CN111803472A

CN111803472A - Novel coronavirus resistant medicine and application thereof

Info

Publication number: CN111803472A
Application number: CN202010636515.6A
Authority: CN
Inventors: 刘映霞; 杨明辉; 韦金丽; 杨扬; 黄婷; 赖锦志; 刘磊
Original assignee: Third Peoples Hospital of Shenzhen
Current assignee: Third Peoples Hospital of Shenzhen
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2020-10-23

Abstract

The invention provides an application of resveratrol and derivatives thereof in preparing a novel coronavirus resistant medicament, and in the application, the resveratrol serving as a human health product can obviously inhibit the in vitro replication of a novel coronavirus, and has low cytotoxicity. Considering the immune regulation function of resveratrol, the resveratrol is recommended to be used for clinical prevention or treatment of patients with new coronary pneumonia. Therefore, the invention also provides a novel anti-coronavirus medicine.

Description

Novel coronavirus resistant medicine and application thereof

Technical Field

The invention belongs to the technical field of antiviral drugs, and particularly relates to a novel coronavirus resistant drug and application thereof.

Background

The new coronavirus is also known as 2019-nCoV, and people infected with the new coronavirus have common signs of respiratory tract symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. At present, no specific treatment method exists for diseases caused by the novel coronavirus, and the production of the antibody and the vaccine also needs a large amount of clinical tests for verification. Therefore, in order to prevent further spread of the epidemic and to cure infected patients as soon as possible, screening of drugs against novel coronaviruses is not easy.

Resveratrol is a typical stilbene polyphenolic compound and is an antitoxin produced when many plants are stimulated. Can be synthesized in grape leaf and grape skin, and is bioactive component in wine and grape juice. It is easy to be absorbed by oral administration, and excreted via urine and feces after metabolism. In vitro experiments and animal experiments show that resveratrol has antioxidant, antiinflammatory, anticancer and cardiovascular protecting effects.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of resveratrol in preparing a medicine for resisting novel coronavirus so as to solve the harm of the novel coronavirus to personal safety and social economy.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: the application of resveratrol and derivatives thereof in preparing a novel coronavirus resistant medicament is disclosed, wherein the resveratrol is shown as the following formula:

on the basis of the technical scheme, the invention can also have the following further specific selection or optimization selection.

Specifically, the resveratrol derivative comprises a derivative obtained by etherifying, esterifying, methoxylating, hydroxylating and/or halogenating resveratrol.

Specifically, the resveratrol and the derivatives thereof play a role in inhibiting virus replication after the virus infects cells, and the role is probably realized by activating SIRT1 signals. In addition, the resveratrol and the derivatives thereof also have a certain inhibiting effect on virus infected cells.

In addition, the invention also provides a novel anti-coronavirus drug, and the preparation of the novel anti-coronavirus drug comprises resveratrol and derivatives thereof and one or more pharmaceutically acceptable carriers; the carrier comprises diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant and synergist which are conventional in the pharmaceutical field.

Specifically, the preparation of the anti-novel coronavirus medicament is selected from injection, tablets, pills, capsules, suppositories, suspending agents or emulsions.

Compared with the prior art, the invention has the beneficial effects that: the new crown pneumonia epidemic outbreak at the end of 2019 brings huge influence to the world, the epidemic is mainly caused by virus spread of 2019-nCoV, and the global epidemic is not controlled yet at present. In the application, Resveratrol (Resveratrol) as a health product for human use can significantly inhibit the in vitro replication of the new coronavirus (half effective dose of EC50 ═ 4.48 μ M), and is less cytotoxic. Further research shows that resveratrol and derivatives thereof mainly play a main role after the new coronavirus infects Vero cells, and after the cells infect the virus, the cells are added with resveratrol to be cultured for 48 hours at 50 mu M, compared with a control group, the replication of the new coronavirus is inhibited by 98 percent. In addition, the cells are infected again after 50 mu M resveratrol is added into the virus liquid, compared with a control group, the replication of the new coronavirus is inhibited by 64 percent, which indicates that the veratryl alcohol not only inhibits the replication of the virus after the virus infects the cells, but also the resveratrol can block the new coronavirus from entering the cells through a receptor approach to a certain extent. Considering that resveratrol has low biological toxicity as a health product and an immunomodulator for human use, the resveratrol can be recommended to be used for clinical prevention or treatment of patients with new coronary pneumonia.

Drawings

FIG. 1 inhibition of the replication of new coronaviruses by resveratrol at different concentrations (nucleic acid content of virus efflux);

FIG. 2 shows the inhibitory effect of resveratrol at different concentrations on the replication of new coronaviruses (intracellular viral protein content);

FIG. 3 Effect of treatment with resveratrol at different concentrations on Vero cytotoxicity;

figure 4 effect of different treatment stages of resveratrol on its inhibition of replication of new coronaviruses;

FIG. 5 Effect of SIRT1 inhibitor Sirtinol and agonist SRT 1720 on replication of new coronaviruses.

Detailed Description

For a better understanding of the present invention, the following further illustrates the present invention with reference to the accompanying drawings and specific examples, but the present invention is not limited to the following examples.

Example 1:

the application selects VERO cells (African green monkey kidney cells) which are susceptible to the new coronavirus (2019-nCoV) as research objects (Vero is a cell line mainly used in the in vitro experiment of the current new coronavirus drug screening). The culture conditions are as follows: DMEM containing 10% serum, incubated at 37 deg.C with 5% CO₂Culturing in the incubator.

The novel coronavirus used for the experiment was isolated from throat swabs of patients in third national hospital, Shenzhen, at 2.2020, and the sequence information was uploaded to the GISAID database (BetacoV/Shenzhen/SZTH-003/2020strain viral GISAID access number: EPI _ ISL _ 406594).

Resveratrol for the experiments was purchased from SIGMA, cat # R5010 (purity 99%), diluted to 0.1M with DMSO and subsequently diluted to (1.56. mu.M, 3.13. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M, 200. mu.M) with DMEM containing 2% serum for later culture.

Vero cells are spread in a 24-well plate after 4-5 generations, and inoculation is started when the cells grow to 80%. The new coronavirus was diluted with serum-free DMEM, and after Vero cells were finally infected with MOI-0.01, the cells were cultured in an incubator for 2 hours. Taking out 24-well plate cells, discarding virus solution, washing with serum-free DMEM for 3 times, and then adding diluted resveratrol for cell culture, wherein each group is repeated for 3 times. After the cells were cultured for 48h, cell culture supernatant was collected and cells were fixed with 4% paraformaldehyde. The levels of nucleic acids in the culture medium and intracellular virus levels were separately detected and observed by immunofluorescence.

Example 2:

in order to clarify the influence of resveratrol on cytotoxicity, the study was carried out using a CCK-8 kit. The CCK-8 reagent used in this experiment was purchased from Solebao, China (cat # CK 04).

Vero cells were plated into 96-well plates at 100. mu.l cell suspension per well after passage to 4-5 passages. The plates were pre-incubated in an incubator for 24 hours until the cells grew to 80%, resveratrol was diluted to 0.1M with DMSO, then resveratrol was diluted with DMEM containing 10% serum to (1.56 μ M, 3.13 μ M, 6.25 μ M,12.5 μ M,25 μ M,50 μ M,100 μ M,200 μ M), DMEM containing 10% serum was used as a drug blank, and DMEM containing 0.05% DMSO was prepared as a blank with three wells containing only basal medium and CCK-8 reagent and no cell suspension was selected as a blank. Add 10. mu.l of the above test substances at different concentrations to the plates, repeat 3 of each, incubate in the incubator for 48 hours, replace fresh medium (remove medium and wash cells twice with medium, then add new medium), remove drug effects, add 10. mu.l CCK-8 solution to each well (care not to generate bubbles in the wells, which would affect the OD reading), incubate the plates in the incubator for 2 hours, and measure absorbance at 450nm with a microplate reader.

And (3) activity calculation:

cell viability (%) ([ a (dosed) -a (blank) ]/[ a (0 dosed) -a (blank) ] × 100

A (dosing): absorbance of wells with cells, CCK-8 solution and drug solution

A (blank): absorbance of wells with media and CCK-8 solution without cells

A (0 dosing): absorbance of wells with cells, CCK-8 solution and no drug solution

Cell viability: cell proliferation Activity or cytotoxic Activity

Example 3:

this study needs to further define at what stage of viral infection resveratrol can act in order to further determine the primary mechanism by which resveratrol may act.

The resveratrol addition is mainly divided into four stages:

1. infection pretreatment: diluting resveratrol to 50 μ M with DMEM containing 10% serum, pretreating the cells for 2h, then discarding the pretreatment solution, washing three times with serum-free DMEM, diluting the virus with serum-free DMEM, and incubating for 2h with MOI-0.01 addition virus, then washing the virus off, and washing three times with serum-free DMEM. The cells were then cultured with DMEM containing 2% serum until 48 hours for harvest;

2. treatment at infection: diluting resveratrol to 50 mu M with serum-free DMEM to prepare solution A, then diluting the virus to MOI-0.01 with the solution A, incubating cells for 2h, then discarding the incubation solution, washing the cells for 3 times with serum-free DMEM, then adding 2% serum-free DMEM to culture the cells for 48h, and collecting samples;

3. and (3) post-infection treatment: diluting the virus with serum-free DMEM to MOI of 0.01, incubating the cells for 2h, discarding the virus solution, washing with DMEM for 3 times, adding DMEM containing 50 μ M resveratrol and 2% serum, and culturing for 48 hours to collect the sample;

4. and (3) whole-process treatment: vero cells overgrowing with 80% Vero cells are firstly treated with DMEM containing 50 mu M resveratrol and 10% serum for 2h, the treatment solution is discarded, the Vero cells are incubated with DMEM containing neocoronaviruses with MOI of 0.01 and 50 mu M resveratrol for 2h after being washed for three times, the incubation solution is discarded, the Vero cells are washed with clean DMEM for three times, and then the Vero cells are cultured with DMEM containing 50 mu M resveratrol and 2% DMEM for 48h and sampled.

Example 4:

in order to initially explore a molecular mechanism of resveratrol for inhibiting the replication of the new coronavirus, the research selects a downstream effector molecule SIRT1 of resveratrol as a potential target, and further defines the downstream target of resveratrol for inhibiting the replication of the new coronavirus through the use of an antagonist and an agonist of SIRT 1.

The SIRT1 agonist SRT 1720 was purchased from MCE (cat. No. HY-10532), and was dissolved in DMSO in pure form and used by diluting with 2% serum-containing DMEM to 0.01. mu.M, 0.1. mu.M, and 1. mu.M, respectively.

The SIRT1 inhibitor Sirtinol was purchased from MCE corporation (cat. No: HY: 13515) and was dissolved in DMSO and used by diluting with DMEM containing 2% serum to 10. mu.M, 50. mu.M, 100. mu.M, respectively.

Vero cells are spread in a 24-well plate after 4-5 generations, and inoculation is started when the cells grow to 80%. The new coronavirus was diluted with serum-free DMEM, and after Vero cells were finally infected with MOI-0.01, the cells were cultured in an incubator for 2 hours. The 24-well plate cells were taken out, virus solution was discarded, and washed 3 times with serum-free DMEM, and then diluted agonists and inhibitors at different concentrations were added separately for cell culture, with 3 replicates per group. After the cells were cultured for 48h, cell culture supernatant was collected and cells were fixed with 4% paraformaldehyde. The nucleic acid levels in the culture broth were measured separately.

The results of the above experiments are shown in figures 1-4, respectively, where resveratrol was effective in inhibiting in vitro replication of 2019-nCoV (as shown in figure 1), at a semi-effective dose (EC50 ═ 4.48 μ M). In addition, resveratrol can significantly reduce intracellular viral protein level, and when the resveratrol concentration reaches 50 μ M, viral protein is not substantially present in cells (as shown in figure 2). As shown in figure 3, the resveratrol has low cytotoxicity to Vero cells, the cell activity can be maintained above 90% by treating the resveratrol with the concentration of less than 100 mu M, and the cell activity can be maintained above 80% by ensuring that the concentration of the resveratrol reaches 200 mu M. Resveratrol mainly plays a role in inhibiting the replication of new coronavirus after the virus infects cells, and the inhibition efficiency can reach more than 98 percent. In addition, resveratrol can also play a certain inhibiting role in the process of infecting cells by viruses, and the inhibiting efficiency reaches 64 percent. Suggesting that it may block the binding of the RBD domain of the S protein of the new coronavirus to the ACE2 receptor of the cell. In view of its specific role, resveratrol may not only be used as a clinical drug for the treatment of new coronavirus infection, but also as a drug for the prevention of infection.

Resveratrol acts as a natural agonist of intracellular SIRT1 and exerts its cell regulatory effect through the SIRT1 pathway, and in order to further determine whether resveratrol can inhibit the replication of new coronaviruses through SIRT1, the present application utilizes agonists and inhibitors of SIRT1 and determines its effect on the replication of new coronaviruses. The result shows that the SIRT1 protein has a certain regulation effect on the replication of the new coronavirus. Viral infection was markedly exacerbated by Sirtinol inhibition of SIRT1 activity, but SRT 1720 did not exhibit comparable viral inhibitory effects to resveratrol, possibly due to improper concentration treatment or a different effect and manner of activating resveratrol. In conclusion, the intracellular SIRT1 protein has a regulating effect on the replication of new coronavirus, the reduction of the activity of the protein can aggravate the replication of the virus in cells, and the resveratrol can obviously inhibit the replication of the virus possibly because the resveratrol activates the SIRT1 pathway, but related researches need to further prove.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The application of resveratrol and derivatives thereof in preparing a novel coronavirus resistant medicament is disclosed, wherein the resveratrol is shown as the following formula:

2. the use of resveratrol and its derivatives in the preparation of anti-coronavirus drugs according to claim 1, wherein: the derivative of resveratrol comprises a derivative of resveratrol obtained by etherification, esterification, methoxylation, hydroxylation and/or halogenation.

3. The use of resveratrol and its derivatives in the preparation of anti-coronavirus drugs according to claim 1, wherein: the resveratrol and the derivatives thereof play a role in inhibiting virus replication after the virus infects cells, and are realized by activating a SIRT1 signal, and the resveratrol and the derivatives thereof have an inhibiting effect on the virus infected cells.

4. An anti-novel coronavirus drug, which is characterized in that: comprises resveratrol and derivatives thereof and one or more pharmaceutically acceptable carriers.

5. The anti-neocoronavirus agent of claim 4, wherein: the carrier comprises a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and a synergist which are conventional in the pharmaceutical field.

6. The anti-neocoronavirus agent of claim 4, wherein: the preparation of the novel coronavirus resistant medicament is selected from injection, tablets, pills, capsules, suppositories, suspending agents or emulsions.