Application of rutin in anti-coxsackie virus medicine

Abstract

The invention relates to application of rutin in an anti-coxsackie virus drug, and relates to the field of drugs. Rutin can be highly and stably combined with a CVB5-3C protein target through Van der Waals force and hydrogen bonds, replication of the coxsackie virus is inhibited, and the effect of treating and / or preventing the coxsackie virus is achieved.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to application of rutin in anti-Coxsackie virus medicines. Background Art

[0002] Coxsackievirus is a common enterovirus that infects the human body through the respiratory tract and digestive tract in most cases. After infection, the human body will have cold symptoms such as fever, sneezing, and coughing. Coxsackievirus is divided into two types: Group A and Group B. Coxsackievirus A infection is more common in children, and adult infection accounts for 21.7%. The incubation period is 1 to 3 days, and upper respiratory tract infection occurs, with an acute onset, runny nose, cough, sore throat, fever, and general discomfort.

[0003] Group B coxsackievirus (CVB) is a non-enveloped small RNA virus of the genus Enterovirus in the family Picornaviridae. CVB includes six serotypes (CVB 1-6) that cause a variety of illnesses ranging from the common cold to severe myocarditis and encephalitis. CVB is the leading cause of enterovirus-induced acute myocarditis and cardiomyopathy. However, effective antiviral therapy remains unavailable.

[0004] Vaccines and antiviral drugs are the most effective means to treat enterovirus. However, the EV71 inactivated vaccine that has been launched in my country is a monovalent vaccine targeting only the EV71 C4 subtype, and has no cross-immune protection against the CVB5 virus. CVB infection causes a variety of symptoms ranging from the common cold to myocarditis, encephalitis and pancreatitis. There are no specific antiviral drugs available to treat CVB infection. In addition, due to the long development time, high difficulty and high R&D funding of vaccines, and the large number of enterovirus species with a high probability of mutation, there have been no reports on the development of other enterovirus vaccines, 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 anti-enterovirus treatment methods and drugs. Summary of the invention

[0005] In view of the above problems, the present invention provides an application of rutin in the preparation of a drug for treating and / or preventing coxsackievirus. Rutin can highly and stably bind to the CVB5-3C protein target through van der Waals forces and hydrogen bonds, and then tightly bind 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] The present invention provides an application of rutin in preparing a drug for treating and / or preventing coxsackie virus. The structure of the rutin is shown below:

[0007]

[0008] Enteroviruses have similar gene structures. Coxsackievirus B5 (CVB5) belongs to enterovirus group B type 5. CVB5 contains a positive single-stranded RNA genome of about 7.4 kb, which consists of a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR) and an open reading frame (ORF). The 5'-UTR is usually folded 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 very important for CVB5 replication. ORF is subdivided into P1, P2 and P3 regions. P1 encodes four structural proteins, namely VP1, VP2, VP3, and VP4. P2 encodes three non-structural proteins 2A, 2B, and 2C. P3 encodes four non-structural proteins 3A, 3B, 3C, and 3D. VP1, VP2 and VP3 are exposed on the surface of the virus shell to form the CVB5 virus particle capsid complex, and VP4 is embedded in the inside of the virus shell and tightly connected to the virus core. The VP1 of CVB5 contains the main antigen binding site that directly determines the antigenicity of the virus and is the main determinant of virus neutralization. The N-terminus of the VP1 capsid protein has an important antigenic region and exhibits high immunogenicity. The C-terminus of the VP1 capsid protein can stimulate the production of neutralizing antibodies. Among the non-structural proteins, 2C, 3C and 3D are the most conserved in evolution, while 2A, 2B and 3A are highly variable in enterovirus. The 3C protease contains 183 amino acids and has proteolytic activity. During the replication of enterovirus, the 3C protein catalyzes the cleavage of viral precursor proteins to form mature structural proteins and non-structural proteins. That is, most enterovirus precursor proteins can only be subsequently replicated and encapsulated after being hydrolyzed by 3C protease to form functional proteins. Therefore, the 3C protease is the core protease for the hydrolysis of enterovirus precursor polyproteins and plays a vital role in the replication of enterovirus. 3C protease also has many functions, such as promoting viral replication and enhancing host cell apoptosis. Inhibiting its catalytic function can effectively inhibit the cleavage of enterovirus precursor protein and block viral replication. It is an important target for enterovirus drug treatment research.

[0009] Rutin is a flavonoid compound widely found in many plants, and has multiple biological activities such as anti-inflammatory, hypoglycemic, anti-oxidant, neuroprotective, renal protective, liver protective and reducing the activity of Aβ oligomers. Based on this, the inventors proposed to use rutin to treat and prevent coxsackievirus, and verified through cell experiments that rutin can be highly stably bound to the CVB5-3C protein target through van der Waals forces and hydrogen bonds, and then tightly bound 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.

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

[0011] In one embodiment, the group B coxsackievirus includes 6 serotypes: coxsackievirus B1-6.

[0012] In one embodiment, the drug for treating and / or preventing Coxsackie virus further comprises a pharmaceutically acceptable excipient and / or a drug compatible with rutin.

[0013] In one embodiment, the excipients include at least one of a solvent, a disintegrant, a flavoring agent, a colorant, a lubricant, an antioxidant, a preservative, a binder, a filler or a thickener.

[0014] In one embodiment, the working concentration of 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 ingredients can achieve the expected effect when used. It can be understood that those skilled in the art can prepare a mother solution or a stock solution with a higher concentration when preparing the above-mentioned rutin-containing medicine, and then dilute it when it is used. The mother solution or the stock solution and its concentration are all within the protection scope of the present 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 administration route of the drug for treating and / or preventing coxsackie virus includes oral administration or intravenous injection.

[0019] The present invention also provides a medicine for treating and / or preventing Coxsackie virus, comprising a medicine active ingredient and an auxiliary material, wherein the medicine 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] The present invention discloses an application of rutin in the preparation of a drug for treating and / or preventing coxsackieviruses. Rutin can be highly stably bound to a CVB5-3C protein target through van der Waals forces and hydrogen bonds, and then tightly bound to the VP1 protein of coxsackievirus B5 containing a major antigen binding site, thereby inhibiting the replication of coxsackievirus B5, and achieving the effect of treating and / or preventing coxsackieviruses. The present invention uses non-toxic concentrations of Rutin to carry out CVB5 virus infection RD cell experiments, and verifies that Rutin can inhibit the replication of CVB5 virus in a dose-dependent manner. Rutin provides a new natural small molecule drug option for the prevention and treatment of coxsackievirus infectious diseases, and has important research and development value and development significance for the prevention and treatment of coxsackieviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the chemical structural formula of rutin in the embodiment ( Figure 1 A) and schematic diagram of the molecular docking analysis of rutin and enterovirus 3C protein ( Figure 1 B);

[0024] Figure 2 This is a graph showing the results of CCK8 detecting the change in viability of RD cells after Rutin treatment in the embodiment;

[0025] Figure 3 This is a graph showing the results of detecting the antiviral activity of Rutin against CVB5 in RD cells using the CCK8 method in the examples;

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

[0027] Figure 5 This is a result diagram showing that Rutin has an inhibitory effect on the transcription level of CVB5 virus in the embodiment;

[0028] Figure 6 This is a result diagram showing that Rutin can inhibit the plaque formation induced by CVB5 virus in the example;

[0029] Figure 7 This is a graph showing the effect of Rutin on CPE caused by CVB5 virus in the example. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0032] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0033] Example

[0034] 1. Chemical structure and molecular docking analysis of Rutin.

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

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

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

[0038] 2. CCK8 method was used to determine cell viability.

[0039] Human malignant embryonic rhabdoid tumor cell line (RD cell) was cultured in DMEM medium containing 10% fetal bovine serum and placed in a 37°C, 5% CO2 incubator for culture. One day in advance, 5000 cells / well were inoculated in 96-well plates in 6 replicates per group. After 24 hours of growth, different final concentrations of Rutin (50, 100, 200, 400, 800, 1600, 3200 μM) were added for incubation. After 48 hours of continuous culture, 10 μL CCK8 was added to each well for incubation, and the absorbance was detected at 450 nm using an ELISA reader after 1 hour. Figure 2 The results showed that Rutin had no toxicity to cells at a concentration of 0-800 μM, and then inhibited the growth of RD cells in a concentration-dependent manner.

[0040] 3. CCK8 detects the effect of Rutin on viral replication.

[0041] RD cells were inoculated into 96-well plates and incubated overnight. RD cells were infected with enterovirus strain CVB5 (MZ826339.1) at MOI = 0.5. CVB5 virus was diluted in DMEM medium without FBS, and 100 μL was added to each well. The cells were incubated in a cell culture incubator at 5% CO2 and 37°C. After 1.5 h, the supernatant was discarded, and different concentrations of Rutin (20, 40, 60, 80, 100 μM) were used to continue incubation for 48 h, and cell viability was determined using the CCK-8 assay.

[0042] The results showed that 20 μM Rutin could significantly reduce the cytopathic effect induced by CVB5 infection, play a significant protective role on infected cells, and increase the cell survival rate ( Figure 3 ).

[0043] 4. Western blot was used to detect the effect of Rutin on viral replication.

[0044] RD cells were inoculated in a 12-well plate, and CVB5 virus (MOI = 0.5) was added for infection 24 hours later. RD cells were incubated with 40 and 80 μM Rutin 2 hours later. Cells were collected 24 hours and 36 hours after infection, and cells were lysed on ice for 10 minutes using PierceTM-IP-Lysis-Buffer (87787) solution (purchased from Thermo-Scientific). Centrifuged at 12,000-rpm for 3 minutes at 4°C using a low-temperature centrifuge, and 5× protein loading buffer (purchased from Beyotime) was added to the supernatant, and the protein sample was boiled in a metal bath for 10 minutes to denature it.

[0045] The SDS-PAGE gel (purchased from Keygen Biotech Co., Ltd.) was loaded into the electrophoresis tank, and after adding the electrophoresis solution, an appropriate amount of protein sample was added to the gel well, and electrophoresis was performed according to the upper concentrated gel constant voltage of 90V, 25min, and the lower separation gel constant voltage of 110V, 50min. The PVDF membrane was soaked in methanol for 15s for activation, and then the gel after electrophoresis was transferred to the membrane at a constant current of 200mA for 60min. After the transfer, the membrane was rinsed once with PBST, and then immersed in 5% skim milk powder prepared with PBST and blocked on a shaker at room temperature for 2h. After that, the primary antibody Mouse anti-CVB5-VP1 (purchased from Jinkairui Company) and Mouse anti-Beta-Tubulin (66240-1) were purchased from Wuhan Sanying Biotechnology Co., Ltd., and the PVDF membrane was incubated at 4°C overnight. After washing with TBST, the corresponding secondary antibody (anti-mouse IgG HRP-linked antibody (#7076) purchased from CST) was added for 1 hour of incubation at room temperature. After washing, the color developer (Super ECL Plus purchased from UElandy) was added and the signal detection and image analysis were performed using a chemiluminescence imaging system in a gel imager. Figure 4 The results showed that compared with the simple virus infection group, Rutin treatment could significantly inhibit the expression of CVB5 virus VP1 protein.

[0046] 5. qRT-PCR quantitatively detects the effect of Rutin on viral replication.

[0047] RD cells were inoculated in a 12-well plate, and CVB5 virus (MOI = 0.5) was added for infection 24 hours later. RD cells were incubated with 40 and 80 μM Rutin 2 hours after infection. Infected cells were collected at 24 and 36 hours, respectively, and total cell RNA was extracted using an RNA extraction kit. ReverTra Ace qPCR RT Kit (TOYOBO) was used for reverse transcription and synthesis of cDNA. qRT-PCR experiments were performed using a 2×SYBR Green Real-time PCR Mix (TOYOBO) kit. The reaction was performed on an LC480 fluorescence quantitative PCR instrument. Figure 5 The results showed that Rutin could significantly inhibit the transcription levels of CVB5 virus VP1 and 3C mRNA.

[0048] 6. Plaque assay was used to determine the inhibitory effect of Rutin on CVB5 virus replication.

[0049] Collect the supernatant of Vero cells infected with CVB5 virus and store at -80°C. Vero cells are cultured in DMEM culture medium containing 10% fetal bovine serum in an incubator at 37C, 5% C02. Veo cells are inoculated into a 12-well plate. After the Vero cells grow into a monolayer, they are washed three times with PBS and incubated for infection after diluting the previously collected supernatant 100 times. After 1.5 hours, replace the Vero cells with a fresh mixed culture medium containing an equal volume of 2× serum-free DMEM and microcrystalline cellulose (2×DMEM: 3% microcrystalline cellulose mixture = 1:1). After 72 hours, fix the cells with 4% paraformaldehyde for 10 minutes and stain with 2% crystal violet for 10 minutes. Discard the crystal violet staining solution and observe and count the number of plaques. Figure 6 Compared with the control group infected with virus alone, the number of cell plaques caused by virus infection was significantly reduced in the Rutin-treated group, 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 inoculated into 6-well plates and placed in a 5% CO2, 37°C incubator for overnight culture. After 1.5 hours of infection of RD cells with CVB5 (MOI = 0.5), the cell supernatant was discarded, and the cells were washed twice with sterile PBS, and then 40 and 80 μM Rutin were added for incubation. The groups without virus or Rutin and with virus alone were used as controls. The 6-well plates were placed in an incubator for continuous culture, and the cell morphology was observed and photographs were taken the next day. Figure 7 As shown in the results, compared with normal RD cells, enterovirus CVB5 infection of RD cells can cause cell shrinkage, detachment and death of cytopathic effect (CPE), while Rutin incubation can significantly reduce this effect, play a significant protective role on infected cells, and increase cell survival rate. This shows that Rutin does have a significant inhibitory effect on CVB5 virus.

[0052] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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 above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A use of rutin in the preparation of a drug for treating and / or preventing coxsackie virus, wherein the structure of the rutin is as follows:

2. The use according to claim 1, characterized in that: The coxsackievirus includes group A coxsackievirus and group B coxsackievirus.

3. The use according to claim 1, characterized in that: The drug for treating and / or preventing Coxsackie virus also includes pharmaceutically acceptable adjuvants and / or drugs compatible with rutin.

4. The use according to claim 3, characterized in that: The auxiliary materials include at least one of solvents, disintegrants, flavoring agents, colorants, lubricants, antioxidants, preservatives, adhesives, fillers or thickeners.

5. The use according to claim 1, characterized in that: The working concentration of rutin is ≥20 μM.

6. The use according to any one of claims 1 to 5, characterized in that: The dosage forms of the drug for treating and / or preventing Coxsackie virus include capsules, granules, tablets, oral liquids, pills, injections, ointments, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.

7. The use according to claim 6, characterized in that: The administration routes of the drug for treating and / or preventing Coxsackie virus include oral administration or intravenous injection.

8. A drug for treating and / or preventing Coxsackie virus, characterized in that: The invention comprises a pharmaceutical active ingredient and auxiliary materials, wherein the pharmaceutical active ingredient is rutin.

9. The drug according to claim 8, characterized in that The drug inhibits the replication of Coxsackievirus B5 by binding to the VP1 protein of Coxsackievirus B5.

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