Application of 1, 2, 3, 4, 6-O-pentagalloylglucose and derivatives thereof in preparation of antiviral drugs

Abstract

The invention provides an application of 1, 2, 3, 4, 6-O-pentagalloylglucose and a derivative thereof in preparation of an antiviral drug, in particular to an application in a pseudorabies virus. Researches prove that the 1, 2, 3, 4, 6-O-pentagalloylglucose can effectively inhibit PRV-GFP infection on Vero cells, and TCID50 tests find that the 1, 2, 3, 4, 6-O-pentagalloylglucose can effectively inhibit proliferation of PRV and shows an obvious dose-dependent effect. The 1, 2, 3, 4, 6-O-pentagalloylglucose is nontoxic to Vero cells in a certain dosage range, so that the 1, 2, 3, 4, 6-O-pentagalloylglucose and the derivative thereof can be used for preparing the pseudorabies virus medicine in a reasonable dosage range, and the pseudorabies virus can be inhibited or prevented.

Description

Technical Field

[0001] The invention relates to the field of 1,2,3,4,6-O-pentagalloyl glucose, and in particular to application of 1,2,3,4,6-O-pentagalloyl glucose and derivatives thereof in the preparation of antiviral drugs. Background Art

[0002] Pseudorabies is an acute, highly contagious disease of pigs caused by pseudorabies virus (PRV). Its main symptoms include reproductive disorders, growth stagnation and respiratory disorders, which have caused huge economic losses to the global pig industry. With the continuous emergence of various PRV variants, existing vaccines can no longer effectively prevent PRV infection. In addition, it has been found clinically that PRV can infect humans. Therefore, it is crucial to find and identify effective antiviral drugs to deal with the threat of PRV.

[0003] 1,2,3,4,6-O-Pentagalloylglucose (PGG) is a polyphenol compound widely found in many plants. It has multiple effects such as antibacterial, antifungal, anti-inflammatory, antioxidant, anticancer, antidiabetic and antiviral. However, there are few reports on the research of PGG against PRV. The structural formula of PGG is as follows: Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of 1,2,3,4,6-O-pentagalloylglucose and its derivatives in the preparation of antiviral drugs, especially in the preparation of pseudorabies virus.

[0005] The object of the present invention is achieved in the following manner: application of 1,2,3,4,6-O-pentagalloylglucose and its derivatives in the preparation of antiviral drugs, the virus is specifically pseudorabies virus, and 1,2,3,4,6-O-pentagalloylglucose is a pharmaceutically acceptable salt or a compound modified based on the same.

[0006] The antiviral here includes antiviral itself and also includes individual anti-strain proteins.

[0007] The pseudorabies virus strain is PRV-GFP, or one of its identical genotypes. 1,2,3,4,6-O-pentagalloylglucose can inhibit PRV-GFP, and can also inhibit strains of the same genotype as PRV-GFP.

[0008] Compared with the prior art, the present invention proves that 1,2,3,4,6-O-pentagalloylglucose can effectively inhibit PRV-GFP infection in Vero cells, and the TCID 50 The experiment found that 1,2,3,4,6-O-pentagalloylglucose can effectively inhibit the proliferation of PRV, and showed a significant dose-dependent effect. 1,2,3,4,6-O-pentagalloylglucose is non-toxic to Vero cells within a certain dose range. Therefore, within a reasonable dose range, 1,2,3,4,6-O-pentagalloylglucose and its derivatives can be used to prepare pseudorabies virus drugs, thereby inhibiting or preventing pseudorabies virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Fig. 1 Toxicity of different concentrations of PGG on Vero cells.

[0010] Fig. 2 Effects of different concentrations of PGG on PRV-GFP infection in Vero cells (Scale bar = 150 μm).

[0011] Fig.3 TCID of PRV-GFP by different concentrations of PGG on Vero cells 50 The effect of titer. DETAILED DESCRIPTION

[0012] The present invention is described in detail below in conjunction with specific embodiments. It is necessary to point out that this embodiment is only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technical personnel skilled in the art can make some non-essential improvements and adjustments based on the above-mentioned contents of the present invention.

[0013] 1 Materials and methods 1.1 Test materials 1.1.1 Cells, strains, and compounds African green monkey kidney cells (Vero) and PRV-GFP strains were provided by the Animal Infectious Disease Laboratory Team of Henan Agricultural University. 1,2,3,4,6-O-pentagalloylglucose (PGG) was purchased from MCE (MedChemExpress) with a purity of 99.77%.

[0014] 1.1.2 Main reagents and instruments 1.1.2.1 Main reagents DMEM high-glucose medium was purchased from Hyclone; serum-free cell freezing medium was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.; 0.25% trypsin EDTA digestion solution (2.5 g porcine trypsin and 0.2 g EDTA dissolved in 1 liter PBS, containing phenol red) was purchased from Solarbio; penicillin-streptomycin double antibody and trypsin were purchased from Solarbio; dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, a subsidiary of Merck; CCK-8 kit was purchased from Abbkine; PBS (phosphate buffered saline) (NaCl 8.0 g; KCl 0.2 g; Na2HPO4 1.44 g; KH2PO4 0.24 g; distilled water to 1 L; pH adjusted to 7.4) used in the experiment was prepared by the laboratory; fetal bovine serum (FBS) was purchased from Gibio.

[0015] 1.1.2.2 Main instruments The -80°C ultra-low temperature refrigerator, clean bench, multifunctional microplate reader and EVOSTM M5000 imaging system were all purchased from Thermo Fisher Scientific Inc., USA; the electric constant temperature water bath was purchased from Shanghai Jinghong Testing Equipment Co., Ltd., China; the cell culture incubator was purchased from Shanghai Precision Instrument Co., Ltd.; the low-speed automatic balancing centrifuge was purchased from Beijing Medical Centrifuge Factory, China; the EP tube was purchased from Zhengzhou Dianjing Technology Co., Ltd.; the cell culture flask and cell culture plate were purchased from Nest; the cryopreservation tube was purchased from Vazyme, and the cell cryopreservation box was purchased from Sigma-Aldrich.

[0016] 1.2 Test methods 1.2.1 Cell recovery Add FBS to DMEM high-glucose medium to obtain DMEM high-glucose medium containing 10% FBS, which is the complete medium.

[0017] (1) First, take out the cryovial containing Vero cells from the -80 ℃ ultra-low temperature freezer, quickly place it in a 37 ℃ electric constant temperature water bath, and gently shake it to complete the cell thawing process within 2 min.

[0018] (2) Next, remove the cryovial containing cells from the water bath, place it in a centrifuge and balance it, and centrifuge it at 800 rpm for 5 min.

[0019] (3) After centrifugation, remove the cryovial from the centrifuge and spray it with 75% alcohol, then place it in the clean bench. Use a 1mL pipette to aspirate the supernatant, retain the cell pellet, add 1mL of DMEM high-glucose medium (complete medium) containing 10% FBS, gently pipette to mix the cell pellet, and then transfer it to a sterile cell culture flask that has been pre-added with 5mL of DMEM high-glucose medium containing 10% FBS.

[0020] (4) Finally, gently shake the cell culture flask to mix well, and then place it in a cell culture incubator at 37°C and 5% CO2. After 6 hours, observe the cell adhesion and change the medium.

[0021] 1.2.2 Cell passaging (1) When the growth density of Vero cells in step (4) of 1.2.1 reaches more than 90%, remove the culture medium in the cell culture flask in an ultra-clean workbench, and then wash the cells three times with PBS to remove the residual culture medium.

[0022] (2) Next, add 1 mL of 0.25% trypsin-EDTA digestion solution to the cell culture flask and place it in a 37°C, 5% CO2 cell culture incubator for 2 min of digestion. Then, gently tap the cell culture flask. When the cells are observed to have completely fallen off the cell flask wall under a microscope, add an equal volume of complete culture medium to the cell culture flask to terminate the digestion process.

[0023] (3) Then, blow the cells off the bottom of the culture flask and transfer them to a 15 mL centrifuge tube. Place the centrifuge tube in a centrifuge and centrifuge at 800 rpm for 5 min.

[0024] (4) After centrifugation, discard the supernatant and keep the cell pellet. Add 2 mL of complete culture medium and gently pipette to mix the cell pellet. Then, culture the cells in separate flasks according to the experimental plan and cell characteristics, and add complete culture medium to make the final volume reach 5 mL.

[0025] 1.2.3 Cell cryopreservation (1) First, select Vero cells with good growth conditions from the Vero cells that have been passaged in 1.2.2. When the cell density reaches 90%, remove the culture medium and wash the cells three times with PBS.

[0026] (2) Then, add 1 mL of 0.25% trypsin EDTA digestion solution to the cell culture flask and place it in a 37°C, 5% CO2 cell culture incubator for 2 min. Observe the changes in cell morphology under a microscope and gently tap the cell culture flask to make the cells fall off to the bottom of the flask. Subsequently, add DMEM high-glucose medium containing 10% FBS to terminate the digestion process.

[0027] (3) Next, use a pipette to gently blow away the liquid and transfer it to a 15 mL centrifuge tube. Place the tube in a centrifuge and centrifuge at 800 rpm for 5 min, then discard the supernatant.

[0028] (4) Finally, add serum-free cell freezing solution, pipette slowly to mix it evenly, and then dispense it into labeled cryotubes and place them in a cell freezing box. Then, store it in an ultra-low temperature freezer at -80°C.

[0029] 1.2.4 Determination of cytotoxicity FBS was added to the DMEM high-glucose medium to obtain a DMEM high-glucose medium containing 2% FBS, which was the cell maintenance medium.

[0030] Preparation of drug dilution solution: dilute PGG drug powder to 100 mM with DMSO, and further dilute it to 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 100 μM with cell maintenance solution; add DMSO to the cell maintenance solution to obtain a cell maintenance solution containing 0.1% DMSO. The PGG concentration is 0 μM. Next, perform the following steps: (1) Select the Vero cells that have been passaged in 1.2.2, and add 1 mL of 0.25% trypsin-EDTA digestion solution to the culture flask for 2 minutes of digestion. After digestion is complete, gently tap the cell flask to remove the cells. Then, add an equal volume of complete culture medium to the cell flask to stop the digestion process. Finally, blow and mix the cells, then transfer them to a 15 mL centrifuge tube, place the centrifuge tube in a centrifuge, centrifuge at 800 rpm for 5 minutes, and then discard the supernatant.

[0031] (2) Add complete culture medium to the cells obtained in step (1) to a cell density of 1×10 5 , which was then recorded as a cell suspension, and then the cell suspension was added to the 96-well plate at 100 μL / well to plate the cells in a 96-well plate, which was then placed in a 37°C, 5% CO2 cell culture incubator for culture.

[0032] (3) After the cells grew for 24 h, the same volume of 0 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 100 μM PGG was added to different wells of the 96-well plate as the drug-addition group with different concentrations of PGG; at the same time, the A (blank) group was set up, which was a cell maintenance fluid with the same volume of PGG but without cells; the drug-addition group with different concentrations of PGG and the A (blank) group were added, and each group was set up with 5 replicates. Then, the 96-well plate was placed in a 37 ℃, 5% CO2 cell culture incubator for 24 h.

[0033] (4) After incubation for 24 h, the toxicity of PGG to cells was determined using a CCK-8 kit. 10 μL of CCK-8 solution was added to each well and incubated for 1 h.

[0034] (5) Finally, a multifunctional microplate reader was used to measure and record the results. The following formula was used to calculate cytotoxicity: Cell viability (%) = [A (drug addition) - A (blank)] ÷ [A (0 drug addition) - A (blank)] × 100, where A (drug addition): OD value of the wells with cells, CCK-8 solution and drug solution, A (drug addition) group is the group with 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 100 μM PGG added respectively; A (0 drug addition): OD value of the wells with cells, CCK-8 solution but no drug solution, A (0 drug addition) group is the group with 0 μM PGG added; A (blank): OD value of the wells with cell maintenance solution and CCK-8 solution but no cells; Cell viability (%): cell proliferation activity or cytotoxicity.

[0035] 1.2.5 Virus amplification (1) First, select Vero cells with good growth conditions from the Vero cells that have undergone cell passaging in 1.2.2, and then wash them three times with PBS to remove the residual complete culture medium.

[0036] (2) Next, 0.1 MOI of PRV-GFP was inoculated into Vero cells and placed in a cell culture incubator at 37°C and 5% CO2.

[0037] (3) Then, the EVOSTM M5000 imaging system was used to observe the infection of PRV-GFP virus. When the lesions reached 80%-90%, the virus culture fluid was collected.

[0038] (4) Finally, freeze and thaw the cells three times in a -80°C refrigerator to release the virus particles in the cells. The virus solution was then dispensed into 1.5 mL EP tubes and stored for subsequent experiments.

[0039] 1.2.6 PGG antiviral test Preparation of drug dilution solution: dilute the PGG drug powder to 100 mM with DMSO, and then further dilute it to 2.5 μM, 5 μM, and 10 μM with cell maintenance solution according to the experimental requirements; add DMSO to the cell maintenance solution to obtain a cell maintenance solution containing 0.01% DMSO, and record the PGG concentration as 0 μM.

[0040] (1) First, select the Vero cells with good growth conditions from the Vero cells that have been passaged in 1.2.2 and plate them in a 24-well plate. When the cell density in the culture plate reaches 90%, remove the culture medium and wash the plate three times with PBS.

[0041] (2) Then, the same volume of 0 μM, 2.5 μM, 5 μM and 10 μM PGG dilutions were added to different wells of the 24-well plate obtained in step (1) as different concentrations of PGG groups; at the same time, 300 μL of cell maintenance solution with the same volume as the PGG dilution was added to the wells of the 24-well plate obtained in step (1) as the CK group. The CK group contained cells and cell maintenance solution but did not contain PGG. There were 4 replicates for each of the CK group and the different concentrations of PGG groups. Then the 24-well plate was placed in a cell culture incubator at 37 °C and 5% CO2 for 4 h.

[0042] (3) After incubation for 4 h, remove the supernatant and wash three times with PBS. Then, add PRV-GFP with an MOI of 0.1 to the wells with PGG at a concentration gradient of 0 μM, 2.5 μM, 5 μM, and 10 μM, place in a cell culture incubator at 37 °C and 5% CO2 for adsorption for 1 h, discard the virus solution, and add the corresponding dilution solution or maintenance solution according to step (2). Subsequently, the 24-well plate was continued to be cultured in a cell culture incubator at 37 °C and 5% CO2.

[0043] (4) 24 hours after virus infection, use the EVOSTM M5000 imaging system to take photos and record. Then, put the 24-well plate into an ultra-low temperature freezer at -80 °C and freeze and thaw three times to release the virus particles in the cells. Finally, transfer the collected virus and drug mixture into a 1.5 mL EP tube for subsequent experiments.

[0044] 1.2.7 TCID 50 Determination of (1) Before the experiment, select Vero cells with good growth condition from the Vero cells that have been passaged in 1.2.2, inoculate the Vero cells in a 96-well plate, and culture them in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0045] (2) After the drug and virus mixture collected in 1.2.6 PGG antiviral test is completely thawed, take 100 μL and add it to 900 μL of DMEM high-glucose medium for dilution to obtain a 10-fold dilution, which is recorded as the first gradient.

[0046] (3) Next, take 100 μL of the first gradient dilution and add it to 900 μL of DMEM high-glucose medium. This will give a 100-fold dilution relative to the original virus solution, which is recorded as the second gradient. Repeat this process for gradient dilutions.

[0047] (4) Then, add the diluted virus dilutions into a 96-well plate, add 100 μL to each well, and place it in a cell culture incubator at 37 °C and 5% CO2 for 72 h.

[0048] (5) During this period, the EVOSTM M5000 imaging system was used to observe and record the pathological changes of the virus.

[0049] (6) Finally, the Reed-Muench method was used to calculate the TCID of the virus. 50 Titer.

[0050] 1.2.8 Data Analysis All experiments were repeated at least three times independently, and GraphPad Prism 9.0 software was used for data analysis and plotting, and the results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used for statistical analysis. In the figure, * is used to indicate significant differences between groups, where * indicates P < 0.05, that is, significant differences; ** indicates P < 0.01, that is, very significant differences; *** indicates P < 0.001, that is, extremely significant differences.

[0051] 2 Results and Analysis 2.1 Determination of the maximum non-toxic concentration of PGG When the concentration of PGG on Vero cells was ≤20 μM, no obvious cytotoxicity was observed, and there was no statistical difference in the cell activity of each group compared with the 0 μM PGG group, with the cell viability ranging from 97.17% to 110.55% (e.g. Figure 1 The maximum non-toxic concentration of PGG in Vero cells is 20 μM.

[0052] 2.2 Effect of PGG on PRV-GFP infection in Vero cells As shown in Figure 2, after treatment with 2.5 μM, 5 μM and 10 μM PGG, the PRV-GFP fluorescence intensity in Vero cells was significantly weakened, and as the PGG concentration increased, the PRV-GFP fluorescence intensity gradually weakened, indicating that PGG has a significant inhibitory effect on the infection of PRV-GFP.

[0053] 2.3 TCID of PGG for PRV-GFP in Vero cells 50 Effect of titer As shown in Figure 3, as the concentration of PGG gradually increased, the TCID 50 The titer gradually decreased, with the maximum decrease being about 2.5 lg (about 316 TCID 50 ), indicating that the ability of PGG to inhibit PRV-GFP increased with the increase of its concentration, showing a dose-dependent effect.

[0054] The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited thereto. It should be pointed out that for those skilled in the art and any technician familiar with the technical field, without departing from the overall concept of the present invention, the technical solution and the inventive concept of the present invention are equivalently replaced or changed, and several changes and improvements are made, which should also be regarded as the protection scope of the present invention.

Claims

1. Application of 1,2,3,4,6-O-pentagalloylglucose and its derivatives in the preparation of antiviral drugs, characterized in that: The virus is specifically pseudorabies virus, and the 1,2,3,4,6-O-pentagalloyl glucose derivative is a pharmaceutically acceptable salt or a modified compound based on the 1,2,3,4,6-O-pentagalloyl glucose derivative.

2. The use of 1,2,3,4,6-O-pentagalloylglucose and its derivatives in the preparation of antiviral drugs according to claim 1, characterized in that: The pseudorabies virus strain is PRV-GFP, or one of its identical genotypes.

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