add 100μL DMSO to each well, incubate for 10min, measure absorbance at 490nm wavelength with microplate reader and calculate half cytotoxic concentration CC 50 (50% Cytotoxity Concentration). The results are as follows Figure 1 As shown, the results showed that 18β-glycyrrhetinic acid CC 50 The concentration of 18β-glycyrrhetinic acid at 50 μM has no obvious cytotoxicity to PK15 cells for 48 h, and it has good safety for drug preparation.

[0041] Example 2 Inhibitory effect of 18β-glycyrrhetinic acid on pseudorabies virus in PK15 cells

[0042] 18β-glycyrrhetinic acid was diluted to 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, and 0.391 μM for later use. PK-15 cells were diluted to 2.5×10 cells / mL in DMEM containing 10% fetal bovine serum. 4 / mL, transferred to 96-well plates at 200 μL per well, cultured in a 37°C, 5% CO2 incubator for 24 h. After the cells grew into a monolayer of cells, 40 μL of PRV was added to each well, incubated for 1 h 30 min, and the virus solution was discarded. The drug treatment group was added with 18β-glycyrrhetinic acid dilutions of 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, and 0.391 μM, 150 μL / well, respectively, and 5 replicates were set for each concentration; 150 μL / well of cell maintenance solution was added to the virus control group, and 150 μL / well of cell maintenance solution was added to the cell control group without virus and drug. After 48 h of culture, the culture medium was discarded, and each cell well was washed twice with 1 mL of PBS. In a light-proof environment, 100 μL of MTT detection reagent was added to each well. After incubation for 4 h, the liquid in the well was removed, and 100 μL of DMSO was added to each well. After the crystals dissolved, the OD490 nm value was detected with an enzyme-labeled instrument and the half effective concentration IC was calculated. 50 (Half inhibitory concentration,IC 50 ). The result is Figure 2 As shown, the results showed that the IC 50 The inhibitory effect on PRV was 8.3 μM, and the inhibition rate of the virus reached 82% at a concentration of 25 μM. The antiviral activity was dose-dependent, and it could inhibit PRV at a concentration of 3.125-50 μM, and the best effect was achieved at 25 μM.

[0043] Example 3 Effect of 18β-glycyrrhetinic acid on pseudorabies virus titer

[0044] PK-15 cells were diluted to 2.5 × 10 cells / mL in DMEM containing 10% fetal bovine serum. 4 / mL, transfer 200μL per well to a 96-well plate, culture in a 37℃, 5% CO2 incubator for 24h, and after it grows into a monolayer of cells, use cell maintenance solution to dilute the drug in gradients, mix the virus solution with drugs of each concentration gradient in equal amounts (final concentration of 18β glycyrrhetinic acid: 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391μM), incubate the mixture with cells for 1h30min, discard the liquid in each well, add 200μL of DMEM maintenance solution without fetal bovine serum to each well, and culture for 48h. The cell plate was repeatedly frozen and thawed 3 times to release the virus particles. Collect the cell suspension in each well, filter with a 0.22μm filter, and collect the filtrate. The cells were inoculated into 96-well plates at 200 μL per well and cultured in a 37°C, 5% CO2 incubator for 24 h. When the cell confluence reached at least 90%, the filtrate collected above was added and diluted 10-fold with DMEM to 10 -1 ~10 -8, a total of 8 dilutions, inoculated into each cell well, each dilution set 8 cell wells repeated. Cultured in 37 ℃, 5% CO2 incubator for 48 hours, observed the CPE of cells in each well, and calculated the 50% tissue culture infectious dose (TCID50) of the virus using the Reed-Muench method. The results are as follows Figure 3 As shown in B, it can be seen that 18β-glycyrrhetinic acid significantly reduced the TCID50 of PRV, indicating that 18β-glycyrrhetinic acid has good inhibitory activity against PRV.

[0045] Example 4 Western blot determination of the inhibitory activity of 18β-glycyrrhetinic acid on pseudorabies virus

[0046] PK-15 cells were evenly inoculated into 6-well plates and cultured at 37°C, 5% CO2 for 24 hours. The experiment was started when the cell density reached 90%. The cell culture medium was discarded, and the virus group, drug plus virus group, and cell control group were set up. The virus group and drug group were inoculated with virus solution, and the cell control group was added with DMEM maintenance solution and incubated for 1h30min; after the virus solution was discarded in the drug group, 1.5mL of drug dilution solution (50, 25μM) was added, and after the liquid was discarded in the virus group and cell group, 1.5mL of maintenance solution was added, and 3 parallel wells were set for each concentration. After culturing in a 37°C, 5% CO2 incubator for 48 hours, the cells were washed three times with PBS and observed under an inverted microscope. The results are as follows Figure 3 As shown in A, the results showed that the cell pathological changes were significantly reduced after treatment with 18β-glycyrrhetinic acid, indicating that 18β-glycyrrhetinic acid can reduce the cell damage caused by PRV.

[0047] Lyse cells: prepare RIPA lysis mixture, RIPA: PMSF (protease inhibitor) = 100:1, add 250μL to each well after mixing, and lyse on ice for 30 minutes. Add an equal volume of 2×SDS Loading buffer and mix well, and boil in a water bath for 10 minutes. Perform SDS-PAGE on the treated samples, and then transfer them to a PVDF membrane. Wash the membrane 3 times with TBST for 5 minutes each time, then block with 5% skim milk powder at room temperature for 2 hours, wash the membrane 3 times with TBST, and add primary antibody (PRV gB antibody or β-Actin antibody) and incubate overnight at 4°C. Wash the membrane 3 times with TBST, and incubate with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody at room temperature for 2 hours. Then wash the membrane 3-5 times with TBST for 5 minutes each time, and shake on a shaker. Add Thermo's ECL color development reagent for color development. Reasonably adjust the exposure time for development according to protein abundance, expression efficiency, etc., and save the picture. The results are as follows Figure 3As shown in Figure C, the results showed that the expression level of PRVgB protein was determined by Western blot, and it was found that the 18β-glycyrrhetinic acid treatment group could significantly reduce the expression of viral protein, which once again verified the anti-PRV activity of 18β-glycyrrhetinic acid.

[0048] Example 5 Effect of 18β-glycyrrhetinic acid on pseudorabies virus replication at different time points

[0049] PK-15 cells were plated in a 24-well plate. When the cells grew to 80%-90%, the culture medium was discarded, and the cells were washed twice with PBS. 100 μL / well of virus solution was added to the 18β-glycyrrhetinic acid-treated group, ribavirin-treated group, and virus control group, and 100 μL / well of maintenance solution was added to the cell control group. The plates were incubated in a 37°C, 5% CO2 incubator for 1 h30 min, and then the virus solution or maintenance solution was discarded. To each well of the 18β-glycyrrhetinic acid treatment group, 500 μL of 18β-glycyrrhetinic acid at a concentration of 50 μM and 25 μM were added; to each well of the ribavirin group, 500 μL of ribavirin at a concentration of 50 μM was added; to each well of the virus control group and the cell control group, 500 μL of maintenance solution was added; each concentration was repeated 3 times, and the cells were cultured in a 37°C, 5% CO2 incubator for 1h30min, and the liquid was discarded; 500 μL of maintenance solution was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 12h, 24h, 36h, and 48h, respectively, and the culture was terminated. The cells were frozen and thawed three times at -80°C, and the samples were collected to extract DNA for fluorescent quantitative PCR detection. The fluorescence quantitative PCR experimental operation was performed according to the method disclosed in Liu H, Zou J, Liu R, Chen J, LiX, Zheng H, Li L, Zhou B. Development of a TaqMan-Probe-Based Multiplex Real-TimePCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022. Vet Sci. 2023 Feb 1; 10(2): 106. The results are as follows Figure 4As shown, the results showed that the number of PRV nucleic acid copies in the 18β-glycyrrhetinic acid (50μM, 25μM) and ribavirin (50μM) treatment groups was significantly lower than that in the virus control group at 12h, 24h, 36h, and 48h. At 12h and 24h, there was no difference in the anti-PRV effect of different concentrations of 18β-glycyrrhetinic acid and ribavirin, but at 36h and 48h, the anti-PRV effect of different concentrations of 18β-glycyrrhetinic acid was significantly better than ribavirin. It can be seen that when 18β-glycyrrhetinic acid is used as an anti-PRV drug, it has an effect better than traditional drugs such as ribavirin.

[0050] Example 6 Evaluation of the protective effect of 18β-glycyrrhetinic acid on pseudorabies virus infection and direct virus killing effect in PK15 cells

[0051] PK-15 cells were plated in a 12-well plate. When the cells grew to 80%-90%, the culture medium was discarded and the cells were washed twice with PBS. 300 μL of 50 μM and 25 μM 18β-glycyrrhetinic acid were added to the 18β-glycyrrhetinic acid group, and 300 μL of 50 μM ribavirin dilution was added to the ribavirin group. 300 μL of maintenance solution was added to each well of the virus control group and the cell control group, and the cells were placed in a 37°C, 5% CO2 incubator for 1h30min and washed twice with PBS. In each test group, 300 μL of virus solution was added to each well, and 300 μL of maintenance solution was added to each well of the cell control group. The cells were placed in a 37°C, 5% CO2 incubator for 1h30min and washed twice with PBS. 1 mL of maintenance solution was added to each well. The culture was terminated after 48 hours, and the cells were frozen and thawed three times at -80°C. The samples were collected to extract DNA for fluorescence quantitative PCR detection. The results are shown in Figure 5 As shown in A, the results showed that in the protective mode, the PRV nucleic acid copy numbers in the 25μM and 50μM 18β-glycyrrhetinic acid treatment groups were significantly lower than that in the virus control group, indicating that 18β-glycyrrhetinic acid has a preventive protective effect against PRV.

[0052] The drug mixture containing PRV and 18β-glycyrrhetinic acid (50μM, 25μM) was placed in a 37°C, 5% CO2 incubator for 1h, and a DMSO solvent control was set up. The treated drug mixture was added to a 12-well cell culture plate with a monolayer of PK-15 cells, and a blank control group, a virus control group, and a ribavirin (50μM) positive control group were set up for adsorption at 37°C for 1h30min, then washed 3 times with PBS, 1mL of maintenance solution was added, and the cell culture plate was placed in a 37°C, 5% CO2 incubator for 48h before culturing was terminated, and the plate was repeatedly frozen and thawed at -80°C for three times, and samples were collected to extract DNA for fluorescence quantitative PCR experiments. The experimental process of fluorescence quantitative PCR method refers to the experimental operation disclosed in Liu H, Zou J, Liu R, Chen J, Li X, Zheng H, Li L, Zhou B. Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022. Vet Sci. 2023 Feb 1; 10(2): 106. The results are as follows Figure 5 As shown in B, the results show that in the direct inactivation mode, the drug groups with different concentrations did not show obvious inhibitory effects on the virus, indicating that 18β-glycyrrhetinic acid has no direct killing effect on PRV.

[0053] Example 7 Effect of 18β-glycyrrhetinic acid on the inhibitory effect of different stages of pseudorabies virus life cycle

[0054] (1) Effect of 18β-glycyrrhetinic acid on PRV adsorption stage

[0055] PK-15 cells were plated in a 12-well plate. When the cells grew to 80%-90%, the cells were precooled at 4°C for 1h, washed twice with PBS, 18β-glycyrrhetinic acid was mixed with the virus solution to make the final drug concentrations of 50μM and 25μM, respectively, and then 300μL was added to each cell well; ribavirin group used virus maintenance solution to dilute ribavirin (50μM) 2-fold, and 300μL was added to each well; 300μL of virus solution was directly added to the virus group, and 300μL of maintenance solution was added to the cell control group. The plates were incubated at 4°C for 1h30min, washed twice with PBS, 1mL of maintenance solution was added to each well, and the plates were repeatedly frozen and thawed at -80°C for 3 times. The samples were collected to extract DNA for fluorescence quantitative PCR experiments. The experimental process of the fluorescence quantitative PCR method refers to the experimental operation disclosed in Liu H, Zou J, Liu R, Chen J, Li X, Zheng H, Li L, Zhou B. Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022. Vet Sci. 2023 Feb 1; 10(2): 106. The results are as follows Figure 6 As shown in A, the results showed that 18β-glycyrrhetinic acid had a strong inhibitory effect on the adsorption stage of PRV, and its antiviral effect was better than that of ribavirin.

[0056] (2) Effect of 18β-glycyrrhetinic acid on the penetration stage of PRV

[0057] PK-15 cells were plated in a 12-well plate. When the cells grew to 80%-90%, the cells were precooled at 4°C for 1 hour and then washed twice with PBS. 300 μL of virus solution was added to each well of the 18β-glycyrrhetinic acid treatment group, ribavirin treatment group and virus control group, and 300 μL of maintenance solution was added to the cell control group. After incubation at 4°C for 1 hour and 30 minutes, the cells were washed twice with PBS. 300 μL of 50 μM and 25 μM 18β-glycyrrhetinic acid were added to the 18β-glycyrrhetinic acid treatment group, respectively; 300 μL of 50 μM ribavirin was added to the ribavirin group; 300 μL of maintenance solution was added to each well of the virus control group and the cell control group, and the cells were placed in a 37°C, 5% CO2 incubator for 3 hours, washed twice with PBS, and 1 mL of maintenance solution was added. The cells were frozen and thawed three times at -80°C, and samples were collected to extract DNA for fluorescence quantitative PCR experiments. The experimental method was the same as step (1). The results are shown in Figure 1. Figure 6As shown in B, the results showed that 18β-glycyrrhetinic acid had no inhibitory effect on the PRV penetration stage.

[0058] (3) Effects of 18β-glycyrrhetinic acid on the synthesis (replication) phase of PRV

[0059] PK-15 cells were plated in a 12-well plate. When the cells grew to 80%-90%, the culture medium was discarded and the cells were washed twice with PBS. 300 μL of virus solution was added to each well of the 18β-glycyrrhetinic acid treatment group, ribavirin treatment group, and virus control group, and 300 μL of maintenance solution was added to the cell control group. The cells were placed in a 37°C, 5% CO2 incubator for 6 hours and washed twice with PBS. 300 μL of 50 μM and 25 μM 18β-glycyrrhetinic acid were added to the 18β-glycyrrhetinic acid group, and 300 μL of 50 μM ribavirin was added to the ribavirin group. 300 μL of maintenance solution was added to each well of the virus control group and the cell control group, and the cells were placed in a 37°C, 5% CO2 incubator for 4 hours. The cells were washed twice with PBS, 1 mL of maintenance solution was added, and the cells were frozen and thawed three times at -80°C. The samples were collected to extract DNA for fluorescent quantitative PCR experiments. The experimental method was the same as step (1). The results are shown in Figure 6 As shown in C, the results showed that 18β-glycyrrhizic acid had a strong inhibitory effect on the synthesis (replication) stage of PRV, and its antiviral effect was better than that of ribavirin.

[0060] (4) Effect of 18β-glycyrrhetinic acid on the release phase of PRV

[0061] PK-15 cells were plated in a 12-well plate. When the cells grew to 80%-90%, the culture medium was discarded and the cells were washed twice with PBS. 300 μL of virus solution was added to each well of the 18β-glycyrrhetinic acid group, ribavirin group, and virus control group, and 300 μL of maintenance solution was added to the cell control group. The cells were incubated in a 37°C, 5% CO2 incubator for 24 hours. The cells were washed twice with PBS. 300 μL of 50 μM and 25 μM 18β-glycyrrhetinic acid dilutions were added to the 18β-glycyrrhetinic acid group, and 300 μL of 50 μM ribavirin dilutions were added to the ribavirin group. 300 μL of maintenance solution was added to each well of the virus control group and the cell control group, and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours. The cells were washed twice with PBS, 1 mL of maintenance solution was added, and the cells were frozen and thawed three times at -80°C. The samples were collected to extract DNA for fluorescence quantitative PCR experiments. The experimental method was the same as step (1). The results are shown in Figure 6 As shown in D, the results showed that 18β-glycyrrhetinic acid did not affect the release of PRV.

[0062] The above is a preferred embodiment of the present invention, and the present invention should not be limited to the contents disclosed in the embodiment. However, any simple modification, equivalent changes and modifications made to the above embodiment according to the technical essence of the present invention without departing from the contents of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. Use of β-glycyrrhizic acid in the preparation of drugs for resisting pseudorabies virus infection in pigs.

2. The use of 18β-glycyrrhetinic acid according to claim 1 in the preparation of a drug for resisting pseudorabies virus infection in pigs, characterized in that: The concentration of 18β-glycyrrhetinic acid is 0.391-50 μM.

3. The use of 18β-glycyrrhetinic acid according to claim 1 in the preparation of a drug for resisting pseudorabies virus infection in pigs, characterized in that: The concentration of 18β-glycyrrhetinic acid is 25 μM.

4. The use of 18β-glycyrrhetinic acid according to claim 1 in the preparation of a drug for resisting pseudorabies virus infection in pigs, characterized in that: The 18β-glycyrrhetinic acid is used for inhibiting the infection of porcine pseudorabies virus.

5. The use of 18β-glycyrrhetinic acid in the preparation of a drug for resisting pseudorabies virus infection in pigs according to claim 1, characterized in that: The 18β-glycyrrhetinic acid inhibits the infection of the pseudorabies virus by inhibiting the adsorption or replication of the pseudorabies virus.

6. The use of 18β-glycyrrhetinic acid in the preparation of a drug for resisting pseudorabies virus infection in pigs according to claim 1, characterized in that: The pseudorabies virus is pseudorabies virus type II.

7. The use of 18β-glycyrrhetinic acid in the preparation of a drug for resisting pseudorabies virus infection according to claim 1, characterized in that: The pseudorabies virus is strain PRV SC15-01.

8. The use of 18β-glycyrrhetinic acid in the preparation of a drug for resisting pseudorabies virus infection according to claim 1, characterized in that: The medicine is a medicine for preventing or treating pseudorabies virus infection in pigs.

9. A pharmaceutical preparation for resisting pseudorabies virus infection in pigs, characterized in that: The composition includes 18β-glycyrrhetinic acid.

10. The pharmaceutical preparation for resisting porcine pseudorabies virus infection according to claim 9, characterized in that: The composition also includes pharmaceutically acceptable excipients.

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