Use of rutin in anti-cosxackie virus drugs

Abstract

The present application relates to a kind of application of rutin in anti-cosavirus drug, it relates to the field of medicine.Rutin can be combined with CVB5-3C protein target highly stable by van der waals force, hydrogen bond, inhibit the replication of cosavirus, reach the effect of treating and / or preventing cosavirus.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and in particular to the use of rutin in anti-coxsackie virus drugs. Background Technology

[0002] Coxsackievirus is a common enterovirus that usually infects humans through the respiratory or digestive tract. After infection, symptoms include fever, sneezing, and coughing, similar to those of a common cold. Coxsackievirus is divided into two types: Group A and Group B. Type A infection is more common in children, while adult infections account for 21.7%. The incubation period is 1-3 days, with symptoms including upper respiratory tract infection, rapid onset, runny nose, cough, sore throat, fever, and general malaise.

[0003] Group B Coxsackieviruses (CVB) are non-enveloped small RNA viruses belonging to the genus Enterovirus in the family Picornaviridae. CVB comprises six serotypes (CVB 1–6), which cause a wide range of illnesses, from the common cold to severe myocarditis and encephalitis. CVB is a leading cause of enterovirus-induced acute myocarditis and cardiomyopathy. However, effective antiviral therapies remain unavailable.

[0004] Vaccines and antiviral drugs are the most effective treatments for enteroviruses. However, the EV71 inactivated vaccine currently available in my country is a monovalent vaccine targeting only the EV71 C4 subtype and offers no cross-immune protection against CVB5 virus. CVB infection causes a variety of illnesses, ranging from the common cold to myocarditis, encephalitis, and pancreatitis. There are no specific antiviral drugs available to treat CVB infection. Furthermore, due to the lengthy, complex, and costly development process of vaccines, and the high mutation rate and diversity of enteroviruses, there are currently no reports of other enterovirus vaccine developments, and no antiviral drugs have been approved by the FDA for the treatment of any enterovirus infection. Therefore, there is an urgent need to develop new treatments and drugs for enteroviruses. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides the application of rutin in the preparation of drugs for the treatment and / or prevention of Coxsackievirus. Rutin can bind stably to the CVB5-3C protein target through van der Waals forces and hydrogen bonds, and then bind tightly to the VP1 protein containing the main antigen binding site, thereby inhibiting the replication of Coxsackievirus and achieving the effect of treating and / or preventing Coxsackievirus.

[0006] This invention provides the use of rutin in the preparation of drugs for treating and / or preventing Coxsackie virus, wherein the structure of rutin is shown below:

[0007]

[0008] Enteroviruses share similar genetic structures. Coxsackievirus B5 (CVB5) belongs to group B5 of enteroviruses. CVB5 contains a forward-stranded single-stranded RNA genome of approximately 7.4 kb, consisting of a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and an open reading frame (ORF). The 5'-UTR typically folds into a specific spatial structure and contains an internal ribosome entry site (IRES) for binding to the 40S ribosomal subunit to initiate cap-independent translation. The 3'-UTR contains a variable poly(A) tail, which is crucial for CVB5 replication. The ORF is subdivided into P1, P2, and P3 regions. P1 encodes four structural proteins: VP1, VP2, VP3, and VP4; P2 encodes three non-structural proteins: 2A, 2B, and 2C; and P3 encodes four non-structural proteins: 3A, 3B, 3C, and 3D. VP1, VP2, and VP3 are exposed on the surface of the viral capsid, forming the CVB5 viral particle capsid complex. VP4 is embedded inside the viral capsid and tightly connected to the viral core. VP1 of CVB5 contains the main antigen-binding site, directly determining the virus's antigenicity and being a major determinant of viral neutralization. The N-terminus of the VP1 capsid protein has an important antigenic region, exhibiting high immunogenicity. The C-terminus of the VP1 capsid protein can stimulate the production of neutralizing antibodies. Among non-structural proteins, 2C, 3C, and 3D are evolutionarily most conserved, while 2A, 2B, and 3A are highly variable in enteroviruses. The 3C protease contains 183 amino acids and has proteolytic activity. During enterovirus replication, the 3C protein catalyzes the cleavage of viral precursor proteins, forming mature structural and non-structural proteins. Most enterovirus precursor proteins can only undergo subsequent replication and encapsulation after being hydrolyzed by the 3C protease to form functional proteins. Therefore, the 3C protease is the core protease for the hydrolysis of enterovirus precursor polymers and plays a crucial role in enterovirus replication. 3C proteases also have many functions, such as promoting viral replication and enhancing host cell apoptosis. Inhibiting their catalytic function can effectively inhibit the cleavage of enterovirus precursor proteins and block viral replication, making them an important target for enterovirus drug therapy research.

[0009] Rutin is a flavonoid compound widely found in various plants, possessing a variety of biological activities including anti-inflammatory, hypoglycemic, antioxidant, neuroprotective, nephroprotective, hepatoprotective, and Aβ oligomer-reducing activities. Based on this, the inventors proposed using rutin for the treatment and prevention of Coxsackievirus. Cellular experiments verified that rutin can bind stably to the CVB5-3C protein target via van der Waals forces and hydrogen bonds, and subsequently bind tightly to the VP1 protein containing the main antigen-binding site, thereby inhibiting Coxsackievirus replication and achieving therapeutic and / or preventative effects.

[0010] In one embodiment, the Coxsackievirus includes Group A Coxsackievirus and Group B Coxsackievirus.

[0011] In one embodiment, the Group B Coxsackievirus includes six serotypes: Coxsackievirus B1-6.

[0012] In one embodiment, the treatment and / or prevention drug for Coxsackie virus also includes pharmaceutically acceptable excipients and / or drugs compatible with rutin.

[0013] In one embodiment, the excipients include at least one of solvents, disintegrants, flavoring agents, colorants, lubricants, antioxidants, preservatives, binders, fillers, or thickeners.

[0014] In one embodiment, the working concentration of the rutin is ≥20 μM.

[0015] In one embodiment, the working concentration of rutin is 20-100 μM.

[0016] The working concentration is the concentration at which the above-mentioned components can achieve the expected effect when used. It is understood that when those skilled in the art prepare the above-mentioned drugs containing rutin, they can prepare a stock solution or stock solution with a higher concentration and then dilute it when used. The stock solution or stock solution and its concentration are both within the protection scope of this invention.

[0017] In one embodiment, the dosage form of the drug for treating and / or preventing Coxsackie virus includes capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents, or dispersible tablets.

[0018] In one embodiment, the route of administration of the treatment and / or prevention drug for Coxsackie virus includes oral or intravenous injection.

[0019] The present invention also provides a medicament for treating and / or preventing Coxsackievirus, comprising a pharmaceutical active ingredient and excipients, wherein the pharmaceutical active ingredient is rutin.

[0020] In one embodiment, the drug inhibits the replication of Coxsackievirus B5 by binding to the VP1 protein of Coxsackievirus B5.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention relates to the application of rutin in the preparation of drugs for the treatment and / or prevention of Coxsackievirus. Rutin can bind stably to the CVB5-3C protein target via van der Waals forces and hydrogen bonds, and then tightly bind to the VP1 protein of Coxsackievirus B5, which contains the major antigen-binding site, thereby inhibiting the replication of Coxsackievirus B5 and achieving the effect of treating and / or preventing Coxsackievirus. This invention verified, through experiments using non-toxic concentrations of rutin to infect RD cells with CVB5 virus, that rutin can dose-dependently inhibit the replication of CVB5 virus. Rutin provides a new natural small molecule drug option for the prevention and treatment of Coxsackievirus infectious diseases, and has significant research and development value and significance for the prevention and control of Coxsackievirus. Attached Figure Description

[0023] Figure 1 The chemical structural formula of rutin in the examples is ( Figure 1 A) and a schematic diagram illustrating the molecular docking results of rutin and enterovirus 3C protein ( Figure 1 B);

[0024] Figure 2 This is a graph showing the results of CCK8 assay on changes in the viability of RD cells after Rutin treatment, as illustrated in the example.

[0025] Figure 3 The image shows the results of CCK8 assay detection of the antiviral activity of Rutin against CVB5 in RD cells in the example.

[0026] Figure 4 This is a diagram showing the inhibitory effect of Rutin on CVB5-VP1 protein expression in the examples;

[0027] Figure 5 This is a graph showing the inhibitory effect of Rutin on the transcriptional level of CVB5 virus in the examples.

[0028] Figure 6 This is a diagram showing the results of Rutin inhibiting CVB5 virus-induced plaque formation in the examples.

[0029] Figure 7 The figure shows the effect of Rutin on CPE caused by the CVB5 virus in the example. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0033] Example

[0034] I. Chemical structure and molecular docking analysis of Rutin.

[0035] Rutin was purchased from medchemexpress (HY-N0148), chemical structure as follows: Figure 1 As shown in Figure A, the 3C protease is a key protease of the CVB5 virus, which is highly conserved and is an important target for drug treatment research on the CVB5 virus.

[0036] To assess the affinity of Rutin for the CVB53C protein, the inventors performed molecular docking analysis. The 3D file of the CVB5-3C protein (PDB:5NFS) was downloaded from the PDB database, and the binding posture and interaction between Rutin and the CVB53C protein were obtained using Autodock Vina v.1.2.2, generating the binding energy of the interaction.

[0037] Figure 1 The results showed that Rutin binds to the CVB5-3C protein target via van der Waals forces and hydrogen bonds, with a low binding energy of -6.59 kcal / mol, indicating highly stable binding.

[0038] II. CCK8 assay for cell viability.

[0039] Human malignant embryonic rhabdomyosarcoma cell line (RD cells) was cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. One day prior to incubation, cells were seeded into 96-well plates at a density of 5000 cells / well (6 replicates per group). After 24 hours of growth, different final concentrations (50, 100, 200, 400, 800, 1600, 3200 μM) of Rutin were added for incubation. After another 48 hours of culture, 10 μL of CCK8 was added to each well for further incubation. The absorbance was measured at 450 nm using a microplate reader after 1 hour. Figure 2 The results showed that Rutin was non-toxic to cells at concentrations of 0-800 μM, and subsequently inhibited the growth of RD cells in a concentration-dependent manner.

[0040] III. The impact of CCK8 assay on Rutin on viral replication.

[0041] RD cells were seeded into 96-well plates and incubated overnight. RD cells were then infected with the enterovirus strain CVB5 (MZ826339.1) at an MOI of 0.5. CVB5 virus was diluted in FBS-free DMEM medium, with 100 μL added to each well, and incubated in a cell culture incubator at 37°C with 5% CO2. After 1.5 h, the supernatant was discarded, and the cells were further incubated with different concentrations of Rutin (20, 40, 60, 80, 100 μM) for 48 h. Cell viability was measured using the CCK-8 assay.

[0042] The results showed that 20 μM Rutin significantly reduced the cytopathic effect induced by CVB5 infection, provided significant protection to infected cells, and improved cell survival. Figure 3 ).

[0043] IV. Western blot analysis of the effect of Rutin on viral replication.

[0044] RD cells were seeded in 12-well plates and infected with CVB5 virus (MOI = 0.5) after 24 h. RD cells were incubated with 40 and 80 μM Rutin after 2 h. Cells were collected 24 h and 36 h post-infection and lysed on ice for 10 min using Pierce™-IP-Lysis-Buffer (87787) (purchased from Thermo Scientific). The cells were centrifuged at 12,000 rpm for 3 min at 4 °C, and the supernatant was collected. 5× protein loading buffer (purchased from Beyotime Biotechnology) was added, and the protein sample was denatured by boiling in a metal bath for 10 min.

[0045] SDS-PAGE gels (purchased from KGI Biotechnology Co., Ltd.) were loaded into the electrophoresis tank. After adding electrophoresis buffer, an appropriate amount of protein sample was added to the gel wells. Electrophoresis was performed at a constant voltage of 90V for 25 min for the upper stacking gel and 110V for 50 min for the lower separating gel. PVDF membranes were activated by immersing them in methanol for 15 s. After electrophoresis, the gels were transferred at a constant current of 200 mA for 60 min. After transfer, the membranes were washed once with PBST and then blocked by shaking in 5% skim milk powder (PBST) at room temperature for 2 h. The PVDF membranes were then incubated overnight at 4°C with the primary antibody Mouse anti-CVB5-VP1 (purchased from Kincare Biotechnology Co., Ltd.) and Mouse anti-Beta-Tubulin (66240-1) (purchased from Wuhan Sanying Biotechnology Co., Ltd.). After washing with TBST, the corresponding secondary antibody (anti-mouse IgG HRP-linked antibody (#7076) purchased from CST) was added and incubated at room temperature for 1 hour for binding. After washing, a chromogenic reagent (Super ECL Plus purchased from UELandy) was added, and signal detection and image analysis were performed using a chemiluminescence imaging system in a gel imaging system. Figure 4 The results showed that Rutin treatment significantly inhibited the expression of CVB5 virus VP1 protein compared with the virus infection group alone.

[0046] V. qRT-PCR quantitative detection of the effect of Rutin on viral replication.

[0047] RD cells were seeded in 12-well plates and infected with CVB5 virus (MOI = 0.5) after 24 hours. Two hours post-infection, RD cells were incubated with 40 and 80 μM Rutin. Infected cells were collected at 24 and 36 hours, respectively, and total RNA was extracted using an RNA extraction kit. cDNA was synthesized by reverse transcription using the ReverTra Ace qPCR RT Kit (TOYOBO). qRT-PCR experiments were performed using a 2×SYBR Green Real-time PCR Mix (TOYOBO) kit. Reactions were performed on an LC480 real-time PCR instrument. Figure 5 The results showed that Rutin could significantly inhibit the transcription levels of CVB5 viral VP1 and 3C mRNA.

[0048] VI. Determination of the inhibitory effect of Rutin on CVB5 virus replication by plaque assay.

[0049] The supernatant of Vero cells infected with CVB5 virus was collected and stored at -80°C. Vero cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Vero cells were seeded into 12-well plates. After the Vero cells formed a monolayer, they were washed three times with PBS and then incubated with the collected supernatant diluted 100-fold. After 1.5 hours, the supernatant was replaced with fresh medium containing an equal volume of 2× serum-free DMEM and microcrystalline cellulose (2× DMEM: 3% microcrystalline cellulose = 1:1) to cover the Vero cells. After 72 hours, the cells were fixed with 4% paraformaldehyde for 10 minutes and stained with 2% crystal violet for 10 minutes. The crystal violet staining solution was discarded, and the number of empty plaques was observed and counted. Figure 6 Compared with the control group infected with the virus alone, the Rutin treatment group significantly reduced the number of cell plaques caused by the virus infection, indicating that Rutin has a significant inhibitory effect on the replication of CVB5 virus.

[0050] 7. Rutin can alleviate the cytopathic effect caused by CVB5 infection.

[0051] RD cells were seeded into 6-well plates and incubated overnight at 37°C with 5% CO2. After infecting RD cells with CVB5 (MOI = 0.5) for 1.5 hours, the cell supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 40 μM and 80 μM Rutin were added for incubation, respectively. Controls were provided for cells without virus and without Rutin, and cells with virus only. The 6-well plates were incubated continuously, and cell morphology was observed and photographed the following day. Figure 7 As shown, compared with normal RD cells, enterovirus CVB5 infection of RD cells causes cytopathic effects (CPE) such as cell shrinkage, detachment, and death. However, incubation with Rutin can significantly alleviate this effect, providing significant protection to infected cells and improving cell survival. This indicates that Rutin does indeed have a significant inhibitory effect on CVB5 virus.

[0052] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0053] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of rutin as the sole active ingredient in the preparation of a drug for treating and / or preventing Coxsackievirus, wherein the Coxsackievirus is Coxsackievirus B5, and the structure of the rutin is shown below: Formula I.

2. The application according to claim 1, characterized in that, The drugs for treating and / or preventing Coxsackievirus also include pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The excipients include at least one of solvents, disintegrants, flavoring agents, colorants, lubricants, antioxidants, preservatives, binders, fillers, or thickeners.

4. The application according to claim 1, characterized in that, The working concentration of rutin is ≥20 μM.

5. The application according to any one of claims 1-4, characterized in that, The dosage forms of the drugs for treating and / or preventing Coxsackievirus include capsules, granules, tablets, oral liquids, pills, injections, or ointments.

6. The application according to claim 5, characterized in that, The routes of administration for the treatment and / or prevention of Coxsackievirus include oral or intravenous injection.

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