Antiviral composition as well as preparation method and application thereof

Abstract

The invention relates to an antiviral composition as well as a preparation method and application thereof. The invention relates to an antiviral composition which comprises the following compounds: forsythin, emodin, baicalin and liquiritin in a molar ratio of (50-250): (1-4): (10-40): (100-500). According to the antiviral composition disclosed by the invention, by inhibiting expression of dengue type 2 virus (DENV-2) mRNA in cells, inhibiting release of offspring viruses of the dengue type 2 virus and inhibiting expression of E and NS1 proteins of the dengue type 2 virus, the replication amount of virus RNA in blood infected with the dengue type 2 virus is effectively reduced, and the effects of preventing and treating dengue fever are achieved; the death rate after the dengue 2 virus infection can be effectively reduced; in addition, by inhibiting protein secretion of inflammatory factors induced by the SARS-CoV-2 virus and the influenza A virus (H1N1) and expression of mRNA (messenger Ribonucleic Acid), the traditional Chinese medicine composition plays a role in treating pneumonia and influenza A, and also plays a role in protecting lung injury caused by the SARS-CoV-2 virus and the influenza A virus.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an antiviral composition and a preparation method and application thereof. Background Art

[0002] Dengue fever is an acute infectious disease caused by dengue virus (DENV), which is mainly transmitted by female Aedes aegypti and female Aedes albopictus. It is a Class B infectious disease in my country and is divided into four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. Clinically, the symptoms of dengue fever patients can develop from asymptomatic to dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF). DSS or DHF develops rapidly and has a mortality rate of up to 10% to 40%. Currently, about 390 million people are diagnosed with dengue virus infection every year worldwide, and about half of the population is at risk of contracting dengue fever. Dengue fever is considered one of the top ten health threats in the world.

[0003] The main clinical manifestations of COVID-19 patients are fever, fatigue, and dry cough. The general population is susceptible, and the severity and mortality rates of the elderly or special populations with serious underlying diseases are higher than those of the general population.

[0004] Influenza virus is also known as influenza virus. Its clinical symptoms mainly include acute high fever, body aches, significant fatigue and respiratory symptoms. Influenza A virus often undergoes antigenic mutations, including the H1N1 subtype, which is one of the main influenza virus subtypes that cause human influenza pandemics.

[0005] Although dengue fever, pneumonia and influenza have brought great social and economic burdens to the global society, there is still a lack of corresponding vaccines and specific antiviral drugs for the clinical treatment of dengue fever, pneumonia and influenza. Therefore, there is an urgent need to seek antiviral drugs with precise efficacy, stability and few side effects in order to better respond to viral outbreaks. Summary of the invention

[0006] Based on this, it is necessary to provide an antiviral composition to address the current problem of lack of corresponding vaccines and specific antiviral drugs for the treatment of dengue fever, pneumonia and influenza in clinical practice.

[0007] An antiviral composition contains the following compounds in a molar ratio: forsythiaside: rhamnolide: baicalin: liquiritin is 50-250:1-4:10-40:100-500.

[0008] The above antiviral composition is a Lianggesan effective ingredient composition with significant anti-inflammatory activity. The composition has little cytotoxicity and side effects, and has significant antiviral and anti-inflammatory activity. By inhibiting the expression of dengue virus type 2 (DENV-2) mRNA in cells, inhibiting the release of dengue virus type 2 progeny viruses, inhibiting the expression of E and NS1 proteins of dengue virus type 2, and effectively reducing the replication of viral RNA in the blood after infection with dengue virus type 2, it plays a preventive and therapeutic role in dengue fever, and can effectively reduce the mortality rate after infection with dengue 2 virus; it also inhibits the protein secretion and mRNA expression of inflammatory factors induced by viruses (SARS-CoV-2) and influenza A virus (H1N1), plays a therapeutic role in pneumonia and influenza A, and can also protect lung damage caused by SARS-CoV-2 and influenza A virus.

[0009] In one embodiment, the antiviral composition contains the following compounds in a molar ratio: forsythiaside: rhamnolide: baicalin: liquiritin is 150-250:1-2:15-25:120-130.

[0010] In one embodiment, the antiviral composition contains the following compounds in a molar ratio of forsythiaside: emodin: baicalin: liquiritin of 200:1:20:125.

[0011] In one embodiment, the forsythiaside, emodin, baicalin, and liquiritin have the following structures respectively:

[0012]

[0013] The present invention also provides a method for preparing the antiviral composition, which comprises weighing each compound according to the formula amount and preparing the compound to a predetermined concentration.

[0014] The present invention also provides a preparation of an antiviral composition, comprising the antiviral composition and pharmaceutical excipients.

[0015] In one embodiment, the pharmaceutical excipient is a pharmaceutical excipient selected from one or two of a solvent, a disintegrant, a flavoring agent, a preservative, a colorant, and a binder.

[0016] The present invention also provides an application of the antiviral composition in preparing antiviral drugs and / or treating diseases caused by viruses.

[0017] In one embodiment, the virus is an RNA virus.

[0018] In one embodiment, the virus is dengue 2 virus, novel coronavirus or H1N1 virus.

[0019] In one embodiment, the antiviral composition achieves its antiviral effect by inhibiting the expression of dengue 2 virus mRNA in cells, inhibiting the protein expression of dengue 2 virus protein E and NS1, inhibiting the release of dengue 2 virus progeny viruses, and reducing the number of viral RNA copies in the blood after infection with dengue 2 virus.

[0020] In one embodiment, the antiviral composition achieves the effect of treating virus-induced diseases by inhibiting the protein secretion and mRNA expression of virus-induced inflammatory factors IL-6, TNF-α and IL-1β, thereby providing memory protection for lung damage caused by viral infection.

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

[0022] The antiviral Chinese medicine monomer composition of the present invention and its preparation method and application,

[0023] It has little cytotoxicity and side effects, and has significant antiviral and anti-inflammatory activity. It can prevent and treat dengue fever by inhibiting the expression of dengue virus type 2 (DENV-2) mRNA in cells, inhibiting the release of dengue virus type 2 progeny viruses, inhibiting the expression of E and NS1 proteins of dengue virus type 2, and effectively reducing the replication of viral RNA in the blood after infection with dengue virus type 2. It can also effectively reduce the mortality rate after infection with dengue virus type 2; it can also treat pneumonia and influenza A by inhibiting the protein secretion and mRNA expression of inflammatory factors induced by SARS-CoV-2 and influenza A virus (H1N1), and can also protect lung damage caused by SARS-CoV-2 virus and influenza A virus.

[0024] The antiviral composition of the present invention has definite and stable curative effects on treating dengue fever, pneumonia and influenza and has little side effects.

[0025] The preparation method of the antiviral composition of the present invention has simple process and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of the composition obtained in Example 1 on the cell viability of Huh7, C6 / 36 and BHK-21 cells;

[0027] Figure 2 The effect of the composition obtained in Example 1 on DENV-2 mRNA expression and cell morphology changes under a microscope;

[0028] Figure 3 The effect of the composition obtained in Example 1 on the expression of DENV-2 mRNA at various stages of the DENV-2 replication cycle;

[0029] Figure 4 This is a comparison chart of the inhibitory effect of the composition obtained in Example 1 on the protein expression of DENV-2 protein E and NS1 detected by western blotting;

[0030] Figure 5 This is a comparison chart of the inhibitory effect of the composition obtained in Example 1 on the release of DENV-2 progeny viruses obtained through a virus plaque experiment;

[0031] Figure 6 This is a comparison chart of the survival percentage (percent survival) of DENV-2 infected AG129 mice after administration of the composition obtained in Example 1;

[0032] Figure 7 This is a comparison chart of the amount of DENV-2 viral RNA replication inhibited by the composition obtained in Example 1;

[0033] Figure 8 The effect of the composition obtained in Example 1 on the relative cell viability of RAW264.7 cells;

[0034] Fig. 9 The effect of the composition obtained in Example 1 on the protein secretion and mRNA expression of inflammatory factors IL-6, TNF-α and IL-1β induced by SARS-CoV-2 and H1N1 viruses;

[0035] Fig.10 H&E staining detection images of the protective effect of the composition obtained in Example 1 on the lungs of hACE2 transgenic C57BL / 6 mice infected with SARS-CoV-2 (A, C) and KM mice infected with H1N1 (B, D) and real-time fluorescence quantitative PCR detection of the mRNA expression of inflammatory factors IL-6, TNF-α and IL-1β.

[0036] In the figure, "#" indicates that there is a statistically significant difference compared with the blank group, and "*" indicates that there is a statistically significant difference compared with the virus-infected (model) group. DETAILED DESCRIPTION

[0037] 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.

[0038] 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.

[0039] The reagents used in the following examples, unless otherwise specified, are all commercially available; the methods used in the following examples, unless otherwise specified, are all achievable by conventional methods.

[0040] The four monomer compositions of forsythiaside, rhamnolide, baicalin and liquiritin used in the embodiments of the present invention are all provided by Shanghai Aladdin Biochemical Technology Co., Ltd., and the purity is measured by HPLC-DAD peak area normalization method to be above 98%.

[0041] Example 1

[0042] The inventors found in preliminary research and preliminary experimental screening that forsythiaside, rhamnolide, baicalin and liquiritin have a good antiviral effect when they are mixed in a molar ratio of 200:1:20:125.

[0043] An antiviral composition with the following active substance contents: 20 μM forsythiaside, 0.1 μM emodin, 2 μM baicalin and 12.5 μM liquiritin was prepared.

[0044] Weigh each compound according to the formula amount, mix the weighed compounds together, add a certain volume of dimethyl sulfoxide (DMSO) to prepare the mixture into a solution with a concentration of 16 mg / mL, and obtain the predetermined concentration. The antiviral composition of the present application can be prepared according to the conventional preparation method of a mixed solution of a specific concentration in the art.

[0045] Experimental Example 1

[0046] Toxicity evaluation test of the antiviral composition on human liver cancer cell Huh7, Aedes albopictus larvae cell C6 / 36 and baby hamster kidney cell BHK-21.

[0047] Experimental method: Huh7 (commercially available), C6 / 36 (commercially available) and BHK-21 (commercially available) cells were cultured at 8×10 3 / well were inoculated in a 96-well plate and cultured for 24 hours, and the composition obtained in Example 1 was diluted to a concentration of 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using DMEM culture medium (Huh7) or 1640 culture medium (C6 / 36 and BHK-21) containing 10% fetal bovine serum, and a solvent was prepared as a blank group (control) as a control, and 200 μL per well was added to the 96-well plate respectively; after the above-mentioned well plate was placed in a cell culture incubator and cultured for 96 hours, 100 μL of MTT (5 mg / mL) solution prepared with phosphate buffered saline (PBS) was added to each well, and then incubated at 37° C. for 4 hours, the supernatant was removed, and then 150 μL of dimethyl sulfoxide (DMSO) was added to each well; according to conventional methods, the absorbance value (OD value) of each group of cells was measured at a wavelength of 490 nm using an ELISA instrument, and the cell survival rate was calculated based on the absorbance value.

[0048] The cell viability calculation formula is as follows:

[0049] Survival rate = OD value of cells in each group / OD value of blank group × 100%

[0050] Test results: Figure 1 As shown, the composition obtained in Example 1 is substantially non-toxic to cells at a concentration below 4 μg / mL. Therefore, in subsequent experiments, 4 μg / mL was used as the highest concentration of the composition used in cells.

[0051] Experimental Example 2

[0052] Evaluation test of the antiviral composition for its anti-DENV-2 effect.

[0053] Experimental method: Huh7, C6 / 36 and BHK-21 cells were cultured at 2×10 5 / well were inoculated in a 6-well plate and cultured for 24 hours; 2 hours before the virus infected the cells, the composition obtained in Example 1 with concentrations of 4, 2, 1, 0.5, and 0.25 μg / mL was added to the 6-well plate in an amount of 1 mL per well, and a solvent control was configured as the DENV-2 group; the cell supernatant was removed, 1 mL of virus solution was added to each well and the composition of the above concentration was added at the same time, incubated at 37°C for 1 hour, the virus solution was removed and washed twice with PBS, the composition of the above concentration was added again, and the cells were placed in an incubator and incubated for 48 hours, and then the effect of the composition on the expression of DENV-2 mRNA was detected by fluorescence quantitative PCR according to conventional methods; at the same time, the changes in the morphology of Huh7, C6 / 36 and BHK-21 cells were observed under a microscope every day to detect the effect of the composition on the cytopathic effect.

[0054] The above real-time fluorescence quantitative PCR detection conditions are: 40 cycles of amplification, pre-denaturation at 95°C for 30s, denaturation at 95°C for 5s, and annealing at 60°C for 30s.

[0055] The primer sequences for the above real-time fluorescence quantitative PCR are shown in the table below.

[0056]

[0057] Test results: Figure 2 As shown, the composition obtained in Example 1 inhibited the expression of DENV-2 mRNA in cells in a dose-dependent manner within a concentration range of 1 to 4 μg / mL, and it was found that Huh7, C6 / 36 and BHK-21 cells showed CPE on the 3rd, 1st and 5th days after virus infection, respectively, and the composition obtained in Example 1 was able to inhibit the production of cytopathic effect.

[0058] Experimental Example 3

[0059] Evaluation test of the antiviral effect of the antiviral composition at various stages of the DENV-2 replication cycle.

[0060] Experimental method: Huh7 cells were cultured at 2×10 5 / well were inoculated in a 6-well plate and cultured for 24 hours; 2 hours before virus infection (pre-treatment), during virus infection (co-treatment), 1 hour after virus infection (post-treatment) and throughout the infection stage (full-treatment), the composition obtained in Example 1 with a concentration of 4 μg / mL was administered in an amount of 1 mL per well, and a solvent control was configured as the DENV-2 group; after incubation in a cell culture incubator for 48 hours, the effect of the monomer composition on DENV-2 mRNA expression was detected by fluorescent quantitative PCR according to the conventional method and the same primers as in Experimental Example 2.

[0061] The above real-time fluorescence quantitative PCR detection conditions are: 40 cycles of amplification, pre-denaturation at 95°C for 30s, denaturation at 95°C for 5s, and annealing at 60°C for 30s.

[0062] This experiment determines at which stage of the DENV-2 replication cycle the composition obtained in Example 1 works by time-divided administration, determines whether the composition has a preventive effect on the virus by pre-administration (pre-treatment), determines whether the composition works in the adsorption and entry stage of virus replication by administration during infection (co-treatment), and determines whether the composition works in the biosynthesis stage of the virus replication cycle by administration after infection (post-treatment).

[0063] Test results: Figure 3 As shown, the composition obtained in Example 1 exerts antiviral effects in both the adsorption entry (co-treatment) and biosynthesis (post-treatment) stages, but the antiviral effect in the adsorption entry (co-treatment) stage is more obvious.

[0064] Experimental Example 4

[0065] A validation test of the antiviral composition's ability to inhibit the expression of dengue 2 virus proteins E and NS1.

[0066] Experimental method: Huh7, C6 / 36 and BHK-21 cells were cultured at 2×10 5 / well were inoculated in a 6-well plate and cultured for 24 hours; during virus infection, different concentrations (4, 2, 1 μg / mL) of the composition obtained in Example 1 were given, heparin (Heparin) (50 μg / mL) was used as a positive control group, and a solvent control was configured as a DENV-2 group in an amount of 1 mL per well, and added to the 6-well plate respectively; after incubation in an incubator for 48 hours, the cells were collected to extract proteins; according to conventional methods, the protein expression levels of viral proteins E and NS1 in the cells were detected by western blotting (Huh7 and C6 / 36 cells used GAPDH as an internal reference, and BHK-21 cells used β-actin as an internal reference).

[0067] Test results: Figure 4 As shown, in Huh7 (GAPDH as an internal reference, C6 / 36 (GAPDH as an internal reference) and BHK-21 (β-action as an internal reference) cells, the composition obtained in Example 1 effectively inhibited the protein expression of E and NS1. Because the E protein mainly mediates the adsorption and entry stage of the DENV-2 virus, and the NS1 protein is the main target for the development of DENV-2 vaccines, the composition of the present invention has an inhibitory effect on the two, which can prove that the composition of the present invention has an inhibitory effect on the DENV-2 virus.

[0068] Experimental Example 5

[0069] Evaluation test of the inhibitory effect of antiviral composition on the release of dengue type 2 virus progeny.

[0070] Experimental method: Huh7 cells were cultured at 2×10 5 / well were inoculated in a 6-well plate and cultured for 24h; different concentrations (4, 2, 1μg / mL) of the composition obtained in Example 1 were given during virus infection, and a solvent control group was configured as the DENV-2 group, heparin (Heparin) (50μg / mL) was used as the positive control group, and a solvent control that was not infected with the virus was set as the Control group, and 1mL was added to each well of the 6-well plate. After the cells were incubated in the incubator for 48h, the cell supernatant was collected to infect BHK-21 cells (2×10 5 / well, 6-well plate); BHK-21 cells were infected at 37°C for 1 h, 3 mL of methylcellulose containing 2% fetal bovine serum was added to each well, and the cells were incubated in an incubator for 120 h. BHK-21 cells were fixed and stained with 1 mL of crystal violet containing 4% paraformaldehyde in each well, and the formation of viral plaques was detected by a viral plaque assay according to conventional methods.

[0071] Test results: Figure 5 As shown, the composition obtained in Example 1 effectively inhibits the formation of DENV-2 progeny virus plaques, indicating that the composition can effectively inhibit the release of DENV-2 progeny viruses and has an anti-DENV-2 virus effect.

[0072] Experimental Example 6

[0073] Evaluation of the protective effect of the antiviral composition on DENV-2 infected AG129 mice.

[0074] Experimental method: AG129 mice are immunodeficient mice lacking IFN-α, β, and γ receptors and are susceptible to DENV-2. AG129 mice aged 6 to 8 weeks (n=8) were randomly divided into a control group, a DENV-2 group, and a composition group (the composition obtained in Example 1 was administered at a weight ratio of 50, 25, and 12.5 mg of active substance / kg of mouse body weight, such as 50 mg / kg, which means that according to the weight of the mouse, 50 mg of active substance was administered per kilogram of mouse body weight, including: 28.902 mg of forsythin, 0.145 mg of emodin, 2.890 mg of baicalin, and 18.064 mg of liquiritin); the mice were intraperitoneally injected with DENV-2 (4×10 5 A mouse model of DENV-2 infection was established by gavage with 100 PFU / mL; mice were gavaged with the drug 2 days before DENV-2 infection. The Control group and the DENV-2 group were gavaged with the same volume of saline, and then the drug was gavaged once a day until the end of the experiment. The survival of the mice within 25 days after virus infection was observed and recorded.

[0075] Test results: Figure 6As shown, all mice in the DENV-2 group died within 9 to 12 days after virus infection; on the 25th day after virus infection, the survival rate of mice in the group dosed with 50 mg / kg of the composition was 62.5%, and the survival rates of mice in the groups dosed with 25 and 12.5 mg / kg of the composition were 50% and 25%, respectively.

[0076] Experimental Example 7

[0077] The test was conducted to evaluate the inhibitory effect of the antiviral composition on viremia in the blood of DENV-2-infected AG129 mice (immunodeficient mice lacking IFN-α, β, and γ receptors and susceptible to DENV-2).

[0078] Experimental method: AG129 mice aged 6 to 8 weeks (n=8) were randomly divided into DENV-2 group and composition group (the composition obtained in Example 1 was administered at a ratio of 50, 25 and 12.5 mg active substance / kg mouse body weight); the mice were intraperitoneally injected (4×10 5 A mouse model of DENV-2 infection was established with 100 PFU / mL; mice were gavaged with the drug 2 days before DENV-2 infection, and the DENV-2 group was gavaged with the same volume of normal saline, and then gavaged with the drug once a day until the end of the experiment; blood was collected through the tail vein on the 1st, 3rd, 5th, and 7th day after virus infection and centrifuged to obtain serum, serum mRNA was extracted, and changes in viremia were detected by real-time fluorescence quantitative PCR according to conventional methods.

[0079] The above real-time fluorescence quantitative PCR detection conditions are: 40 amplification cycles in FAM mode, denaturation at 95°C for 2 minutes, annealing at 95°C for 10 seconds, and extension at 60°C for 30 seconds.

[0080] The primer and probe sequences are as follows:

[0081]

[0082] Test results: Figure 7 As shown, after mice were infected with DENV-2, the viremia in their blood reached a peak on the third day after infection and then gradually decreased. The composition obtained in Example 2 inhibited the copy amount of NS1 mRNA in DENV-2 in a dose-dependent manner.

[0083] Experimental Example 8

[0084] Toxicity evaluation test of the antiviral composition on mouse peritoneal mononuclear macrophage RAW264.7 cells.

[0085] Experimental method: RAW264.7 (mouse peritoneal mononuclear macrophages) cells (commercially available) were plated at 8×10 3The cells were inoculated at a density of 100 μg / mL in a 96-well plate and cultured for 24 h; the composition obtained in Example 1 was diluted to a concentration of 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL using DMEM culture medium containing 10% fetal bovine serum, and a solvent was prepared as a control group and added to the 96-well plate at 100 μL per well; after culturing in a cell culture incubator for 48 h, MTT solution was added; after incubation at 37° C. for 4 h, the supernatant was removed, and then 150 μL DMSO was added to each well, and the plate was placed in a microplate reader according to a conventional method, and the absorbance value was measured at a wavelength of 490 nm, and the cell survival rate was calculated based on the absorbance value.

[0086] The cell viability calculation formula is as follows:

[0087] Survival rate = OD value of cells in each group / OD value of blank group × 100%

[0088] Test results: Figure 8 As shown, the composition obtained in Example 1 is substantially non-toxic to cells at a concentration below 4 μg / mL within 48 h, and therefore 4 μg / mL was used as the highest concentration in cells in subsequent experiments.

[0089] Experimental Example 9

[0090] Evaluation test of the antiviral composition's anti-inflammatory effect against SARS-CoV-2 and H1N1 induced inflammation.

[0091] Experimental method: RAW264.7 cells were cultured at 2×10 5 The cells were inoculated at a density of 4, 2, and 1 μg / mL in a 6-well plate and cultured for 24 hours; 12 hours before the virus infected the cells, the composition obtained in Example 1 was added to the 6-well plate at a concentration of 1 mL per well, and a SARS-CoV-2 group, an H1N1 group, and a blank control group were set; the cell supernatant was removed, the virus solution was added, and the monomer composition of the above concentration was added at the same time; after incubation at 37° C. for 1 hour, the virus solution was removed and washed twice with PBS, and the monomer composition of the above concentration was added again; after the cells were incubated in an incubator for 48 hours, the cell supernatant was collected, and the effect of the composition on the secretion of virus-induced inflammatory factors IL-6, TNF-α, and IL-1β proteins was detected by ELISA according to conventional methods, and the cells were collected, and the effect of the monomer composition on the expression of virus-induced inflammatory factors IL-6, TNF-α, and IL-1β mRNA was detected by real-time fluorescence quantitative PCR according to conventional methods.

[0092] The above real-time fluorescence quantitative PCR detection conditions are: 40 cycles of amplification, pre-denaturation at 95°C for 30s, denaturation at 95°C for 5s, and annealing at 60°C for 30s.

[0093] The primer sequences for the above real-time fluorescence quantitative PCR are shown in the table below.

[0094]

[0095] Test results: Fig. 9 As shown, the composition obtained in Example 1 has an inhibitory effect on the protein secretion and mRNA expression of inflammatory factors IL-6, TNF-α and IL-1β, indicating that the composition has a significant inhibitory effect on acute macrophage inflammation caused by SARS-CoV-2 and H1N1.

[0096] Experimental Example 10

[0097] An experiment to evaluate the protective effect of an antiviral composition on lung damage caused by SARS-CoV-2 infection and H1N1 infection.

[0098] Experimental method: hACE2 transgenic C57BL / 6 mice aged 5 to 6 weeks (n=8) were randomly divided into a normal group (CTL), a SARS-CoV-2 infection group and a composition group (the composition obtained in Example 1 was administered at a mass ratio of 40 mg active substance / kg mouse body weight); the mice were infected by intranasal drops for 10 4 A mouse model of SARS-CoV-2 infection was established with SARS-CoV-2 virus at a concentration of 5 PFU / mL. The normal group was given PBS buffer by nasal drops. The mice were gavaged with the drug 5 days before SARS-CoV-2 infection. The normal group and the SARS-CoV-2 group were gavaged with the same volume of normal saline once a day. The lungs were dissected 5 days after infection, and the left lung was fixed with 4% paraformaldehyde. The pathological damage of the lung tissue was detected by H&E staining. The right lung was homogenized and the expression of mRNA of inflammatory factors IL-6, TNF-α and IL-1β was detected by real-time fluorescence quantitative PCR.

[0099] KM mice aged 5 to 6 weeks (n=8) were randomly divided into a normal group (CTL), an H1N1 infection group and an example group (the composition obtained in Example 1 was administered at a mass ratio of 40 mg active substance / kg mouse body weight); the mice were infected with 104 PFU / mL of H1N1 virus by intranasal drops to establish an H1N1 infected mouse model, and the normal group was administered PBS by intranasal drops; the mice were gavaged 5 days before H1N1 infection, and the normal group and the H1N1 group were gavaged with the same volume of physiological saline, once a day; 5 days after infection, the lungs were dissected and fixed with 4% paraformaldehyde, and the pathological damage of the lung tissue was detected by HE staining according to conventional methods. After the right lung was homogenized, the expression of mRNA of inflammatory factors IL-6, TNF-α and IL-1β was detected by real-time fluorescence quantitative PCR according to conventional methods.

[0100] The above real-time fluorescence quantitative PCR detection conditions are the same as those in Experimental Example 9.

[0101] Test results: Fig.10 As shown, the composition obtained in Example 1 has a protective effect on lung damage in hACE2 transgenic C57BL / 6 mice infected with SARS-CoV-2 (A, C) and KM mice infected with H1N1 (B, D).

[0102] 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.

[0103] 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. An antiviral composition, characterized in that: The compound contains the following molar ratio: forsythiaside: rhamnolide: baicalin: liquiritin is 50-250:1-4:10-40:100-500.

2. The antiviral composition according to claim 1, characterized in that The compound contains the following molar ratio: forsythiaside: rhamnolide: baicalin: liquiritin is 150-250:1-2:15-25:120-130.

3. The antiviral composition according to claim 1, characterized in that The compound contains the following molar ratio: forsythiaside: rhamnolide: baicalin: liquiritin is 200:1:20:

125.

4. The antiviral composition according to claim 1, characterized in that The forsythiaside, emodin, baicalin and liquiritin have the following structures respectively:

5. The method for preparing the antiviral composition according to any one of claims 1 to 4, characterized in that: Weigh each compound according to the formula amount and prepare it to a predetermined concentration.

6. Use of the antiviral composition according to any one of claims 1 to 4 in the preparation of antiviral drugs and / or the treatment of diseases caused by viruses.

7. The use according to claim 6, characterized in that: The virus is an RNA virus.

8. The use according to claim 6, characterized in that: The virus is dengue type 2 virus, new coronavirus or H1N1 virus.

9. The use according to claim 6, characterized in that: The antiviral composition achieves the antiviral effect by inhibiting the expression of dengue 2 virus mRNA in cells, inhibiting the protein expression of dengue 2 virus protein E and NS1, inhibiting the release of dengue 2 virus progeny viruses, and reducing the copy amount of viral RNA in the blood after infection with dengue 2 virus.

10. The use according to claim 6, characterized in that: The antiviral composition inhibits the protein secretion and mRNA expression of virus-induced inflammatory factors IL-6, TNF-α and IL-1β, thereby protecting the memory of lung damage caused by viral infection and achieving the effect of treating diseases caused by viruses.

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