Antiviral traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention relates to an antiviral traditional Chinese medicine composition as well as a preparation method and application thereof. Active components of the antiviral traditional Chinese medicine composition are prepared from the following raw materials in parts by weight: 20-28 parts of fructus forsythiae, 5-7 parts of fructus gardeniae, 5-7 parts of radix scutellariae, 10-14 parts of liquorice root, 10-14 parts of radix et rhizoma rhei, 5-7 parts of herba menthae and 10-14 parts of mirabilite. The antiviral traditional Chinese medicine composition disclosed by the invention has good anti-inflammatory activity and antiviral effect, has small influence on cell viability, plays a role in preventing and treating DENV-2 induced dengue fever, can effectively reduce the death rate after DENV-2 infection, can play a role in repairing brain injury caused after DENV-2 infection, and can be used for preventing and treating DENV-2 induced dengue fever. The case injury of liver and large intestine caused by DENV-2 infection can also be effectively relieved; the traditional Chinese medicine composition has a remarkable treatment effect on pneumonia and H1N1 type influenza, and also has a protective effect on lung injury caused by SARS-CoV-2 infection and H1N1 infection.

Description

Technical Field

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

[0002] Dengue fever is an acute infectious disease caused by dengue virus (DENV). It is transmitted by female Aedes aegypti and female Aedes albopictus. It is divided into four serotypes, DENV-1, DENV-2, DENV-3 and DENV-4, based on antigenicity. DENV-2 has a higher transmission range and mortality rate than other types. The clinical symptoms of dengue fever include asymptomatic, mild dengue fever and severe dengue fever, including dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). DSS or DHF develops rapidly and has a mortality rate of up to 10% to 40%. According to the World Health Organization, 390 million people are infected with dengue virus each year worldwide, of which about 96 million are severe patients.

[0003] COVID-19 refers to pneumonia caused by infection with the new coronavirus (SARS-CoV-2).

[0004] Influenza (flu) is an acute respiratory infectious disease caused by influenza virus. Influenza A and B viruses are seasonally prevalent every year, and influenza A virus can cause a global outbreak. Currently, the main influenza A virus that infects humans is the H1N1 subtype.

[0005] Currently, there is still a lack of specific antiviral drugs for the clinical treatment of DENV-2, SARS-CoV-2 and H1N1, so there is an urgent need to seek antiviral drugs with definite efficacy, stability and few side effects.

[0006] Dengue fever, COVID-19 and influenza belong to the category of "epidemic fever" in traditional Chinese medicine. In ancient times, doctors used it to treat epidemic fever. For example, in "Treatise on Febrile Diseases and Epidemics", Yang Lishan, a doctor in the Qing Dynasty, proposed "a total of 15 prescriptions for febrile diseases, which can be used to clear the mild and purge the severe", with Liangge San with modified flavor as the representative prescription. Yu Zhen, a doctor in the Qing Dynasty, pointed out in "Ancient and Modern Medical Cases" that Liangge San should be used for those with dry stools after the symptoms of epidemic fever subside. Liangge San originated from the "Taiping Huimin Hejiju Fang" in the Song Dynasty. Traditional Chinese medicine theory believes that Liangge San has the effects of nourishing yin and relieving yang, cooling the diaphragm and purging heat, quenching thirst and relieving restlessness, purging fire and laxatives, and clearing the upper and lower parts. It is composed of rhubarb, mirabilite, gardenia, forsythia, scutellaria, licorice, mint and bamboo leaves, and is mainly used to treat the heat syndrome of the upper and middle burners.

[0007] Therefore, it is a scientific priority to develop specific antiviral Chinese medicine preparations for dengue fever, COVID-19 and H1N1 influenza based on the traditional prescription Liangge San, which have the advantages of definite efficacy, stability and small nuclear side effects. Summary of the invention

[0008] Based on this, it is necessary to provide an antiviral Chinese medicine composition to address the problem of lack of specific antiviral drugs for the treatment of infectious diseases caused by DENV-2, SARS-CoV-2 and H1N1 viruses in clinical practice.

[0009] An antiviral Chinese medicine composition, wherein the active ingredients are prepared from the following raw materials in parts by weight: 20-28 parts of forsythia, 5-7 parts of gardenia, 5-7 parts of scutellaria, 10-14 parts of liquorice, 10-14 parts of rhubarb, 5-7 parts of mint and 10-14 parts of mirabilite.

[0010] In the process of studying traditional prescriptions, the above-mentioned antiviral Chinese medicine composition was found to have certain anti-inflammatory and antiviral effects. After in-depth research and exploration, the optimal raw material compatibility relationship and ratio were found. The improved antiviral Chinese medicine composition has good anti-inflammatory activity and antiviral effects, and has little effect on cell activity. It has the effects of inhibiting the expression of DENV-2mRNA in cells, inhibiting the protein and mRNA expression of DENV-2 protein E and NS1, inhibiting the release of DENV-2 progeny viruses, and effectively reducing the number of viral RNA copies in the blood after DENV-2 infection, inhibiting the formation of CPE (cytopathic effect) caused by DENV-2 infection, and inhibiting the transfer of cytoplasmic Hsp70 (heat shock protein with a molecular weight of about 70kD) caused by DENV-2 infection to the cell membrane. It can repair brain damage caused by DENV-2 infection, and also It can effectively alleviate the pathological damage of the liver and large intestine caused by DENV-2 infection, prevent and treat dengue fever induced by DENV-2, and effectively reduce the mortality rate after DENV-2 infection; it also has the effect of inhibiting the protein secretion and mRNA expression of inflammatory factors IL-6, TNF-α and IL-1β induced by SARS-CoV-2 and H1N1, thereby playing a significant therapeutic effect on pneumonia and H1N1 influenza, and at the same time has a protective effect on lung damage caused by SARS-CoV-2 infection and H1N1 infection.

[0011] In one embodiment, the antiviral Chinese medicine composition, whose active ingredients are made of the following raw materials in parts by weight: 23-24 parts of Forsythia suspensa, 5-7 parts of Gardenia jasminoides, 5-7 parts of Scutellaria baicalensis, 11-13 parts of Licorice, 11-13 parts of Rhubarb, 5-7 parts of Menthus fructus, and 11-13 parts of Sodium sulfate.

[0012] The invention also provides a method for preparing an antiviral Chinese medicine composition, which comprises weighing various raw materials according to the formula amount, extracting with water as a solvent, and obtaining a medicinal solution.

[0013] In one embodiment, the preparation method comprises the following steps:

[0014] (1) Weighing forsythia suspensa, gardenia jasminoides, scutellaria baicalensis, and liquorice according to the formula, adding water to boil and taking out the liquid medicine to obtain liquid medicine a;

[0015] (2) Weigh rhubarb and mint according to the formula, add them to liquid a, decoct and take out the liquid to obtain liquid b;

[0016] (3) Weigh sodium sulfate according to the formula and add it to solution b.

[0017] In one embodiment, in step (1), Forsythia suspensa, Gardenia jasminoides, Scutellaria baicalensis, and Licorice root are weighed according to the formula amount, water is added according to the material-liquid ratio of 10±2 mL water / g medicinal material, soaked for 30±10 min, and boiled for 20±5 min to obtain medicinal solution a;

[0018] In step (2), rhubarb and mint are weighed according to the formula amount, added to the medicinal solution a, and continued to be decocted for 10±5 minutes, filtered, and obtained medicinal solution b1; water is added to the obtained medicinal residue according to the material-liquid ratio of 6+2mL water / g medicinal material, and boiled for 10±5 minutes to obtain medicinal solution b2; and medicinal solutions b1 and b2 are combined to obtain medicinal solution b;

[0019] In step (3), after filtering the medicinal solution b, adding the sodium sulfate weighed according to the formula, stirring and dissolving, filtering, and obtaining the final medicinal solution.

[0020] In one embodiment, the method further comprises the step (4), wherein the final medicinal solution obtained in the step (3) is concentrated into an extract form, and then dried to obtain the antiviral Chinese medicine composition.

[0021] The present invention also provides an application of the antiviral Chinese medicine composition in preparing antiviral drugs and / or treating diseases caused by viruses.

[0022] In one embodiment, the virus is an RNA virus.

[0023] In one embodiment, the virus is DENV-2 virus, novel coronavirus or H1N1 virus.

[0024] In one of the embodiments, the antiviral Chinese medicine composition achieves its antiviral effect by inhibiting the expression of mRNA in cells, inhibiting the protein and mRNA expression of DENV-2 protein E and NS1, inhibiting the release of DENV-2 progeny viruses, reducing the number of viral RNA copies in the blood after infection, inhibiting the formation of cytopathic effects caused by viral infection, and inhibiting the transfer of cytoplasmic Hsp70 to the cell membrane caused by viral infection.

[0025] In one of the embodiments, the antiviral Chinese medicine 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 protecting the memory of lung damage caused by viral infection.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The antiviral Chinese medicine composition of the present invention has good anti-inflammatory activity and antiviral effect, and has little effect on cell activity. It has the functions of inhibiting the expression of DENV-2 mRNA in cells, inhibiting the protein and mRNA expression of DENV-2 protein E and NS1, inhibiting the release of DENV-2 progeny viruses, and effectively reducing the copy amount of viral RNA in the blood after DENV-2 infection, inhibiting the formation of CPE (cytopathic effect) caused by DENV-2 infection, and inhibiting the transfer of cytoplasmic Hsp70 (heat shock protein with a molecular weight of about 70kD) caused by DENV-2 infection to the cell membrane, etc., which can repair brain damage caused by DENV-2 infection, and also It can effectively alleviate the damage to the liver and large intestine caused by DENV-2 infection, prevent and treat dengue fever induced by DENV-2, and effectively reduce the mortality rate after DENV-2 infection; it also has the effect of inhibiting the protein secretion and mRNA expression of inflammatory factors IL-6, TNF-α and IL-1β induced by SARS-CoV-2 and H1N1, thereby playing a significant therapeutic effect on pneumonia and H1N1 influenza, and at the same time has a protective effect on lung damage caused by SARS-CoV-2 infection and H1N1 infection.

[0028] The antiviral Chinese medicine monomer composition of the present invention has definite and stable curative effects on treating dengue fever, pneumonia and influenza and has little side effects.

[0029] The preparation method of the antiviral Chinese medicine composition of the present invention is simple and easy to implement and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The figure is a comparison of cell viability after adding different concentrations of LGS prepared in Example 1 to Huh7, C6 / 36 and BHK-21 cells;

[0031] Figure 2 DENV-2 mRNA expression and BHK-21 cell activity (cell viability) after adding different concentrations of LGS prepared in Example 1 to the cells before virus infection (pre-treatment), during virus infection (co-treatment), and after virus infection (post-treatment);

[0032] Figure 3 For Western blotting ( Figure 3 A) and real-time fluorescence quantitative PCR ( Figure 3 BD) Two methods were used to detect the protein expression levels of DENV-2 viral proteins E and NS1, and the gene expression levels of E protein and NS1 protein (E mRNA expression, NS1 mRNA expression) in cells after administration of LGS prepared in Example 1;

[0033] Figure 4 This is a comparison diagram of the cell pathological changes under a microscope after administration of LGS prepared in Example 1;

[0034] Figure 5 The comparison of the inhibition of DENV-2 progeny virus by LGS prepared in Example 1;

[0035] Figure 6 The comparison of the process of LGS prepared in Example 1 inhibiting DENV-2 infection and causing the transfer of cytoplasmic Hsp70 to the cell membrane in host cells;

[0036] Figure 7 This is a comparison chart showing that the LGS prepared in Example 1 prolongs the survival time, reduces the clinical score, and delays weight loss in ICR suckling mice infected with DENV-2;

[0037] Figure 8 This is a comparison diagram of the pathological damage of the brain of suckling mice observed by hematoxylin-eosin (H&E) staining after administration of LGS prepared in Example 1;

[0038] Fig. 9 The LGS prepared in Example 1 inhibits the expression of viral proteins E and NS1 in the brain, liver, spleen and serum of ICR suckling mice infected with DENV-2;

[0039] Fig.10The LGS prepared in Example 1 prolongs the survival time of DENV-2-infected AG129 mice and reduces viremia;

[0040] Fig.11 This is a comparison diagram showing that LGS prepared in Example 1 alleviates the pathological damage of the liver, brain and large intestine of AG129 mice infected with DENV-2;

[0041] Fig.12 The comparison of the LGS prepared in Example 1 inhibiting the copy number of DENV-2 gene in the brain, liver, spleen and kidney of DENV-2 infected AG129 mice;

[0042] Fig.13 The figure is a comparison of the cell viability (relative cell viability) after adding different concentrations of LGS prepared in Example 1 to RAW264.7 cells;

[0043] Fig.14 The LGS prepared in Example 1 has a comparative effect on the inflammation induced by SARS-CoV-2 and H1N1;

[0044] Fig.15 The protective effect of LGS prepared in Example 1 on lung inflammation in hACE2 transgenic C57BL / 6 mice infected with SARS-CoV-2 and KM mice infected with H1N1;

[0045] Fig.16 The figure shows a comparison of the anti-SARS-CoV-2 and H1N1 induced inflammatory effects of the LGS prepared in Example 1 and the pharmaceutical composition of the prior art.

[0046] 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

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The selection of each medicinal material in the formula of this application complies with the provisions of the 2015 edition of the "Chinese Pharmacopoeia", including rhubarb (Rheumpalmatum L), produced in Gansu; Glauber's salt (Natrii Sulfas), produced in Henan; Licorice (Glycyrrhiza uralensis Fisch.), produced in Xinjiang; Mint (Mentha haplocalyx Briq), produced in Jiangsu; Gardenia jasminoides Ellis, produced in Guangdong; Scutellaria baicalensis Georgi, produced in Shanxi; Forsythia suspense (Thunb.) Vahl, produced in Zhejiang.

[0051] Example 1

[0052] Formula of antiviral Chinese medicine composition: 24g of Forsythia suspensa, 6g of Gardenia jasminoides, 6g of Scutellaria baicalensis, 12g of Licorice, 12g of Rhubarb, 6g of Menthus fragrans, and 12g of Glauber's salt.

[0053] Preparation of antiviral Chinese medicine composition:

[0054] (1) Weigh forsythia suspensa, gardenia jasminoides, scutellaria baicalensis, and liquorice according to the formula, add distilled water at a material-liquid ratio of 10 mL water / g medicinal material, soak for 30 minutes, and boil for 20 minutes to obtain liquid a;

[0055] (2) Weigh rhubarb and mint according to the formula amount, add them to the medicinal solution a, continue to decoct for 10 minutes, pour out the decoction, and obtain the medicinal solution b1; add distilled water to the obtained medicinal residue according to the material-liquid ratio of 6 mL water / g medicinal material, boil for 10 minutes to obtain the medicinal solution b2; combine the medicinal solutions b1 and b2 to obtain the medicinal solution b;

[0056] (3) Passing the drug solution b through three layers of gauze, adding sodium sulfate weighed according to the formula while it is still hot, stirring to dissolve, and filtering with three layers of filter paper to obtain an antiviral Chinese medicine composition (i.e., improved Liangge San, abbreviated as: LGS);

[0057] (4) The antiviral Chinese medicine composition obtained in step (3) is concentrated by rotary evaporation to an extract form, freeze-dried by a freeze dryer, and stored in a dry place to obtain an antiviral Chinese medicine composition preparation (abbreviated as: LGS preparation).

[0058] Experimental Example 1

[0059] The toxicity evaluation test of LGS on human hepatoma cell Huh7, Aedes albopictus larvae cell C6 / 36 and baby hamster kidney cell BHK-21.

[0060] 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. After culturing for 24 hours, the LGS preparation prepared in Example 1 was diluted to a concentration of 800, 400, 200, 100, 50, 25, and 12.5 μg / mL using DMEM culture medium (Huh7) or 1640 culture medium (C6 / 36 and BHK-21) containing 10% fetal bovine serum, and a blank group (control) of solvent was prepared and added to the 96-well plate at an amount of 200 μL per well; after culturing the 96-well plate in a cell culture incubator for 96 hours, 100 μL of MTT (5 mg / mL) solution prepared with phosphate buffered saline (PBS) was added to each well; after incubating at 37°C for 4 hours, the supernatant was removed, and then 150 μL of DMSO (dimethyl sulfoxide); according to conventional methods, each group of cells was placed in an ELISA reader, the absorbance value (OD value) was measured at a wavelength of 490 nm, and the cell survival rate was calculated based on the absorbance value.

[0061] The cell viability calculation formula is as follows:

[0062] Survival rate = OD value of cells in each group / OD value of blank group × 100%.

[0063] Test results: Figure 1 As shown, the LGS prepared in Example 1 is basically non-toxic to Huh7 and C6 / 36 cells at a concentration below 200 μg / mL, and is basically non-toxic to BHK-21 cells at a concentration below 100 μg / mL. Therefore, in subsequent experiments, 200 μg / mL was used as the highest concentration of LGS prepared in Example 1 used in Huh7 and C6 / 36 cells, and 100 μg / mL was used as the highest concentration of LGS prepared in Example 1 used in BHK-21 cells. This shows that the LGS prepared in Example 1 has no obvious liver and kidney toxicity, and has no obvious toxicity to mosquito cells.

[0064] Experimental Example 2

[0065] Evaluation test of LGS's anti-DENV-2 effect at various stages of DENV-2 infection.

[0066] Experimental method: Huh7 and C6 / 36 were plated at 1×10 5 BHK-21 cells were seeded at a density of 8 × 10 per well in a 12-well plate. 3 / well were inoculated in a 96-well plate and then cultured in a cell culture incubator for 24 h; different concentrations of LGS prepared in Example 1 were given 2 h before virus infection (pre-treatment), during virus infection (co-treatment), 1 h (C6 / 36 and BHK-21) or 2 h (Huh7) after virus infection (post-treatment) (Huh7 and C6 / 36: 200, 100, 50, 25, 12.50, 6.25, 3.13 μg / mL, and a solvent control group was configured as a DENV-2 group; BHK-21: 100, 50, 25, 12.50, 6.25, 3.13, 1.57 μg / mL, and a solvent control group was configured as a DENV-2 group, and a solvent control not infected with the virus was set as a Control group), and the infection titer of DENV-2 was 2×10 5 PFU / mL (Huh7) and 4×10 2 PFU / mL (C6 / 36 and BHK-21); 500 μL of the above solution was added to each well of Huh7 and C6 / 36 cells, and 100 μL of the above solution was added to each well of BHK-21 cells. Huh7 and C6 / 36 were incubated in a cell culture incubator for 48 hours, and the effect of LGS on DENV-2 mRNA expression was detected by real-time fluorescence quantitative PCR according to conventional methods; BHK-21 was incubated in a cell culture incubator for 120 hours, 100 μL of CCK8 reagent was added to each well, and the absorbance value (OD value) was measured at a wavelength of 490 nm using an ELISA reader after incubation at 37°C for 1 hour to detect cell viability.

[0067] The cell viability calculation formula is as follows:

[0068] Survival rate = OD value of cells in each group / OD value of blank group × 100%.

[0069] 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.

[0070] The primer sequences for the above real-time fluorescence quantitative PCR are shown in the table below.

[0071]

[0072] Test results: Figure 2 As shown in A and B, the LGS prepared in Example 1 has no antiviral effect in both the pre-treatment and post-treatment stages. Figure 2As shown in Figure C, the LGS prepared in Example 1 inhibited the expression of DENV-2 genes in Huh7 and C6 / 36 cells during the co-treatment stage, and enhanced the cell activity of BHK-21 after virus infection, indicating that the LGS prepared in Example 1 exerted an antiviral effect in the early stage of DENV-2 infection.

[0073] Experimental Example 3

[0074] Evaluation test of LGS inhibiting the protein and gene expression levels of DENV-2 proteins E and NS1.

[0075] Experimental method: Huh7, C6 / 36 and BHK-21 cells were cultured at 2×10 5 / well were inoculated in a 6-well plate, and different concentrations of LGS prepared in Example 1 (Huh7 and C6 / 36: 100, 50, 25 μg / mL; BHK-21: 25, 12.50, 6.25 μg / mL) were given during virus infection, and a solvent control group was configured as a DENV-2 group, and heparin (Heparin) (50 μg / mL) was used as a positive control group, wherein Huh7 and C6 / 36 cells used GAPDH as an internal reference, and BHK-21 cells used β-actin as an internal reference, and 1 mL was added to each well of the 6-well plate; after incubation in an incubator for 48 hours, Western blotting and real-time fluorescence quantitative PCR were used to detect the expression levels of viral protein E and NS1 protein and gene in the cells according to conventional methods.

[0076] 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.

[0077] The primer sequences for the above real-time fluorescence quantitative PCR are shown in the table below.

[0078]

[0079] Test results: Figure 3 As shown, the LGS prepared in Example 1 inhibits the expression of viral proteins E and NS1 in cells ( Figure 3 A) and genes ( Figure 3 Because the E protein mainly mediates the adsorption and entry stage of DENV-2 virus, and the NS1 protein is the main target of DENV-2 vaccine development, the inhibitory effect of LGS on both can prove that LGS has an anti-DENV-2 virus effect.

[0080] Experimental Example 4

[0081] Evaluation test of LGS's ability to inhibit the formation of CPE (cytopathic effect) caused by DENV-2 infection.

[0082] Experimental method: Huh7, C6 / 36 and BHK-21 cells were cultured at 2×10 5 / well were inoculated in a 6-well plate for 24 hours, and different concentrations of LGS prepared in Example 1 (Huh7 and C6 / 36: 100, 50, 25 μg / mL; BHK-21: 25, 12.50, 6.25 μg / mL) were given during virus infection, and a solvent control group was configured as a DENV-2 group, heparin (Heparin, 50 μg / mL) was used as a positive control group, and a solvent control not infected with the virus was set as a Control group, and 1 mL was added to each well of the 6-well plate; and the changes in cell morphology were observed under a microscope every day.

[0083] Test results: Figure 4 As shown, by observing the cell morphology of Huh7, C6 / 36 and BHK-21 cells after virus infection under a microscope, it was found that Huh7, C6 / 36 and BHK-21 cells showed CPE on the 3rd day, 1st day and 5th day after virus infection, respectively, and the LGS obtained in Example 1 could inhibit the formation of CPE.

[0084] Experimental Example 5

[0085] Evaluation test of LGS inhibiting the release of DENV-2 progeny viruses.

[0086] Experimental method: Huh7 cells were cultured at 2×10 5 / well were inoculated in a 6-well plate, and different concentrations of LGS (100, 50, 25 μg / mL) obtained in Example 1 were given during virus infection, and a solvent control group was configured as a DENV-2 group, and heparin (Heparin) (50 mg / mL) was used as a positive control group, and was added to the 6-well plate at an amount of 2 mL / well; after incubation in an incubator for 48 hours, the cell supernatant was collected and detected according to a conventional method; 300 μL of the supernatant was used to detect the gene expression levels of E and NS1 proteins by real-time fluorescence quantitative PCR, and the remaining supernatant was used to infect BHK-21 cells (2×10 5 / well, 6-well plate); after infection at 37°C for 1 hour, methylcellulose containing 2% fetal bovine serum was added and incubated in an incubator for 120 hours. The cells were fixed and stained with crystal violet containing 4% paraformaldehyde, and the formation of virus plaques was detected by virus plaque assay according to conventional methods.

[0087] The real-time fluorescence quantitative PCR detection conditions are: 40 amplification cycles in FAM mode, denaturation at 95° C. for 2 min, annealing at 95° C. for 10 s, and extension at 60° C. for 30 s. The primer and probe sequences are the same as those in Experimental Example 3.

[0088] Test results: Figure 5 As shown, the LGS obtained in Example 1 inhibited the gene expression levels of E and NS1 in the cell supernatant in a dose-dependent manner ( Figure 5 A), and inhibit the formation of viral plaques ( Figure 5 B).

[0089] Experimental Example 6

[0090] An experiment to evaluate the process by which LGS inhibits the transfer of cytoplasmic Hsp70 to the cell membrane in host cells caused by DENV-2 infection.

[0091] Experimental method: Huh7 cells were cultured at 6×10 5 The cells were inoculated at a density of 100 μg / mL in a 6 cm culture dish, and LGS prepared in Example 1 was administered at a concentration of 100 μg / mL during virus infection, and a solvent control group was configured as a DENV-2 group, and 2 mL of the LGS was added to the culture dishes respectively, and detected according to a conventional method; two hours after virus infection, the cells were collected to extract cytoplasmic proteins and membrane proteins, and western blotting was used to detect the expression of Hsp70 and E protein on the membrane, with ATP1A1 and GAPDH as internal references for membrane protein and cytoplasmic protein, respectively; at the same time, the cells that were similarly infected and administered with LGS prepared in Example 1 (a solvent control that was not infected with the virus was set as the Control group) were used to detect the position of Hsp70 expression by immunofluorescence, and ATP1A1 was used as a membrane protein marker.

[0092] Test results: Figure 6 As shown, the western blotting results showed that the LGS prepared in Example 1 inhibited the transfer of cytoplasmic Hsp70 to the cell membrane ( Figure 6 A), and immunofluorescence also obtained the same results ( Figure 6 B).

[0093] Experimental Example 7

[0094] An evaluation experiment was conducted to show that LGS can prolong the survival time, reduce clinical scores, and delay weight loss in ICR suckling mice infected with DENV-2.

[0095] Experimental method: 6-day-old ICR suckling mice (n=10) were randomly divided into a control group, a DENV-2 group, and an example group (LGS prepared in Example 1 was administered at a raw drug amount of 10, 5, and 2.5 mg / kg of mouse body weight, such as 10 mg / kg, according to the mouse body weight, and a total weight of 10 mg of LGS medicinal materials (including 3.077 mg of Forsythia suspensa, 0.769 mg of Gardenia jasminoides, 0.769 mg of Scutellaria baicalensis, 1.54 mg of Licorice, 1.54 mg of Rhubarb, 0.769 mg of Mentha arvense, and 1.54 mg of Sodium bicarbonate) was administered per kilogram of mouse body weight to obtain the corresponding LGS); the mice were intraperitoneally injected (4×10 5 PFU / mL) and intracranial injection of DENV-2 (4×10 2 A mouse model of DENV-2 infection was established with 100 μg / mL (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 normal saline, and then the drug and normal saline were gavaged every two days; the survival rate was observed on the 10th day after virus infection, and the clinical scores and weight changes of the mice were recorded.

[0096] The clinical score standard is: 0 points for health, 1 point for weight loss and hunched back, 2 points for slow movement, 3 points for weakness of four limbs or unilateral paralysis, 4 points for quadriplegia, and 5 points for death.

[0097] Test results:

[0098] like Figure 7 As shown in A, all suckling mice infected with DENV-2 died at 7.5 dpi. The LGS 10 mg / kg group could prolong the survival time to 9 dpi, and the LGS 5 mg / kg and 2.5 mg / kg groups could prolong the survival time to 8.5 dpi, and the LGS 10 mg / kg and 5 mg / kg groups were statistically significant.

[0099] Since suckling mice mainly show neurological symptoms after being infected with DENV-2, these symptoms were clinically scored. The clinical score standard is: 0 points for health, 1 point for weight loss and hunched back, 2 points for slow movement, 3 points for limb weakness or unilateral paralysis, 4 points for quadriplegia, and 5 points for death.

[0100] like Figure 7As shown in B, after suckling mice were infected with DENV-2, the clinical scores increased rapidly during the period of 5-7.5 dpi. Compared with the DENV-2 group, the clinical scores of the LGS 10 mg / kg group and the 5 mg / kg group increased at a slower rate. At 6.5 dpi, the clinical scores of the three LGS groups were all lower than those of the DENV-2 group, and all were statistically significant. During 7 dpi and 7.5 dpi, only the 10 mg / kg group and the 5 mg / kg group significantly reduced the clinical scores of DENV-2 infected mice.

[0101] For the changes in the body weight of suckling mice, Figure 7 As shown in C, the body weight of suckling mice in the DENV-2 group decreased rapidly during 5-7 d.pi, while the body weight loss rate of the Example group was slowed down during 5-6 d.pi.

[0102] Experimental Example 8

[0103] Evaluation experiment of LGS in alleviating brain pathological damage in ICR suckling mice infected with DENV-2.

[0104] Experimental method: 6-day-old ICR suckling mice (n=10) were randomly divided into a control group, a DENV-2 group, and a LGS group (LGS prepared in Example 1 was administered at a ratio of 10, 5, and 2.5 mg of crude drug / kg of mouse body weight); the mice were intraperitoneally injected (4×10 5 PFU / mL) and intracranial injection of DENV-2 (4×10 2 A mouse model of DENV-2 infection was established with 100 μg / mL (PFU / mL); mice were gavaged with drugs 2 days before DENV-2 infection. The Control group and the DENV-2 group were gavaged with the same volume of saline, and then drugs and saline were gavaged every two days; each group of suckling mice (n=10) were anesthetized and killed on the 7th day after DENV-2 infection, and the suckling mouse brains were collected. The pathological damage of the suckling mouse brains was observed by hematoxylin-eosin (H&E) staining according to conventional methods.

[0105] Test results: Figure 8 As shown, the hippocampus is severely damaged, the neurons are necrotic and fall off, the nuclei are broken, the cells are arranged irregularly, and a large number of lymphocytes can be seen around the blood vessels to form "vascular cuffs" (red arrows); then in the cortical area (Cerebrum), neurons are punctately necrotic, the nuclei are broken, and the cells are arranged irregularly. After the DENV-2-infected suckling mice were treated with LGS prepared in Example 1, the pathological damage of these brain tissues was repaired to varying degrees.

[0106] Experimental Example 9

[0107] Evaluation of LGS's ability to inhibit the expression of viral proteins E and NS1 in the brain, liver, spleen, and serum of DENV-2 infected ICR suckling mice.

[0108] Experimental method: 6-day-old ICR suckling mice (n=10) were randomly divided into a control group, a DENV-2 group, and an example group (LGS prepared in Example 1 was administered at a raw drug dosage of 10, 5, and 2.5 mg / kg mouse body weight). The mice were intraperitoneally injected (4×10 5 PFU / mL) and intracranial injection of DENV-2 (4×10 2 A mouse model of DENV-2 infection was established with 100 μg / mL (PFU / mL); mice were gavaged with drugs 2 days before DENV-2 infection, and the Control group and the DENV-2 group were gavaged with the same volume of normal saline, and then the drugs were gavaged once every two days; each group of suckling mice (n=10) were anesthetized on the 7th day after DENV-2 infection, and the anesthetized suckling mice were bled through the heart to obtain serum, and the suckling mice were euthanized, and the brain, spleen and liver of the suckling mice were collected, and the expression levels of E and NS1 genes and proteins in each tissue were detected by real-time fluorescence quantitative PCR and Western blotting according to conventional methods.

[0109] The above real-time fluorescence quantitative PCR detection conditions are the same as those in Experimental Example 3

[0110] Test results: Fig. 9 As shown in A, LGS significantly inhibited the gene expression of E and NS1 in brain, spleen, liver and serum, and inhibited the protein expression levels of E and NS1 in brain, spleen and liver ( Fig. 9 B).

[0111] Experimental Example 10

[0112] Evaluation experiment of LGS prolonging the survival time and reducing viremia of DENV-2 infected AG129 mice.

[0113] 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 LGS group (LGS prepared in Example 1 was administered at a ratio of 50, 25, and 12.5 mg of crude drug / kg of mouse body weight); the mice were intraperitoneally injected (4×10 5A mouse model of DENV-2 infection was established with 100 μg / mL (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 normal saline, and then the drug was gavaged once a day until the end of the experiment. During the period, the changes in the survival rate and body weight of the mice were recorded, and blood was collected through the tail vein on the 1st day, 3rd day, 5th day, and 7th day after virus infection. The changes in viremia were detected by real-time fluorescence quantitative PCR according to conventional methods.

[0114] The above real-time fluorescence quantitative PCR detection conditions are the same as those of Experimental Example 3

[0115] Test results: Fig.10 As shown in A, the experimental results of survival analysis showed that AG129 mice began to die at 8d.pi after being infected with DENV-2 and died at 12d.pi. After LGS treatment, doses of 50mg / kg and 25mg / kg extended the initial death time of mice to 16d.pi, and 50% of the mice remained alive at the end of the experiment. Although the 12.5mg / kg dose did not keep the mice alive, it extended the final death time of the mice to 20d.pi, indicating that LGS improved the survival rate of mice and protected mice from DENV-2-induced death.

[0116] For the changes in mouse body weight, Fig.10 As shown in B, DENV-2 infection does not cause significant changes in mouse body weight.

[0117] Regarding the changes in viremia during DENV-2 infection, Fig.10 In C, we found that the number of viral E protein gene copies in the serum of mice in the DENV-2 group reached a peak at 3d.pi, and then gradually decreased. By 7d.pi, the number of copies was already at a very low level, while the LGS group prepared in Example 1 significantly inhibited the replication of the viral E protein gene in mice.

[0118] Experimental Example 11

[0119] Evaluation of LGS in alleviating pathological damage in the liver, brain, and colon of DENV-2-infected AG129 mice.

[0120] 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 LGS group (LGS prepared in Example 1 was administered at a ratio of 50, 25, and 12.5 mg of crude drug / kg of mouse body weight); the mice were intraperitoneally injected (4×10 5A mouse model of DENV-2 infection was established with 100 μg / mL (PFU / mL); mice were gavaged with drugs 2 days before DENV-2 infection, and the Control group and the DENV-2 group were gavaged with the same volume of normal saline, and then the drugs were gavaged once a day; AG129 mice (n=8) were anesthetized and killed on the 8th day after DENV-2 infection, and the brain, liver and large intestine were collected. The pathological damage of the organs and the therapeutic effect of LGS were observed by H&E staining according to conventional methods.

[0121] Test results: Fig.11 As shown in A, after mice were infected with DENV-2, the pathological damage of the liver was manifested as liver swelling, hepatocyte nucleus shrinkage, and cytoplasmic vacuoles; Fig.11 B The pathological damage of the brain is manifested as no obvious abnormality in the hippocampus, but a small amount of neuronal punctate necrosis and nuclear fragmentation (black arrows) can be seen in the cortex, moderate proliferation of glial cells in multiple places, vascular cuffing (yellow arrows), and a small amount of granulocytes (red arrows) can be seen; Fig.11 C The pathological damage of the large intestine was manifested by the increase in the number and size of goblet cells at the base of the crypts. The experimental results showed that LGS alleviated the pathological damage of the liver, brain and large intestine of AG129 mice infected with DENV-2.

[0122] Experimental Example 12

[0123] Evaluation experiment of LGS inhibiting the copy number of DENV-2 gene in the brain, liver, spleen and kidney of DENV-2 infected AG129 mice.

[0124] 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 DENV-2 group and a LGS group (LGS prepared in Example 1 was administered at a ratio of 50, 25, and 12.5 mg of crude drug / kg of mouse body weight); the mice were intraperitoneally injected (4×10 5 The DENV-2 infected mouse model was established with 100 μg / mL (PFU / mL); mice were gavaged with drugs 2 days before DENV-2 infection, and the DENV-2 group was gavaged with the same volume of normal saline, and then gavaged with drugs once a day; AG129 mice (n=8) were anesthetized and killed on the 8th day after DENV-2 infection, and the brain, liver, spleen and kidney were collected, and the copy number of viral genes in these organs was detected by real-time fluorescence quantitative PCR according to conventional methods.

[0125] The above real-time fluorescence quantitative PCR detection conditions are the same as those in Experimental Example 3

[0126] Test results: Fig.12As shown, compared with the DENV-2 group, LGS dose-dependently reduced the copy number of the viral E protein gene in the brain, liver, spleen and kidney.

[0127] Experimental Example 13

[0128] Toxicity evaluation test of LGS on mouse peritoneal mononuclear macrophage RAW264.7 cells.

[0129] Experimental method: Mouse peritoneal mononuclear macrophage RAW264.7 cells (commercially available) were plated at 8×10 3 The cells were inoculated at a density of 100 μg / mL in a 96-well plate and cultured for 24 h. The LGS preparation prepared in Example 1 was diluted to a concentration of 768, 384, 192, 96, 48, 24, and 12 μg / mL using DMEM culture medium containing 10% fetal bovine serum. A solvent was prepared as a control group and added to the 96-well plate at 200 μL per well. After culturing in a cell culture incubator for 48 h, 100 μL of MTT solution (5 mg / mL) prepared with PBS was added to each well. After incubation at 37° C. for 4 h, the supernatant was removed by aspiration, 150 μL of DMSO was added to each well, and the absorbance (OD value) was measured at a wavelength of 490 nm on a microplate reader according to a conventional method, and the cell survival rate was calculated based on the absorbance.

[0130] The cell viability calculation formula is as follows:

[0131] Survival rate = OD value of cells in each group / OD value of blank group × 100%.

[0132] Test results: Fig.13 As shown, LGS is substantially non-toxic to RAW264.7 cells at a concentration below 192 μg / mL. Therefore, in subsequent experiments, 192 μg / mL was used as the highest concentration of LGS prepared in Example 1 used in RAW264.7 cells.

[0133] Experimental Example 14

[0134] Evaluation test of LGS's ability to inhibit inflammation induced by SARS-CoV-2 and H1N1.

[0135] Experimental method: RAW264.7 cells were plated at 2×10 5The cells were inoculated at a density of 192, 96, and 48 μg / mL in a 6-well plate and cultured for 24 h; 12 h before the virus infected the cells, LGS prepared in Example 1 at a concentration of 192, 96, and 48 μg / mL was added to the 6-well plate in an amount of 2 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 LGS at the above concentration was added at the same time, incubated at 37° C. for 1 h, the virus solution was removed and washed twice with PBS, and the LGS at the above concentration was added again. The cells were incubated in an incubator for 48 h, and the cell supernatant was collected. According to conventional methods, the effect of LGS on the secretion of virus-induced inflammatory factors interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and interleukin-1β (IL-1β) protein was detected by ELISA; the cells were collected, and according to conventional methods, the effect of LGS on the expression of virus-induced inflammatory factors IL-6, TNF-α, and IL-1β mRNA was detected by real-time fluorescence quantitative PCR.

[0136] 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.

[0137] The primer sequences for the above real-time fluorescence quantitative PCR are shown in the table below.

[0138]

[0139] Test results: Fig.14 As shown in the figure, LGS has a significant inhibitory effect on acute macrophage inflammation caused by SARS-CoV-2 (A, C) and H1N1 (B, D).

[0140] Experimental Example 15

[0141] Evaluation of the protective effect of LGS against lung injury in hACE2 transgenic C57BL / 6 mice infected with SARS-CoV-2 and KM mice infected with H1N1.

[0142] 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 an LGS group (administered with LGS prepared in Example 1 at a mass ratio of 25 g crude drug / kg mouse body weight); the mice were infected by intranasal drip for 10 4A mouse model of SARS-CoV-2 infection was established with SARS-CoV-2 virus at a concentration of 1 PFU / mL. The normal group was given PBS by intranasal 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 according to the conventional method. 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 according to the conventional method.

[0143] The above real-time fluorescence quantitative PCR detection conditions are the same as those in Experimental Example 14.

[0144] KM mice aged 5 to 6 weeks (n=8) were randomly divided into a normal group, an H1N1 infection group and an LGS group (administered with LGS prepared in Example 1 at a mass ratio of 25 g of raw drug / kg of 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 intranasally administered with PBS; the mice were gavaged with the drug 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; the lungs were dissected out 5 days after infection, and the left lung was fixed with 4% paraformaldehyde and HE staining was used to detect the pathological damage of the lung tissue. 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.

[0145] The above real-time fluorescence quantitative PCR detection conditions were the same as the above experimental conditions.

[0146] Test results: Fig.15 As shown, LGS has a protective effect on the lungs of SARS-CoV-2-infected mice (A) and H1N1-infected mice (B), and inhibits the increase in mRNA of inflammatory factors IL-6, TNF-α, and IL-1β caused by SARS-CoV-2 (C) and H1N1 (D) infection.

[0147] Experimental Example 16

[0148] Comparison of the anti-SARS-CoV-2 and H1N1-induced inflammatory effects of LGS and the pharmaceutical compositions of the prior art.

[0149] The pharmaceutical composition of the prior art: weigh 1.69g of epimedium, 1.69g of honeysuckle, 1.69g of polygala, 1.69g of costusroot, 1.69g of chuanxiong, 1.69g of siler, 1.69g of angelica, 1.69g of peony, 1.69g of forsythia, 1.69g of peppermint leaf, 1.69g of ephedra, 1.69g of sodium sulfate, 1.69g of rhubarb, 2.62g of gypsum, 2.62g of platycodon, 2.62g of scutellaria, 1.31g of atractylodes, 1.31g of gardenia, 1.31g of schizonepeta, 4.6g of ginger, 6.37g of talc, and 4.5g of licorice, add 1L of water, and leave at room temperature for 1 hour; perform hot water extraction at 100°C for 3 hours to obtain 100mL of the crude drug extract, which is the pharmaceutical composition of the prior art.

[0150] Experimental method: RAW264.7 cells were plated at 2×10 5 The cells were inoculated at a density of 192 μg / mL in a 6-well plate and cultured for 24 h; 12 h before the virus infected the cells, LGS prepared in Example 1 at a concentration of 192 μg / mL and the above-mentioned pharmaceutical composition at a concentration of 200 μg / mL were added to the cells respectively; the cell supernatant was removed, the virus solution was added and the LGS and the pharmaceutical composition at the above-mentioned concentrations were added at the same time, incubated at 37°C for 1 h, the virus solution was removed and the cells were washed twice with PBS, and the LGS and the pharmaceutical composition at the above-mentioned concentrations were added again; the cells were incubated in an incubator for 48 h, the cell supernatant was collected, and the effects of LGS and the pharmaceutical composition on the secretion of virus-induced inflammatory factors IL-6, TNF-α and IL-1β proteins were detected by ELISA according to conventional methods, and the cells were collected, and the effects of LGS on the expression of virus-induced inflammatory factors IL-6, TNF-α and IL-1β mRNA were detected by real-time fluorescence quantitative PCR according to conventional methods.

[0151] The above real-time fluorescence quantitative PCR detection conditions are the same as those in Experimental Example 14

[0152] Test results: Fig.16 As shown, LGS and the drug combination have significant inhibitory effects on acute macrophage inflammation caused by SARS-CoV-2 (A) and H1N1 (B), respectively, but the inhibitory effect of LGS on inflammation is significantly better than that of the drug combination.

[0153] 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.

[0154] 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 Chinese medicine composition, characterized in that: The active ingredients are prepared from the following raw materials in parts by weight: 20-28 parts of forsythia, 5-7 parts of gardenia, 5-7 parts of scutellaria, 10-14 parts of liquorice, 10-14 parts of rhubarb, 5-7 parts of mint and 10-14 parts of sodium sulfate.

2. The antiviral Chinese medicine composition according to claim 1, characterized in that: The active ingredients are prepared from the following raw materials in parts by weight: 23-24 parts of forsythia, 5-7 parts of gardenia, 5-7 parts of scutellaria, 11-13 parts of liquorice, 11-13 parts of rhubarb, 5-7 parts of mint and 11-13 parts of sodium sulfate.

3. The method for preparing the antiviral Chinese medicine composition according to any one of claims 1 to 2, characterized in that: Weigh each raw material according to the formula amount, extract with water as solvent to obtain the medicinal solution.

4. The preparation method according to claim 3, characterized in that: The following steps are involved: (1) Weighing forsythia suspensa, gardenia jasminoides, scutellaria baicalensis, and liquorice according to the formula, adding water to boil and taking out the liquid medicine to obtain liquid medicine a; (2) Weigh rhubarb and mint according to the formula, add them to liquid a, decoct and take out the liquid to obtain liquid b; (3) Weigh sodium sulfate according to the formula and add it to solution b.

5. The preparation method according to claim 4, characterized in that: In step (1), forsythia suspensa, gardenia jasminoides, scutellaria baicalensis and liquorice are weighed according to the formula amount, water is added according to the material-liquid ratio of 10±2mL water / g medicinal material, soaked for 30±10min, and boiled for 20±5min to obtain medicinal solution a; In step (2), rhubarb and mint are weighed according to the formula amount, added to the medicinal solution a, and continued to be decocted for 10±5 minutes, filtered, and obtained medicinal solution b1; water is added to the obtained medicinal residue according to the material-liquid ratio of 6+2mL water / g medicinal material, and boiled for 10±5 minutes to obtain medicinal solution b2; and medicinal solutions b1 and b2 are combined to obtain medicinal solution b; In step (3), after filtering the medicinal solution b, add the sodium sulfate weighed according to the formula, stir to dissolve, and filter to obtain the final medicinal solution.

6. The preparation method according to claim 5, characterized in that: The method further comprises a step (4), wherein the final medicinal solution obtained in the step (3) is concentrated into an extract form, and after drying, the antiviral Chinese medicine composition is obtained.

7. Use of the antiviral Chinese medicine composition according to any one of claims 1 to 2 in the preparation of antiviral drugs and / or the treatment of diseases caused by viruses.

8. The use according to claim 7, characterized in that: The virus is an RNA virus, a DENV-2 virus, a new coronavirus or an H1N1 virus.

9. The use according to claim 7, characterized in that: The antiviral Chinese medicine composition achieves its antiviral effect by inhibiting the expression of mRNA in cells, inhibiting the protein and mRNA expression of DENV-2 protein E and NS1, inhibiting the release of DENV-2 progeny viruses, reducing the copy amount of viral RNA in the blood after infection, inhibiting the formation of cytopathic effect caused by viral infection, and inhibiting the transfer of cytoplasmic Hsp70 to the cell membrane caused by viral infection.

10. The use according to claim 7, characterized in that: The antiviral Chinese medicine 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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