Preparation for inhibiting coronavirus infection

Abstract

The invention discloses a preparation for inhibiting coronavirus infection. The invention provides an application of a preparation in the following (1) or (2): (1) preparing a product for inhibiting coronavirus infection; (2) inhibiting coronavirus infection; the preparation is mainly prepared by mixing chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide. The mass ratio of the chlorogenic acid to the ginsenoside Rb1 to the astragalus polysaccharide is (0.5 to 1.0) to (0.5 to 1.0) to (0.5 to 1.5). The cytotoxicity of the compound formula preparation provided by the invention is not higher than that of a control sample ribavirin which has obtained security permission, so that the compound formula preparation is relatively safe. Under a safe use concentration, the preparation provided by the invention can be used for effectively inhibiting coronavirus infection in a prophylactic manner, and has a commercial value of being further researched and developed into a coronavirus infection inhibitor.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a preparation for inhibiting coronavirus infection. Background Art

[0002] Coronavirus is a kind of RNA virus that is widely present in nature, can cause interspecies transmission and can cause zoonosis. It is one of the main pathogens of the common cold in humans, with a higher infection rate in children. It is mainly an infection of the upper respiratory tract, and rarely affects the lower respiratory tract. A few can cause diarrhea, bronchitis, pneumonia, and pleural effusion. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), SARS-CoV-2 (commonly known as the new coronavirus), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) that have appeared since the 21st century have obvious damage to the human respiratory tract, gastrointestinal tract, and central nervous system. Clinically, severe patients will experience symptoms such as dyspnea, inflammation, shock, and even death, which seriously affects human health and social development.

[0003] Emerging infectious diseases have been an important cause of public health problems worldwide in recent years. SARS-CoV and MERS-CoV were once locally prevalent around the world and caused a large number of deaths. Coronavirus has become the main type of pathogen that spreads, spreads and causes disease in humans. Its large family system, the general susceptibility and carriage in animals, and several serious outbreaks in human society all show the advantages of this type of virus in evolution and transmission. Although people have developed corresponding vaccines and therapeutic drugs for coronaviruses, when the virus mutates or a new virus appears and causes an outbreak of infectious diseases, the original vaccines or specific preventive drugs are difficult to play the expected prevention and control effect, or even have no effect. The new generation of specific preventive vaccines and therapeutic drugs are limited by the scientific research cycle and technological level, and it is difficult to launch them in a short period of time, which can easily lead to the outbreak of infectious diseases and the rapid spread of the epidemic. Not only does it cause great damage to local public health services, but it also has a huge impact on the society's politics and economy. Therefore, based on the basic biological characteristics of the virus and the common targets of the virus-infected host cells, the development of new virus infection inhibitors is of great significance to meet the challenges of viral infectious disease epidemics.

[0004] Plant compounds are widely present in daily food and are called "the seventh type of nutrients". They are a class of non-nutritional organic chemicals that have special effects on human health. Plant compounds are rich in resources, have powerful biological functions, and have low toxicity to normal cells. Related studies have found that some plant compounds have biological activity that inhibits viral infection and have gradually become one of the hot spots in antiviral research. Preliminary research results show that plant compounds mainly exert their biological functions by affecting the biological characteristics of cell membranes and changing the balance of local microenvironment. Some plant compounds can directly embed into the lipid bilayer structure of cell membranes to change the normal functional flow and potential difference of cell membranes, thereby exerting certain biological effects. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation for inhibiting coronavirus infection and application thereof.

[0006] The purpose of the present invention is to provide a preparation for inhibiting coronavirus infection and application thereof.

[0007] The application provided by the present invention is specifically the application of a preparation in the following (1) or (2): (1) Preparation of products for inhibiting coronavirus infection; (2) Used to inhibit coronavirus infection; The preparation is mainly composed of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide; the mass ratio of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide is (0.5-1.5): (0.5-1.0): (0.5-1.0).

[0008] In one embodiment of the present invention, the preparation for inhibiting viral infection is specifically composed of a mixture of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide; the mass ratio of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide is 1:1:1.5.

[0009] In the present invention, the inhibition of coronavirus infection is as follows (a) or (b): (a) Prevention of coronavirus infection; (b) when acting on a host or a host cell simultaneously with a coronavirus, inhibiting the infection of the coronavirus on the host or the host cell.

[0010] More specifically, in an embodiment of the present invention, the inhibitory effect of the preparation on the coronavirus infection is specifically embodied as follows: taking mammalian cells as the subject of coronavirus infection, the preparation is administered at the same time as or before the coronavirus infects the cells, and the half-maximal inhibitory concentration of the preparation on the coronavirus is significantly lower than that of the positive control drug that inhibits viral infection. The positive control drug is specifically ribavirin.

[0011] In the present invention, the mammalian cell (or the host cell described in (b)) is specifically a mouse fibroblast (17Cl-1 cell).

[0012] In the present invention, the coronavirus is mouse hepatitis coronavirus. More specifically, the coronavirus is mouse hepatitis coronavirus A59 strain.

[0013] The present invention uses coronavirus (mouse hepatitis virus, MHV) as the experimental object, adopts the method of observing cytopathic effects, and analyzes the rules and characteristics of the three plant compound monomers, chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide, and their mixed preparations in inhibiting viral infection under different infection conditions. The results confirmed that the cytotoxicity of the three plant compound monomers and the composite formula mixture provided by the present invention is no higher than that of the control sample ribavirin that has obtained safety approval, and is relatively safe. At a safe concentration of use, the preventive use of plant compound monomers such as chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide, as well as a composite formula mixture of plant extracts, can effectively inhibit coronavirus infection. The preparation provided by the present invention has commercial value for further development as a coronavirus infection inhibitor. DETAILED DESCRIPTION

[0014] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0016] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0017] The quantitative experimental data involved in the following examples are expressed as mean ± standard deviation (±s), and the inter-group comparison data were statistically processed using SPSS 19.0 statistical software using the one-way horizontal variance analysis method.

[0018] Cell line: mouse fibroblasts (17Cl-1 cells), preserved in the laboratory, recorded in the article "Chang Guohui, Liu Jingmei, Yang Yi, et al. A broad-spectrum viral infection inhibitor [P]. Beijing: CN201610056795.7, 2018-03-13.", which can be obtained by the public from the applicant and is only used to repeat the experiments of the present invention. The cells were subcultured according to conventional methods.

[0019] Virus: Coronavirus (murine hepatitis coronavirus A59 strain, abbreviated as MHV-A59) is recorded in the article "Chang Guohui, Liu Jingmei, Yang Yi, et al. A broad-spectrum viral infection inhibitor [P]. Beijing: CN201610056795.7, 2018-03-13." The public can obtain it from the applicant and is only used to repeat the experiments of the present invention.

[0020] Culture medium: 1) Cell growth culture medium: DMEM culture medium (Hyclone product) is used as the mother liquid, and 10% (volume fraction) fetal bovine serum (Glibco product), 0.2mg / ml glutamine (Glibco product), 100U / ml penicillin, streptomycin (Hyclone product) are added respectively. 2) Cell maintenance culture medium, the fetal bovine serum content is 2% (volume fraction), and the rest is the same as 1). 3) Virus proliferation culture medium: the same as cell growth culture medium.

[0021] Chlorogenic acid: a product of Sigma (analytical purity>95%), product number C3878, CAS#327-97-9.

[0022] Ginsenoside Rb1: product of Solebao (HPLC>98%), product number SG8260, CAS# 41753-43-9.

[0023] Astragalus polysaccharide: product of Solebold (analytical purity >90%), product number IA0570, CAS#89250-26-0.

[0024] Positive control drug: Ribavirin, a product of Solebao, product number SR8570. Since there is no standard positive control drug that inhibits viral infection by changing the host cell membrane properties, this study uses ribavirin, the most commonly used antiviral drug, as a control. Dissolve it in PBS buffer before the experiment, filter it to 2560μg / ml, sterilize it, and store it at -20℃.

[0025] Cytotoxicity detection kit: Cell Counting Kit-8 (CCK8), a product of Yisheng Biotechnology Co., Ltd., product number 40203ES80. Inverted phase contrast microscope: product of Olympus Corporation, Japan.

[0026] Cell culture incubator: product of Thermo Corporation, USA.

[0027] Multifunctional enzyme reader: SpectraMax M5 multifunctional enzyme reader produced by American Molecular Devices.

[0028] 1. Cell recovery and passaging Take out 17Cl-1 cells from liquid nitrogen, thaw quickly in a 37℃ water bath, centrifuge at 1000rpm for 5min, discard the supernatant, add an appropriate amount of cell growth medium, and repeatedly blow and mix until the cell density is about 2×10 5 / ml, 10ml / bottle, in T25 cell culture flask, in a cell carbon dioxide incubator. After 24h, observe the cell adhesion under a microscope. After 48h, or when the cells grow to a density of about 95%, discard the original culture medium, add PBS buffer, rinse twice, add 0.5ml0.25% (0.25g / 100ml) Trypsin-EDTA, incubate and digest in a 37℃ incubator. When the cells shrink and become round, quickly add cell growth culture medium to stop digestion. After repeated blowing and mixing, centrifuge at 1000rpm for 5min, discard the supernatant, and make the concentration of suspended cells about 1×10 5 / ml, 10ml / bottle, transferred into T25 cell culture flask, placed in cell culture incubator for routine subculture.

[0029] 2. Virus proliferation Take a T25 cell culture flask with a cell growth density of about 80%, discard the stock solution, add PBS, rinse twice to remove serum residues, add diluted virus (diluent is PBS buffer), inoculation amount MOI = 0.01, and 1ml cell maintenance culture medium, shake well, place in a cell culture incubator for adsorption for 1h, discard the supernatant, and add 10ml of the corresponding virus proliferation culture medium to each bottle. Observe the cytopathic effect, after 48-72h, or when the cytopathic effect reaches more than 75%, collect the virus, take the supernatant, put it in a liquid nitrogen tank and then take it out, freeze and thaw it repeatedly 3 times, centrifuge at 3000rpm, 4℃, for 5min, aliquot the supernatant, and measure the virus titer TCID 50 .

[0030] 3. Virus titer TCID 50 Determination The single cell suspension was added to a 96-well microplate using the cytopathic effect assay (CPE) to a volume of 2 × 10 cells per well. 5 / ml. Culture in a cell culture incubator for 48-72h until the cell monolayer density is about 80%, then remove and discard the culture medium, rinse twice with PBS. Use virus proliferation culture medium to dilute the virus stock solution 10 times to 10 -3 ~10 -13Each concentration gradient has 8 wells in one column, 100μl in each well, and two columns of blank controls are set up. Adsorb in a cell culture incubator for 1 hour, discard the supernatant, and then add 200μl of maintenance culture medium. Observe under a microscope every day, and record the experimental results when the cell lesion rate of the virus control group is "++++". Calculate the virus titer TCID according to the Reed-Muench method 50 Each group of strains was repeated three times.

[0031] lgTCID 50 =(percentage of lesion rates above 50% - 50%) / (percentage of lesion rates above 50% - percentage of lesion rates below 50%) x difference between logarithms of dilutions + lg (dilution of lesion rates above 50%).

[0032] Example 1. Preparation of a plant extract composite formula virus infection inhibitor Chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide were mixed evenly in a mass ratio of 1:1:1.5 to obtain a plant extract compound formulation. Before the experiment, it was dissolved in PBS buffer to prepare 2560 μg / ml (the total concentration of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide in the solution), filtered and sterilized, and stored at -20°C for later use.

[0033] Example 2: Cytotoxicity test This example uses a cytotoxicity assay to measure the cytotoxicity of the composite formulation prepared in Example 1 to mammalian cells. The specific operation is as follows: The compound formula preparation solution (2560μg / ml) prepared in Example 1 was diluted in multiple ratios to obtain 6 concentrations of 20μg / ml, 40μg / ml, 80μg / ml, 160μg / ml, 320μg / ml, 640μg / ml, and 1280μg / ml. Then the dilutions of different dilutions were added to a 96-well culture plate of 17Cl-1 cells with a cell density of about 80%, 100μl per well, and 4 duplicate wells were made for each dilution, with normal cells (i.e., no compound formula preparation was added) as a control. After 2h of action, the test solution was discarded, and cell maintenance culture medium was added, 200μl was added to each well, and placed in a cell culture incubator for culture. After 48h, 100μl of CCK8 reagent was added to each well, and after 1.5h of action at 37°C, the OD value was determined using a SpectraMax M5 multifunctional microplate reader (Molecular Devices, USA), with the absorption wavelength set to 492nm. By one-way analysis of variance, the statistical differences between the OD values ​​of each concentration group and the control group (normal cells, i.e., no composite formula preparation was added) were compared to determine the maximum non-toxic concentration of the composite formula preparation prepared in Example 1 (i.e., the maximum concentration that was not statistically different from the OD value of the control group).

[0034] The experiment simultaneously set up the monomers of three compounds, namely chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide, as monomer controls of the compound formula preparation; and set up the antiviral infection positive control drug ribavirin as a positive control.

[0035] The results are shown in Table 1. It can be seen that the maximum non-toxic concentration of the composite formula preparation to 17Cl-1 cells is 640 μg / ml, which is the same as the maximum non-toxic concentration of the marketed drug ribavirin. It can be seen that the plant compound composite formula preparation prepared in Example 1 has good safety.

[0036] Table 1 Results of the maximum cell non-toxicity test of plant compound monomers and compound formulations

[0037] Example 3, Coronavirus infection inhibition test This example uses the CCK8 method to determine the inhibitory effect of the composite formula prepared in Example 1 on coronavirus infection. The test coronavirus is the mouse hepatitis coronavirus A59 strain.

[0038] Take a 96-well culture plate containing 17Cl-1 cells grown into a monolayer with a cell growth density of about 80%, pour out the culture medium, rinse the cells three times with PBS, and then add the composite formula prepared in Example 1 under three conditions A, B, and C respectively: A. While the virus is adsorbed: add an equal volume of 2×100 TCID 50 After mixing the coronavirus virus solution with the test substance solution of 2 times concentration, add 100μl / well of the mixture to the cell culture plate, place it in the cell culture incubator, and discard it after the virus is adsorbed for 1 hour. After washing the cell surface with PBS for 3 times, add the cell maintenance culture solution.

[0039] B. Before virus adsorption: add 100 μl of the test solution of corresponding dilution to each well, discard the test solution, rinse the cell surface with PBS for 3 times, and add 100 TCID 50 100 μl of coronavirus virus solution was placed in a cell culture incubator and discarded after the virus was adsorbed for 1 hour. After washing the cell surface with PBS for 3 times, the cell maintenance culture medium was added.

[0040] C. After virus adsorption: add 100 TCID 50 100μl of coronavirus virus solution was placed in a cell culture incubator and discarded after the virus was adsorbed for 1 hour. 100μl of the test solution of the corresponding dilution was added to each well, and the test solution was discarded. After adsorption in a cell culture incubator for 1 hour, it was discarded. After washing the cell surface with PBS 3 times, the cell maintenance culture medium was added.

[0041] The test substances are monomers of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide, the composite formula prepared in Example 1, or ribavirin, a positive control drug for antiviral infection. The concentration of the test substance solution is set to the following concentration gradient: 20 μg / ml, 40 μg / ml, 80 μg / ml, 160 μg / ml, 320 μg / ml, 640 μg / ml, 1280 μg / ml.

[0042] Each group of experiments was set up with a cell control and a positive control group (the positive control was a cell control group with only 100 TCID 50 Virus, blank control only added cell maintenance culture medium), cultured in a cell culture incubator, observed under an inverted microscope every day, and CCK8 kit was used to detect cell proliferation when the cytopathic effect of the virus control group (i.e. positive control group) was "++++". Each group had 4 replicate wells, and the experiment was repeated 3 times.

[0043] The virus inhibition rate was calculated as follows: Virus inhibition rate (%) = (drug treatment group <h2 style=";text-align:left;direction:ltr">D492nm - Virus control group <h2 style=";text-align:left;direction:ltr"> D492nm ) / (cell control group <h2 style=";text-align:left;direction:ltr"> D492nm - Virus control group <h2 style=";text-align:left;direction:ltr"> D492nm )×100% The half inhibitory concentration (IC50) was calculated by Probit regression method.

[0044] The results showed that under the three administration conditions, chlorogenic acid, plant compound formula preparations and ribavirin can interfere with coronavirus infection of 17Cl-1 cells. Among them, the plant extract compound formula preparation had the best effect in inhibiting virus infection when the inhibitor was added at the same time and before virus adsorption, and its virus half-inhibitory concentration was 127±17μg / ml and 107±12μg / ml, respectively. Under the condition of adding inhibitors after virus adsorption, the control drug ribavirin had a better effect in inhibiting virus infection, and its virus half-inhibitory concentration was 257±19μg / ml. Among the monomer inhibitors, chlorogenic acid had the best effect in inhibiting virus infection when added before virus adsorption, and the virus half-inhibitory concentration was 172±14μg / ml. When ginsenoside Rb1 and astragalus polysaccharide were added after virus adsorption, they could not effectively inhibit coronavirus infection. The specific results are shown in Table 2.

[0045] Table 2 The half inhibitory concentration (IC) of each inhibitor on coronavirus infection 50 )

[0046] Note: * indicates comparison with ribavirin group, p<0.05.

[0047] Based on the experimental results of Examples 2 and 3, it can be seen that the cytotoxicity of the composite formulation provided by the present invention is no higher than that of the control sample ribavirin, which has obtained safety approval, and is relatively safe. The plant extract compound formulation provided by the present invention can effectively inhibit the infection of coronavirus. The compound formulation provided by the present invention has commercial value for further development as a coronavirus infection inhibitor.

[0048] Comparison of the inhibitory effects of plant extract compound formulations with different ratios on coronavirus infection The experimental method is as shown in Example 3, and the test substances are the composite formula preparation prepared in Example 1, the control composite formula preparation 1, the control composite formula preparation 2, the control composite formula preparation 3, or the antiviral infection positive control drug ribavirin, and the rest of the operations are the same as in Example 3. Among them, the formulas of the control composite formula preparation 1, the control composite formula preparation 2 and the control composite formula preparation 3 are as follows: Control composite formula preparation 1: Chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide were mixed in a mass ratio of 0.5:1:0.5.

[0049] Control composite formula preparation 2: Chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide were mixed in a mass ratio of 1:0.5:1.

[0050] Control composite formula preparation 3: Chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide were mixed in a mass ratio of 1:1:1.

[0051] The results showed that the half inhibitory concentration (IC50) of the control compound formulation 1, the control compound formulation 2 and the control compound formulation 3 against coronavirus infection was significantly lower than that of the compound formulation prepared in Example 1 under the three conditions of A, B and C (p < 0.05). See Table 3 for specific results.

[0052] Table 3 Half inhibition concentration (IC) of different compound formulations on coronavirus infection 50 )

[0053] Note: * indicates p<0.05 compared with the ribavirin group. # indicates p<0.05 compared with the compound formula preparation group of Example 1. The present invention is described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present invention and without unnecessary experiments. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that are out of the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following claims.

Claims

1. A preparation for inhibiting coronavirus infection, characterized in that: The preparation is prepared by mixing chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide; the mass ratio of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide is (0.5-1.5): (0.5-1.0): (0.5-1.0).

2. The preparation according to claim 1, characterized in that: The preparation is prepared by mixing chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide; the mass ratio of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide is 1:1:1.

5.

3. The preparation according to claim 2, characterized in that: The coronavirus is mouse hepatitis coronavirus.

4. The preparation according to any one of claims 1 to 3, characterized in that: The inhibition of viral infection is as follows (a) or (b): (a) Prevention of viral infection; (b) when acting on a host or a host cell simultaneously with a virus, inhibiting the infection of the host or the host cell by the virus.

5. A method for preparing a preparation for inhibiting coronavirus infection, comprising the following steps: mixing chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide in a mass ratio of (0.5-1.5): (0.5-1.0): (0.5-1.0) to obtain the preparation.

6. The method according to claim 5, characterized in that: The mass ratio of chlorogenic acid, ginsenoside Rb1 and astragalus polysaccharide is 1:1:1.

5.

7. The method according to claim 6, characterized in that: The coronavirus is mouse hepatitis coronavirus.

8. The method according to any one of claims 5 to 7, characterized in that: The inhibition of viral infection is as follows (a) or (b): (a) Prevention of viral infection; (b) when acting on a host or a host cell simultaneously with a virus, inhibiting the infection of the host or the host cell by the virus.

9. Use of the inhibitor according to any one of claims 1 to 4 or the method according to any one of claims 5 to 8 in the preparation of a product for inhibiting coronavirus infection.

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