Application of natural flower glycoside substances in preparation of antiviral drugs

Abstract

The invention discloses an application of a natural anthocyanin substance in preparation of an antiviral drug, which is characterized in that anthocyanin is used as a potential effective component for inhibiting the activity of herpes virus DNA polymerase, and the activity of the DNA polymerase is inhibited through the combination of the anthocyanin and the DNA polymerase, so that a good anti-herpes virus effect is realized.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of a natural anthocyanin in the preparation of antiviral drugs. Background Technology

[0002] Currently, nucleoside analogues such as acyclovir and its derivatives are commonly used to treat herpesvirus infections (such as HSV1, HSV2, VZV, CMV, HHV6, HHV7, EBV, and KSHV). These drugs mainly work by inhibiting viral DNA polymerase. However, excessive and prolonged use of these drugs can lead to drug resistance in the virus and can also cause side effects such as kidney damage and neurotoxicity.

[0003] Natural anthocyanins possess diverse structures and rich biological activities, making them a valuable resource for antiviral drug development. However, there is currently no technical solution for preparing non-nucleoside drugs from natural anthocyanins to inhibit viral DNA polymerase activity. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of natural anthocyanins in the preparation of antiviral drugs. It uses anthocyanins as a potential effective component for inhibiting the activity of herpesvirus DNA polymerase. By binding anthocyanins to DNA polymerase, the activity of DNA polymerase is inhibited, thereby achieving a good anti-herpesvirus effect.

[0005] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem:

[0006] On the one hand, it provides an application of anthocyanins in the preparation of drugs against double-stranded DNA viruses.

[0007] On the other hand, it also provides drugs that combat double-stranded DNA viruses.

[0008] Preferably, the double-stranded DNA virus includes herpesvirus.

[0009] Preferably, the herpesvirus includes any one of HSV1, EBV, and KSHV.

[0010] Preferably, anthocyanins are used to inhibit viral DNA polymerase activity.

[0011] Preferably, the concentration of anthocyanins in the antiviral drug is 0.3-50 μM.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention uses anthocyanins as a potential effective component for inhibiting the activity of herpesvirus DNA polymerase. The ability of anthocyanins to directly inhibit DNA polymerase activity was detected by fluorescence method. In vitro cell experiments showed that anthocyanins have low toxicity and good safety characteristics. At low concentrations, they can inhibit the activity of herpesvirus DNA polymerase and further inhibit the synthesis of viral DNA to achieve a good anti-herpesvirus effect. Attached Figure Description

[0014] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The figures show the molecular dynamics simulation results of anthocyanin docking with HSV1 DNA polymerase (a), EBV DNA polymerase (b), and KSHV DNA polymerase (c), respectively.

[0016] Figure 2 This is a diagram showing the protein sample purification and analysis of HSV1 DNA polymerase (POL_HSV1), EBV DNA polymerase (POL_EBV), and KSHV DNA polymerase (POL_KSHV) in this invention.

[0017] Figure 3 This is a graph showing the test results of anthocyanins inhibiting the activity of HSV1 DNA polymerase, EBV DNA polymerase, and KSHV DNA polymerase in this invention.

[0018] Figure 4 The anthocyanin CC in this invention 50 The dose-response curve of the value.

[0019] Figure 5 The anthocyanins in this invention have anti-HSV1 EC 50 The dose-response curve of the value.

[0020] Figure 6 This is a diagram showing the docking results of HSV1 DNA polymerase and anthocyanins in this invention. In the diagram, the dashed lines represent hydrogen bond interactions, and the interacting residues are yellow stick-shaped structures.

[0021] Figure 7 This is a diagram showing the docking results of EBV DNA polymerase and anthocyanins in an embodiment of the present invention. In the diagram, the dashed lines represent hydrogen bond interactions, and the interacting residues are yellow stick-shaped structures.

[0022] Figure 8This is a diagram showing the docking results of HSKV DNA polymerase and anthocyanins in an embodiment of the present invention. In the diagram, the dashed lines represent hydrogen bond interactions, and the interacting residues are yellow stick-shaped structures. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0025] Example 1

[0026] Targeted natural product screening

[0027] Natural products with potential anti-herpesvirus activity were initially selected by literature review. Virtual screening and binding energy analysis were performed using Glide Dock and Prime. Molecular dynamics simulations were then conducted to determine the reliability of the docking results between different natural products and three herpesvirus DNA polymerases (HSV1 DNA polymerase POL_HSV1, EBV DNA polymerase POL_EBV, and KSHV DNA polymerase POL_KSHV).

[0028] like Figure 1 As shown, this application performed molecular dynamics simulations on the docking results of anthocyanins with POL_HSV1, POL_EBV, and POL_KSHV, respectively. The results showed that within a time interval of 100 ns, the RMSD (root mean square deviation) of the three DNA polymerases (i.e., "Protein") and anthocyanins (i.e., "Ligand") did not change significantly after binding. This indicates that the binding of DNA polymerases and anthocyanins is stable. Therefore, anthocyanins were ultimately identified as a potential molecule that can achieve anti-herpes virus effects by inhibiting DNA polymerase activity.

[0029] Expression and purification of the target protein

[0030] Using an insect cell (sf9 cell) expression system, protein samples of HSV1, EBV, and KSHV DNA polymerases were expressed and purified. The specific procedures are as follows:

[0031] 1. Using HieffClone Universal II One Step Cloning Homologous Recombination Kit from Yisheng Biotechnology, the DNA polymerase genes of HSV1, EBV and KSHV herpesviruses were cloned into the expression vector pFastBac 1 respectively (that is, each gene was cloned into a separate expression vector pFastBac 1, rather than cloning all three genes into the same expression vector pFastBac 1), and a Strep affinity purification tag was added to the C-terminus of each protein.

[0032] 2. The expression plasmid obtained in step 1 was transformed into DH10Bac competent cells to prepare recombinant rod granules. The obtained rod granules were used to transfect sf9 cells to obtain P1 generation virus. Then, the P1 generation virus was used to infect sf9 cells again to obtain P2 generation virus with higher titers suitable for protein expression. At this time, the P2 generation virus was added to sf9 cells to express the target protein. After 48-72 hours of expression, the cells were collected and the protein was purified.

[0033] 3. Protein purification

[0034] Cells were resuspended in 20 mL of Lysis buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 4 mM MgCl2, 2 mM DTT, 0.04% NP-40, 10% glycerol), and 1 mM PMSF, 220 μL of protease inhibitor (100×), and 200 μL of BiOlock were added. Cells were then vortexed. Cells were then disrupted by sonication. The disrupted cells were transferred to a sonication tube, balanced on a balance, and centrifuged in a Type 70Ti rotor at 4°C and 40,000 rpm for 1 hour.

[0035] After centrifugation, the supernatant was transferred to an adsorption column, bound to column powder, and slowly swirled on a mixer for 1 hour. The column powder was washed with 40 mL of Lysis buffer to remove any residual sterile solution. The column powder was resuspended in 4 mL of Wash buffer and transferred to a 10 mL column. A sample was taken, and 60 μL of LTEV protease solution was added for enzymatic digestion. The protein was recovered after 4 hours of digestion.

[0036] The recovered protein was further purified by molecular sieve using a buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 4 mM MgCl2, and 2 mM DTT. Finally, SDS-PAGE electrophoresis was performed to analyze and select protein samples with high purity and concentration of target protein bands, such as... Figure 2As shown, the three herpesvirus DNA polymerases POL_HSV1, POL_EBV and POL_KSHV isolated all had high purity. Finally, the target protein was concentrated and preserved using a 30kD ultrafiltration tube.

[0037] Figure 2 In the diagram, M represents the protein marker, W represents the whole-cell sample, Su represents the supernatant after centrifugation, FT represents the breakthrough sample after affinity chromatography, and R represents the sample after protein binding to the column.

[0038] Detection of the inhibitory activity of anthocyanins on viral DNA polymerase from natural products

[0039] Detection was performed using Biotium's EvaEZ fluorescent polymerase activity assay kit, etc. Natural product anthocyanins The inhibition of DNA polymerase activity of three herpesviruses—HSV1, EBV, and KSHV—is demonstrated through the following procedure:

[0040] A 100 mM anthocyanin solution was prepared using dimethyl sulfoxide (DMSO) as the solvent. A 10 mM anthocyanin solution and a 10% DMSO solution (negative control) were prepared by diluting the DMSO solution with buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 4 mM MgCl2). In a 96-well quantitative PCR plate, 10 μL of 2× reaction solution, 7 μL of H2O, 2 μL of anthocyanin solution (experimental group) or 10% DMSO solution (control group), and 1 μL of herpesvirus DNA polymerase solution were mixed thoroughly. Three replicates were set up for each sample. After sample addition, the plate was quickly sealed and placed in a real-time quantitative PCR instrument. The plate was run at an isothermal temperature of 37°C for 15 min, and fluorescence changes in the wavelength range of 465-510 nm were measured.

[0041] The results are as follows Figure 3 As shown, compared with the control group, anthocyanins inhibited the activities of three DNA polymerases, POL_HSV1, POL_EBV and POL_KSHV, especially EBV. The fluorescence value ΔF of POL_EBV did not change significantly over time.

[0042] Analysis of the anti-herpes virus activity of anthocyanins

[0043] I. CC 50 Determination of value

[0044] 1. Vero cells were cultured in complete medium at approximately 2 × 10⁻⁶. 4 Seed 100 μL of cells per well into a 96-well plate and incubate overnight at 37°C with 5% CO2 to form a cell monolayer. Observe under an inverted microscope. When the cells have covered about 80%-90% of the plate, discard the supernatant and set aside for later use.

[0045] 2. Dilute the anthocyanin solution with DMSO according to the concentrations listed in Table 1, resulting in eight concentration gradients: 50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, and 0.02 μM. Each concentration was prepared in duplicate (wells A and B). Two wells were also prepared as a blank control group (containing cells, culture medium, and CCK-8 solution, but without anthocyanin solution) and two wells as a CCK-8 blank control group (containing culture medium and CCK-8 solution, but without cells and anthocyanin solution). Add 100 μL of both the anthocyanin solution and the control group solution to the corresponding wells and incubate at 37°C with 5% CO2 for 48 hours.

[0046] Table 1. Schematic diagram of cytotoxic plating

[0047]

[0048] 3. After culturing for 48 hours, discard the supernatant, add 100 μL of CCK-8 solution to each well, and incubate for 1 hour in a cell culture incubator at 37°C and 5% CO2.

[0049] 4. After incubation for 1 hour, the absorbance of each well was measured at 450 nm and 630 nm using an ELISA reader. The results were recorded, and cell viability was calculated to determine the maximum non-toxic concentration range of the anthocyanin solution for cells. The CC value was also calculated. 50 value.

[0050] The formula for calculating cell viability is as follows:

[0051]

[0052] In formula (1):

[0053] As represents the absorbance (OD) of the anthocyanin solution experimental group (including cells, culture medium, CCK-8 solution, and anthocyanin solution) at each well. 450 -OD 630 );

[0054] Ac(Blank) is a blank control group for cells (containing cells, culture medium, CCK-8 solution, but without anthocyanin solution). The absorbance (OD) of each well is... 450 -OD 630 );

[0055] Ab (CCK-8 control) is the CCK-8 blank control group (containing culture medium and CCK-8 solution, but without cells and anthocyanin solution) absorbance (OD). 450 -OD 630 ).

[0056] CC50 The value calculation process is as follows:

[0057] Import the data into GraphPad Prism 8.0 software, use "log(inhibitor) vs. Response-Variable slope(four parameters)" to fit the curve, select the nonlinear regression fitting option, and the results are as follows. Figure 4 As shown, and according to Figure 4 Determine CC 50 >500μM.

[0058] II. EC 50 Determination of value

[0059] 1. Vero cells were cultured in a medium containing 10% fetal bovine serum at a concentration of approximately 2 × 10⁻⁶. 4 Seed 100 μL of cells per well into a 96-well plate and cultured overnight at 37°C to form a cell monolayer. When the cells have spread to approximately 90%-100% confluence, discard the supernatant and set aside for later use.

[0060] 2. Dilute the anthocyanin solution with DMSO according to the concentrations in Table 2, with a total of 8 concentration gradients, namely 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 μM.

[0061] Table 2. Schematic diagram of antiviral cladding

[0062]

[0063] 3. Dilute the HSV1 virus to the required titer using DMEM medium based on the total number of cells per well, so that the MOI value is 0.02.

[0064] 4. Take out the 96-well plate containing Vero cells. First, add 100 μL of diluted anthocyanin solution to the corresponding culture wells, and then add 100 μL of virus diluent to the corresponding culture wells (two replicates for each concentration, i.e., well A and well B) to serve as the experimental group. Separately set up a positive control group (add 100 μL of medium containing 1% DMSO and 100 μL of virus diluent) and a negative control group (add 100 μL of 2% medium and 100 μL of medium containing 1% DMSO). Place the culture plate in an incubator at 37°C and 5% CO2 and incubate for 48 hours.

[0065] 5. After culturing for 48 hours, discard the supernatant and rinse once with PBS. Then add 100 μL of CCK-8 solution to each well and incubate for 1 hour in an incubator at 37°C and 5% CO2.

[0066] 6. After 1 hour of incubation, the absorbance of each well was measured at 450 nm and 630 nm using an ELISA reader. The results were recorded, and the cell inhibition rate and EC50 were calculated. 50 value.

[0067] The formula for calculating the inhibition rate is as follows:

[0068]

[0069] In equation (2): Bs is the absorbance of the experimental group pore (OD). 450 -OD 630 Bc represents the absorbance (OD) of the negative control group. 450 -OD 630 Bb represents the absorbance (OD) of the positive control group. 450 -OD 630 ).

[0070] EC 50 The value calculation process is as follows:

[0071] Import the data into GraphPad Prism 8.0 software, use "log(inhibitor) vs. Response-Variable slope(four parameters)" to fit the curve, select the nonlinear regression fitting option, and the results are as follows. Figure 5 As shown, and according to Figure 5 Determine EC 50 =26.53μM.

[0072] In this invention, the term "CC" is used. 50 "50% cytotoxic concentration" refers to the concentration at which a compound can cause 50% of cells to lose activity or die in in vitro experiments. It is used as an important indicator for assessing the cytotoxicity of drugs or compounds. 50 The lower the value, the stronger the toxicity of the compound to cells.

[0073] The term "EC" used 50 "EC" refers to the concentration required for a compound to produce 50% of its maximum effect in in vitro experiments. This effect can be, for example, inhibiting the activity of a certain enzyme, activating a certain receptor, or promoting cell proliferation. 50 The lower the value, the more significant the biological effect of the compound at a lower concentration, which usually indicates that the compound has high activity.

[0074] The term used is "titer," which refers to the number of virus particles in a given volume of medium and is usually used to measure the concentration of a virus suspension.

[0075] The term "MOI value" refers to the ratio of the number of viral particles to the number of host cells in an infection experiment, reflecting the average number of viral particles that each cell is exposed to.

[0076] Simultaneously, the docking effect between anthocyanins and herpesvirus DNA polymerase was analyzed based on the crystal structures of HSV1 DNA polymerase, EBV DNA polymerase, and KSHV DNA polymerase, respectively. Figure 6-8 As shown, anthocyanins interact extensively with all three DNA polymerases, indicating that anthocyanins and DNA polymerases can bind stably.

[0077] Example 2:

[0078] This embodiment provides a A drug for treating double-stranded DNA viruses, comprising anthocyanins for inhibiting viral DNA polymerase activity, wherein the double-stranded DNA virus includes herpesviruses, specifically any one of HSV1, EBV, and KSHV, and wherein the concentration of chebulin tannic acid in the drug is 0.3-50 μM.

[0079] In summary, this invention utilizes anthocyanins, a natural product, as a potentially effective component for inhibiting herpesvirus DNA polymerase activity. Furthermore, its ability to directly inhibit DNA polymerase activity was detected using fluorescence methods. In vitro cell experiments demonstrated that anthocyanins have a significant inhibitory effect on CC... 50 It can maintain low cytotoxicity at >500 μM while exhibiting moderate antiviral activity (EC50). 50 =26.53μM).

[0080] This demonstrates that anthocyanins have low toxicity and good safety, and can inhibit the activity of herpesvirus DNA polymerase at low concentrations, further inhibiting the synthesis of viral DNA to achieve good anti-herpesvirus activity.

[0081] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The application of an anthocyanin in the preparation of drugs against double-stranded DNA viruses.

2. The application as described in claim 1, characterized in that, The double-stranded DNA virus includes herpesviruses.

3. The application as described in claim 2, characterized in that, The herpesviruses include any one of HSV1, EBV, and KSHV.

4. The application as described in claim 1, characterized in that, Anthocyanins are used to inhibit viral DNA polymerase activity.

5. A drug for combating double-stranded DNA viruses, characterized in that, The antiviral drug contains anthocyanins.

6. The drug as described in claim 5, characterized in that, The double-stranded DNA virus includes herpesviruses.

7. The drug as described in claim 5, characterized in that, The herpesviruses include any one of HSV1, EBV, and KSHV.

8. The drug as described in claim 5, characterized in that, Anthocyanins are used to inhibit viral DNA polymerase activity.

9. The drug as described in claim 5, characterized in that, The concentration of anthocyanins in the antiviral drug is 0.3-50 μM.

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