Application of traditional Chinese medicine composition in preparation of medicine for preventing and / or treating influenza A and related diseases

Abstract

The invention provides application of a traditional Chinese medicine composition in preparation of medicines for preventing and / or treating influenza A and related diseases thereof. The traditional Chinese medicine composition comprises the following components or is prepared from the following raw material medicines in parts by weight: 20-40 parts of gypsum, 10-30 parts of wild buckwheat rhizome, 10-30 parts of herba houttuyniae, 3-15 parts of roasted herba ephedrae, 3-15 parts of fried semen armeniacae amarae, 3-15 parts of bulbus fritillariae cirrhosae, 5-25 parts of herba ardisiae japonicae, 3-15 parts of folium eriobotryae, 3-15 parts of fried fructus perillae and 3-15 parts of roasted radix glycyrrhizae. The traditional Chinese medicine composition disclosed by the invention has a relatively good inhibition effect on influenza A viruses, is low in toxicity and high in safety, also has a good anti-influenza A virus infection effect, and can provide a basis for clinically treating pulmonary infection caused by the influenza A viruses.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine. Specifically, the present invention relates to the use of a traditional Chinese medicine composition in preparing a medicine for preventing and / or treating influenza A and related diseases. Background Art

[0002] Influenza A refers to an acute respiratory infectious disease caused by influenza A virus. The main differences between influenza A and common influenza are different pathogens, different infectivity and different symptoms. There are many types of viruses that cause common influenza, including rhinovirus, coronavirus, parainfluenza virus, adenovirus and respiratory syncytial virus. The pathogen that causes influenza A is influenza A virus. The virus was first isolated in humans in April 2009. It is a new influenza virus formed by hybridization and recombination of gene fragments of human influenza virus, swine influenza virus and avian influenza virus. The infectivity of common influenza virus is generally weak and the transmission speed is slow. Influenza A virus is highly contagious and spreads quickly. Common influenza is mainly characterized by upper respiratory tract symptoms, such as runny nose, sneezing, sore throat, cough, etc. The fever symptoms are relatively mild, and the symptoms of headache and muscle aches are generally mild. In addition to nasal congestion, runny nose, sore throat and other symptoms, most influenza A patients will also have high fever symptoms, and may also have significant headaches and muscle aches. Moreover, influenza A is more likely to cause complications, such as viral myocarditis and pneumonia, and severe cases may be life-threatening due to complications such as respiratory failure. The above complications are relatively rare in common influenza, and in most cases they can be recovered on their own.

[0003] In addition, the treatment methods for influenza A and common influenza are also significantly different. Common influenza is self-limiting and generally does not require special treatment. It can get better in about a week. Those with obvious symptoms can use symptomatic treatment drugs appropriately. If influenza A is not treated in time, it may cause viral pneumonia. Conventional drugs used to treat common influenza include Shuanghuanglian oral liquid. The research of Liang Xing et al. showed that the treatment of influenza is mainly antiviral treatment, that is, by inhibiting the neuraminidase on the viral envelope to inhibit the proliferation of the virus in living cells, Shuanghuanglian oral preparations combined with oseltamivir phosphate can show good treatment effects for influenza ("Efficacy Analysis of Shuanghuanglian Oral Liquid Combined with Oseltamivir Phosphate in the Treatment of Influenza-like Cases", Electronic Journal of Clinical Medical Literature, Issue 94, 2019). However, Shuanghuanglian oral liquid has not shown good efficacy in the treatment of influenza A, and there is currently no literature that clearly shows that the drugs used in the traditional Chinese medicine diagnosis and treatment plan have targeted sensitivity to H1N1, H3N2 and H1N1-OC influenza strains.

[0004] The drugs currently used to treat influenza A mainly include oseltamivir phosphate and ribavirin, which usually need to be administered in the early stage of the disease (within 48 hours after the onset of symptoms), but due to the single target, drug resistance has emerged. For example, the H275Y mutation of H1N1 leads to oseltamivir resistance. In addition, oseltamivir phosphate can cause adverse reactions of digestive tract discomfort in patients, including nausea, vomiting, diarrhea, abdominal pain, etc., followed by adverse reactions of the respiratory system, including bronchitis, cough, etc., in addition to adverse reactions of the central nervous system, such as dizziness, headache, insomnia, fatigue, etc. Inhaled use of ribavirin can lead to deterioration of lung function, bacterial pneumonia, pneumothorax and cardiovascular reactions (decreased blood pressure and cardiac arrest), etc., as well as conjunctivitis and rash. The main adverse reactions after intravenous or oral administration are hemolytic anemia, hemoglobin reduction, anemia, fatigue, etc. Therefore, it is urgent to develop new, efficient and less toxic and side-effect anti-influenza A virus drugs. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a use of a Chinese medicine composition in the preparation of a medicament for preventing and / or treating influenza A and its related diseases. The Chinese medicine composition provided by the present invention has a good inhibitory effect on influenza A virus, low toxicity, high safety, and good anti-infection effect of influenza A virus, providing a basis for its clinical use in the treatment of lung infection caused by influenza A virus.

[0006] The above object of the present invention is achieved through the following technical solutions:

[0007] The invention provides a use of a traditional Chinese medicine composition in preparing a medicine for preventing and / or treating influenza A and related diseases. The traditional Chinese medicine composition comprises the following components or is prepared from raw materials comprising the following components in parts by weight: 20-40 parts of gypsum (Gypsum Fibrosum), 10-30 parts of golden buckwheat (Fagopyrum dibotrys (D.Don) Hara), 10-30 parts of houttuynia cordata Thunb, 3-15 parts of roasted ephedra (Ephedrae Herba), 3-15 parts of stir-fried bitter almonds (Armeniacae Semen Amarum), 3-15 parts of Fritillaria cirrhosa D.Don, 5-25 parts of dwarf tea (Herbσ Ardisiae Japonicae), 3-15 parts of loquat leaves (Eriobotryajaponica Thunb), 3-15 parts of stir-fried perilla seeds (Perillae Fructus) 3-15 parts and Radix Glycyrrhizae Preparata 3-15 parts.

[0008] According to some embodiments of the present invention, the Chinese medicine composition is the only active ingredient in the medicine.

[0009] According to some embodiments of the present invention, the traditional Chinese medicine composition comprises the following components or is prepared from the raw materials comprising the following components, by weight: 25-35 parts of gypsum, 15-25 parts of golden buckwheat, 15-25 parts of houttuynia cordata, 8-12 parts of roasted ephedra, 8-12 parts of stir-fried bitter almonds, 8-12 parts of Fritillaria cirrhosa, 10-20 parts of dwarf camellia, 8-12 parts of loquat leaves, 8-12 parts of stir-fried perilla seeds and 8-12 parts of roasted licorice, and optional pharmaceutically acceptable excipients.

[0010] Preferably, in parts by weight, the traditional Chinese medicine composition comprises the following components or is prepared from raw materials comprising the following components: 30 parts of gypsum, 20 parts of golden buckwheat, 20 parts of houttuynia cordata, 9 parts of roasted ephedra, 9 parts of stir-fried bitter almonds, 9 parts of Fritillaria cirrhosa, 15 parts of dwarf camellia, 9 parts of loquat leaves, 9 parts of stir-fried perilla seeds and 9 parts of roasted licorice.

[0011] According to some embodiments of the present invention, the influenza A is caused by one or more influenza A virus strains selected from the group consisting of H1N1 subtype, H3N2 subtype and drug-resistant influenza A virus strains; preferably, the influenza A is caused by H1N1 subtype influenza A virus strain and / or oseltamivir-resistant influenza A virus strain; more preferably, the H1N1 subtype influenza A virus strain is A / FM / 1 / 47 (H1N1); the H3N2 subtype influenza A virus strain is A / Beijing / 32 / 92 (H3N2); and the oseltamivir-resistant influenza A virus strain is H1N1-OC.

[0012] According to some embodiments of the invention, the influenza A-related disease is pneumonia.

[0013] According to some embodiments of the present invention, the Chinese medicine composition or the raw material drug further comprises a pharmaceutically acceptable excipient; preferably, the pharmaceutically acceptable excipient is selected from one or more of a diluent, an excipient, a binder, a preservative and a flavoring agent.

[0014] According to some embodiments of the present invention, the Chinese medicine composition is prepared by a method comprising the following steps:

[0015] (1) crushing the Fritillaria cirrhosa and then percolating with an ethanol aqueous solution, collecting the percolation liquid and the Fritillaria cirrhosa residue respectively, and then recovering the ethanol in the percolation liquid to obtain a Fritillaria cirrhosa extract for later use;

[0016] (2) distilling Houttuynia cordata by steam, collecting the distillate, the extract and the Houttuynia cordata residue respectively, and then extracting the volatile oil in the distillate for later use;

[0017] (3) After gypsum is boiled with water for the first time, the residue of Fritillaria cirrhosa, the residue of Houttuynia cordata, and the water-soaked roasted ephedra, golden buckwheat, stir-fried bitter almond, dwarf tea, loquat leaf, stir-fried perilla seed, and roasted liquorice are added, and water is added and boiled again, the decoction is collected, filtered, and the filtrate is collected;

[0018] (4) combining the filtrate obtained in step (3) with the extracted drug solution obtained in step (2), and concentrating the mixture to obtain a concentrated solution;

[0019] (5) The concentrated solution is mixed with the Fritillaria cirrhosa extract, dried, crushed, and then a pharmaceutically acceptable excipient is added, mixed well, and made into granules. After drying, the volatile oil obtained in step (2) is sprayed into the granules to obtain the traditional Chinese medicine composition.

[0020] Preferably, in step (1), the volume fraction of the ethanol aqueous solution is 60-80%, preferably 70%.

[0021] Preferably, in step (2), the steam distillation time is 1-5 hours, preferably 3 hours.

[0022] Preferably, in step (3), the soaking time is 0.5-2 hours, preferably 1 hour.

[0023] Preferably, in step (3), the first decoction time is 0.1-2 hours, preferably 0.5 hours.

[0024] Preferably, in step (3), the re-decocting is carried out under the following conditions: the number of decoctions is 1-5 times, preferably 3 times; the decoction time each time is 0.5-5 hours, preferably 2 hours.

[0025] The present invention has at least the following beneficial effects:

[0026] 1. The Chinese medicine composition provided by the present invention shows a good inhibitory effect on influenza A virus in in vitro experiments, and has low toxicity and high safety. Specifically, the present invention evaluates the inhibitory effect of the Chinese medicine composition of the present invention on H1N1, H3N2 and H1N1-OC on MDCK cells. The results show that the Chinese medicine composition of the present invention has a certain inhibitory effect on the above-mentioned tested virus strains, especially showing a good inhibitory effect on H1N1-OC. At the same time, the present invention determines the toxicity of the Chinese medicine composition of the present invention to MDCK cell lines, and the results show that the Chinese medicine composition of the present invention has low cytotoxicity, high safety, and has a high selection index for H3N2 and H1N1-OC.

[0027] 2. The Chinese medicine composition provided by the present invention shows good anti-influenza A virus infection effect in in vivo experiments, which can provide a basis for its treatment of lung infection caused by influenza A virus. Specifically, the present invention observed the weight, survival days and survival rate of mice in the H1N1 infected mouse model, and found that the treatment with the Chinese medicine composition of the present invention can improve the survival rate of virus-infected mice, prolong the survival time of mice, and reverse the weight loss of mice caused by virus infection. Further analysis shows that the Chinese medicine composition of the present invention can significantly reduce the virus titer in the lung tissue of infected mice, inhibit the activation of the TLR-7-My88-NF-κB signaling pathway in the lung tissue, and reduce the production of pro-inflammatory factors IL-6, IL-1β and TNF-α, thereby alleviating the symptoms of infectious pneumonia such as pulmonary edema, congestion and tissue damage in mice induced by viral infection.

[0028] 3. The Chinese medicine composition provided by the present invention exhibits more significant efficacy in inhibiting influenza A virus compared with various medicinal compositions disclosed in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0030] Figure 1 The results show that the Chinese medicine composition of the present invention and oseltamivir phosphate have a protective effect on the body weight of mice infected with influenza A virus H1N1.

[0031] Figure 2 The results show that the Chinese medicine composition and oseltamivir phosphate of the present invention improve the survival of mice infected with influenza A virus H1N1, wherein (A) the survival rate of infected mice; and (B) the survival time of infected mice.

[0032] Figure 3 The effects of the Chinese medicine composition of the present invention and oseltamivir phosphate on lung tissue edema in virus-infected mice are shown, (A) ratio of lung wet weight to body weight; (B) ratio of lung wet weight to lung dry weight; (C) lung tissue of virus-infected mice; *p<0.05; **p<0.01; ***p<0.001.

[0033] Figure 4 It shows that the Chinese medicine composition of the present invention and oseltamivir phosphate reduce the virus titer of the lung tissue of mice infected with influenza A virus H1N1, *p<0.05; **p<0.01; ***p<0.001.

[0034] Figure 5The Chinese medicine composition of the present invention and oseltamivir phosphate alleviate the lung damage of mice infected with influenza A virus H1N1 (×200 magnification), blue arrow: eosinophil and lymphocyte infiltration; orange arrow: necrotic and sloughing of bronchial mucosal epithelial cells, partial disappearance of the structure, and only the tube wall is visible; yellow arrow: eosinophil and macrophage infiltration can be seen in the alveolar cavity; black arrow: congestion of alveolar wall capillaries; brown arrow: eosinophilic flocs attached to the alveoli to form a transparent membrane.

[0035] Figure 6 It shows that the Chinese medicine composition of the present invention and oseltamivir phosphate reduce the content of pro-inflammatory factors IL-1β, IL-6 and TNF-α in the serum of mice infected with influenza A virus H1N1, * p<0.05; ** p<0.01; *** p<0.001.

[0036] Figure 7 The results show that the Chinese medicine composition of the present invention and oseltamivir phosphate inhibit the activation of TLR-7-MyD88-NF-κB pathway in lung tissue of mice infected with influenza A virus H1N1, wherein the upper figure shows the expression levels of MyD88, TLR7 and p-NF-κB-p65 proteins detected by Western blot, and the lower figure shows the semi-quantitative analysis of protein bands using Image J software.

[0037] Figure 8 It shows that the Chinese medicine composition of the present invention and oseltamivir phosphate inhibit the expression level of TLR-7 and MyD88 mRNA in the lung tissue of mice infected with influenza A virus H1N1. * p<0.05; ** p<0.01. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0039] The raw gypsum, golden buckwheat, houttuynia cordata, roasted ephedra, stir-fried bitter almond, fritillaria cirrhosa, dwarf tea, loquat leaf, stir-fried perilla seed and roasted licorice used in the following examples and comparative examples were all purchased from Anhui Haixin Pharmaceutical Co., Ltd.

[0040] Example 1

[0041] The following components were weighed in parts by weight: 30 parts of gypsum, 20 parts of golden buckwheat, 20 parts of houttuynia cordata, 9 parts of roasted ephedra, 9 parts of stir-fried bitter almonds, 9 parts of Fritillaria cirrhosa, 15 parts of dwarf tea, 9 parts of loquat leaves, 9 parts of stir-fried perilla seeds and 9 parts of roasted licorice, and a Chinese medicine composition was prepared according to the following method:

[0042] (1) grinding Fritillaria cirrhosa into coarse powder and then percolating with a 70% by volume ethanol aqueous solution, collecting the percolation liquid and Fritillaria cirrhosa residue respectively, and then recovering the ethanol in the percolation liquid until it is tasteless, thereby obtaining a Fritillaria cirrhosa extract for standby use;

[0043] (2) steam distilling Houttuynia cordata for 3 hours, collecting the distillate, the extract and the Houttuynia cordata residue respectively, and then extracting the volatile oil in the distillate for later use;

[0044] (3) After boiling gypsum with water for half an hour, add the residue of Fritillaria cirrhosa, the residue of Houttuynia cordata, and the roasted ephedra, golden buckwheat, fried bitter almond, dwarf tea, loquat leaf, fried perilla seed and roasted liquorice that have been soaked in water for 1 hour, boil three times with water, each time for 2 hours, combine the decoctions, filter and collect the filtrate;

[0045] (4) combining the filtrate obtained in step (3) with the extracted drug solution obtained in step (2), and concentrating the mixture to obtain a concentrated solution;

[0046] (5) The concentrated solution is mixed with the Fritillaria cirrhosa extract, dried, crushed, and then an appropriate amount of sucrose and dextrin are added, mixed, made into granules, dried, and sprayed with the Houttuynia cordata volatile oil obtained in step (2) to obtain a traditional Chinese medicine composition.

[0047] Example 2

[0048] The following components were weighed in parts by weight: 20 parts of gypsum, 25 parts of golden buckwheat, 30 parts of houttuynia cordata, 3 parts of roasted ephedra, 15 parts of stir-fried bitter almonds, 3 parts of Fritillaria cirrhosa, 22 parts of dwarf camellia, 3 parts of loquat leaves, 15 parts of stir-fried perilla seeds and 3 parts of roasted licorice.

[0049] The Chinese medicine composition was prepared according to the method of Example 1.

[0050] Example 3

[0051] The following components were weighed in parts by weight: 40 parts of gypsum, 15 parts of golden buckwheat, 10 parts of houttuynia cordata, 15 parts of roasted ephedra, 3 parts of stir-fried bitter almonds, 15 parts of Fritillaria cirrhosa, 8 parts of dwarf camellia, 15 parts of loquat leaves, 3 parts of stir-fried perilla seeds and 15 parts of roasted licorice.

[0052] The Chinese medicine composition was prepared according to the method of Example 1.

[0053] Comparative Example 1

[0054] The following components were weighed in parts by weight: 5 parts of gypsum, 40 parts of golden buckwheat, 40 parts of houttuynia cordata, 1 part of roasted ephedra, 2 parts of stir-fried bitter almonds, 2 parts of Fritillaria cirrhosa, 35 parts of dwarf camellia, 25 parts of loquat leaves, 1 part of stir-fried perilla seeds and 1 part of roasted licorice.

[0055] The Chinese medicine composition was prepared according to the method of Example 1.

[0056] Comparative Example 2

[0057] The following components were weighed in parts by weight: 50 parts of gypsum, 5 parts of golden buckwheat, 4 parts of houttuynia cordata, 3 parts of roasted ephedra, 40 parts of stir-fried bitter almonds, 19 parts of Fritillaria cirrhosa, 3 parts of dwarf camellia, 1 part of loquat leaf, 19 parts of stir-fried perilla seeds and 1 part of roasted licorice.

[0058] The Chinese medicine composition was prepared according to the method of Example 1.

[0059] Comparative Example 3

[0060] The following components were weighed in parts by weight: 30 parts of gypsum, 9 parts of stir-fried bitter almonds, 9 parts of Fritillaria cirrhosa and 9 parts of roasted liquorice.

[0061] The Chinese medicine composition is prepared according to the following method:

[0062] (1) grinding Fritillaria cirrhosa into coarse powder and then percolating with a 70% by volume ethanol aqueous solution, collecting the percolation liquid and Fritillaria cirrhosa residue respectively, and then recovering the ethanol in the percolation liquid until it is tasteless, thereby obtaining a Fritillaria cirrhosa extract for standby use;

[0063] (2) After decocting gypsum with water for half an hour, add the residue of Fritillaria cirrhosae and stir-fried bitter almonds and roasted licorice that have been soaked in water for 1 hour, add water and decoct three times, each time for 2 hours, combine the decoctions, filter, and collect the filtrate;

[0064] (3) concentrating the filtrate obtained in step (2) to obtain a concentrated solution;

[0065] (4) The concentrated solution is mixed with the extract of Fritillaria cirrhosae, dried, crushed, and then appropriate amounts of sucrose and dextrin are added, mixed well, made into granules, and dried to obtain a traditional Chinese medicine composition.

[0066] Comparative Example 4

[0067] The following components were weighed in parts by weight: 30 parts of gypsum, 20 parts of golden buckwheat, 20 parts of houttuynia cordata, 9 parts of roasted ephedra, 9 parts of stir-fried bitter almonds and 9 parts of roasted licorice.

[0068] The Chinese medicine composition is prepared according to the following method:

[0069] (1) steam distilling Houttuynia cordata for 3 hours, collecting the distillate, the extract and the Houttuynia cordata residue respectively, and then extracting the volatile oil in the distillate for later use;

[0070] (2) After gypsum was boiled in water for half an hour, Houttuynia cordata residue and roasted ephedra, golden buckwheat, fried bitter almonds and roasted licorice that had been soaked in water for 1 hour were added, and water was boiled for three times, each time for 2 hours, the decoctions were combined, filtered, and the filtrate was collected;

[0071] (3) combining the filtrate obtained in step (2) with the extract obtained in step (1), and concentrating the mixture to obtain a concentrated solution;

[0072] (4) Drying and crushing the concentrated solution, adding appropriate amounts of sucrose and dextrin, mixing well, making granules, drying, and spraying the Houttuynia cordata volatile oil obtained in step (1) to obtain a traditional Chinese medicine composition.

[0073] Comparative Example 5

[0074] The following components were weighed in parts by weight: 30 parts of gypsum, 20 parts of houttuynia cordata, 9 parts of roasted ephedra, 9 parts of stir-fried bitter almonds and 9 parts of roasted licorice.

[0075] The Chinese medicine composition is prepared according to the following method:

[0076] (1) steam distilling Houttuynia cordata for 3 hours, collecting the distillate, the extract and the Houttuynia cordata residue respectively, and then extracting the volatile oil in the distillate for later use;

[0077] (2) After decocting gypsum with water for half an hour, add houttuynia cordata residue and roasted ephedra, stir-fried bitter almonds and roasted licorice that have been soaked in water for 1 hour, add water and decoct three times, each time for 2 hours, combine the decoctions, filter and collect the filtrate;

[0078] (3) combining the filtrate obtained in step (2) with the extract obtained in step (1), and concentrating the mixture to obtain a concentrated solution;

[0079] (4) Drying and crushing the concentrated solution, adding appropriate amounts of sucrose and dextrin, mixing well, making granules, drying, and spraying the Houttuynia cordata volatile oil obtained in step (1) to obtain a traditional Chinese medicine composition.

[0080] Comparative Example 6

[0081] The following components were weighed in parts by weight: 30 parts of raw gypsum, 9 parts of roasted ephedra, 9 parts of stir-fried bitter almonds, 9 parts of stir-fried perilla seeds, and 9 parts of roasted licorice.

[0082] The Chinese medicine composition is prepared according to the following method:

[0083] (1) After gypsum is boiled in water for half an hour, roasted ephedra, stir-fried bitter almonds, stir-fried perilla seeds and roasted licorice root that have been soaked in water for 1 hour are added, and water is added to boil three times, each time for 2 hours, the decoctions are combined, filtered, and the filtrate is collected;

[0084] (2) concentrating the filtrate obtained in step (1) to obtain a concentrated solution;

[0085] (3) Drying and crushing the concentrated solution, adding appropriate amounts of sucrose and dextrin, mixing well, forming granules, and drying to obtain a traditional Chinese medicine composition.

[0086] Test 1 In vitro activity test

[0087] 1. Experimental Materials

[0088] 1. Drugs, reagents and preparation methods thereof

[0089] 1.1 Drugs and reagents

[0090] The Chinese medicine compositions prepared in the above embodiments and comparative examples.

[0091] Oseltamivir hydroxy acid (oseltamivir acid) was purchased from MCE Company, batch number: 112376.

[0092] 1.2 Tested virus strains and cell lines

[0093] Influenza A virus strains A / FM / 1 / 47 (H1N1), A / Beijing / 32 / 92 (H3N2) and oseltamivir-resistant influenza A virus strain (H1N1-OC) were cultured and amplified in nine-day-old SPF-grade chicken embryos and then packaged. The chicken embryo allantoic fluid was frozen in a -80℃ refrigerator in the laboratory and preserved by the Microbiology and Immunology Laboratory of China Pharmaceutical University.

[0094] Canine kidney epithelial cells (MDCK) were purchased from ATCC cell bank and maintained by the Microbiology and Immunology Laboratory of China Pharmaceutical University.

[0095] 1.3 Drug preparation method

[0096] The Chinese medicine compositions prepared in the above examples and comparative examples were: 4.0 mg of the drug was weighed using an analytical balance, dissolved in complete culture medium to a final concentration of 4.0 mg / mL, and diluted in a 1:2 ratio when used.

[0097] Oseltamivir acid: Weigh 2.0 mg of the drug and dissolve it in complete culture medium to a final concentration of 2.0 mg / mL. When using, perform gradient dilution according to the final concentration of the drug and set aside.

[0098] 2 Statistical methods

[0099] All data were analyzed using GraphPad Prism 6.0 statistical software package. One-way analysis of variance was used for comparison among multiple groups, and Student's t test was used for pairwise comparison among groups. p < 0.05 was considered statistically significant.

[0100] (II) Experimental methods and results

[0101] 1 Experimental methods

[0102] 1.1 Drug CC on cells 50 Determination of value

[0103] The MDCK cells in good growth state were digested with trypsin, centrifuged, counted, and adjusted to the appropriate density (5000 cells / well). Then the cell suspension was evenly added to a 96-well plate, and then the cell culture plate was placed in a 37°C, 5% CO2 incubator for overnight culture until a cell monolayer was formed. The original culture medium was discarded, and the drug (100 μL / well) diluted in a gradient with complete culture medium was added. Three replicates were set for each gradient, and the cells were placed in a 37°C, 5% CO2 incubator for further culture for 48 hours, and photographed. After replacing the serum-free culture medium, a commercially available CCK8 solution (Biyuntian Co., Ltd.) was added, and the cells were placed in a 37°C, 5% CO2 incubator for incubation for 2 hours, and the absorbance value was measured at a wavelength of 450nm using an ELISA reader.

[0104] 1.2 TCID of the tested virus strains on MDCK cells 50

[0105] The MDCK cells in good growth state were digested with trypsin, centrifuged, counted, and adjusted to an appropriate cell density (5000 cells / well). The cell suspension was then evenly added to a 96-well plate. The cell culture plate was then placed in a 37°C, 5% CO2 incubator and cultured overnight until a cell monolayer was formed. Chicken embryo allantoic fluid of influenza virus or cell supernatant containing the virus was diluted 10-fold in a serum-free medium (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 ). Discard the original cell culture medium, add virus dilutions of different concentration gradients, and discard the virus solution after co-culturing with the cells for 2 hours. Replace the culture medium with cell maintenance medium, place the cell culture plate in a 37°C, 5% CO2 incubator for continued culture, and observe the cytopathogenic effect (CPE) every day. Positive results are those with cytopathic effect, and negative results are those without cytopathic effect. The cytopathic effect refers to the direct result of influenza virus infecting host cells and proliferating in large numbers in the cells, which is mainly manifested as cell necrosis, rounding, fragmentation, and floating. Refer to the Reed-Muench method to calculate the virus half-cell infectious dose (TCID 50 ).

[0106] Distance ratio = (percentage of lesions above 50% - 50%) / (percentage of lesions above 50% - percentage of lesions below 50%)

[0107] lgTCID 50 = distance ratio × difference between dilution logarithms + dilution logarithms with a lesion rate higher than 50%

[0108] 1.3 EC of drug for viral suppression50 Value determination

[0109] The MDCK cells in good growth state were digested with trypsin, centrifuged, counted, and adjusted to an appropriate density (5000 cells / well), and then the cell suspension was evenly added to a 96-well plate, and then the cell culture plate was placed in a 37°C, 5% CO2 incubator overnight to form a cell monolayer. The chicken embryo allantoic fluid or cell supernatant containing the virus was diluted to 100 times TCID with serum-free medium. 50 , added to the test cells, and the virus-infected cells were placed in a 37°C, 5% CO2 incubator for incubation for 2 hours, and the virus-containing cell supernatant was discarded after 2 hours. The Chinese medicine composition of Example 1 with different concentration gradients (1000, 500, 250, 125, 62.5 and 31.25 μg / mL) was added to the cell culture wells, and a positive drug group, a blank control group and a virus model group were set up at the same time. After adding the corresponding drugs, the cells were placed in a 37°C, 5% CO2 incubator for 48 hours. The original culture solution was discarded, CCK8 solution was added, and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours, and the absorbance value was measured at a wavelength of 450nm on an enzyme marker.

[0110] 2 Experimental results

[0111] 2.1 Effects of drugs on CC of MDCK cells 50 value

[0112] The toxicity of the Chinese medicine composition of each embodiment and comparative example and the positive control drug oseltamivir acid to MDCK cells was determined by CCK8 method. The results are shown in Table 4.

[0113] CC 50 It refers to the drug concentration required to kill 50% of the host cells in an in vitro test. The higher the half-cell toxicity concentration of the drug, the lower the toxicity of the drug to the host cells. As shown in Table 4, the CC of oseltamivir acid and the Chinese medicine compositions of Comparative Examples 1 to 6 50 The values ​​are significantly lower than those in Examples 1 to 3, which indicates that the Chinese medicine composition of the present invention is more advantageous than oseltamivir acid and Comparative Examples 1 to 6 in reducing MDCK cell toxicity.

[0114] 2.2 TCID of the tested virus strains on cells 50

[0115] Determine the TCID of cells infected with the test virus 5o , that is, the virus half-cell infection dose, using 100 times the TCID 50The infection concentration of the virus was used for subsequent experiments. The infection toxicity of the virus was calculated with reference to the Reed-Müench formula. The results showed that the TCID 50 The values ​​are 10 -3.23 / 100μL, 10 -2.75 / 100μL and 10 -3.09 100μL. In the in vitro cytopathic inhibition assay, the test virus solution was diluted to 100TCID 50 As the infectious dose of the virus in the formal test.

[0116] 2.2.1 TCID of H1N1 strains on MDCK cells 50 (10 -3.23 )

[0117] Table 1 TCID of influenza A virus (H1N1) on MDCK cells 50

[0118]

[0119]

[0120] Distance ratio = 0.23; TCID 50 =10 -3.23 ; 100TCID 50 =10 -1.23

[0121] 2.2.2 TCID of H3N2 strains on MDCK cells 50 (10 -2.75 )

[0122] Table 2 TCID of influenza A virus (H3N2) on MDCK cells 50

[0123]

[0124] Distance ratio = 0.75; TCID 50 =10 -2.75 ; 100TCID 50 =10 -0.75

[0125] 2.2.3 TCID of H1N1-OC on MDCK cells 50 (10 -3.09 )

[0126] Table 3 TCID of oseltamivir-resistant influenza virus H1N1-OC on MDCK cells 50

[0127]

[0128] Distance ratio = 0.9; TCID 50 =10 -3.09 ; 100TCID 50 =10 -1.09 2.3 EC of drug inhibition on the tested strain 50 value

[0129] By observing the protective effect of the Chinese medicine composition of the present invention on the pathological effect of cells infected by different virus strains, the inhibitory effect of the drug on influenza virus in vitro was evaluated. The results showed that the Chinese medicine composition of the present invention showed a certain protective effect on the pathological effect of MDCK cells infected by the tested virus strains, indicating that it has a good in vitro antiviral effect.

[0130] 2.3.1 Protective effect of drugs on MDCK cells infected with H1N1 strain

[0131] In MDCK cells infected with H1N1 strain, the EC of the Chinese medicine composition of Example 1 50 =133.43 μg / mL, and the selectivity index (SI) for H1N1 is 20.31. It can be seen that the Chinese medicine composition of Example 1 exhibits a certain inhibitory effect on influenza virus strain H1N1.

[0132] In addition, the EC of the drug 50 The lower the value, the lower the concentration required for the drug to inhibit 50% of the virus in the in vitro test, which generally means that the drug is more effective. As shown in Table 4, the EC values ​​of the Chinese medicine compositions of Comparative Examples 1 to 6 are 50 The values ​​are significantly higher than those of Examples 1 to 3, which indicates that the Chinese medicine composition of the present invention has obvious advantages over Comparative Examples 1 to 6 in inhibiting influenza virus strain H1N1.

[0133] In addition, the higher the drug selectivity index (SI), the more effective the drug is in inhibiting the virus at a lower concentration (EC 50 low value), and less toxic to host cells (CC 50 Such drugs effectively inhibit viruses while causing less damage to normal cells, and thus have higher safety and effectiveness. As shown in Table 4, the SI of the Chinese medicine compositions of Examples 1 to 3 are significantly higher than those of Comparative Examples 1 to 6, indicating that the Chinese medicine compositions of the present invention have higher safety and effectiveness in inhibiting influenza virus strain H1N1 than Comparative Examples 1 to 6.

[0134] 2.3.2 Protective effect of drugs on MDCK cells infected with H1N1-OC strain

[0135] In MDCK cells infected with oseltamivir-resistant influenza virus strain H1N1-OC, the EC of the traditional Chinese medicine composition of Example 1 50 =137 μg / mL. Oseltamivir acid treatment had no significant protective effect on cells infected with the resistant influenza virus H1N1-OC strain. The selectivity index of the Chinese medicine composition of Example 1 for H1N1-OC was 19.78 (Table 4), indicating that the Chinese medicine composition of Example 1 had a certain in vitro antiviral activity against the oseltamivir-resistant virus strain H1N1-OC.

[0136] In addition, as shown in Table 4, the EC of the Chinese medicine compositions of Comparative Examples 1 to 6 50 The values ​​are significantly higher than those of Examples 1 to 3, which indicates that the Chinese medicine composition of the present invention has significant advantages over Comparative Examples 1 to 6 in inhibiting the virus strain H1N1-OC.

[0137] In addition, as shown in Table 4, the SI of the Chinese medicine composition of Examples 1 to 3 is significantly higher than that of Comparative Examples 1 to 6, indicating that the Chinese medicine composition of the present invention has higher safety and effectiveness in inhibiting the virus strain H1N1-OC than Comparative Examples 1 to 6.

[0138] 2.3.3 Protective effect of drugs on MDCK cells infected with H3N2 strain

[0139] In MDCK cells infected with H3N2 strain, the EC of the Chinese medicine composition of Example 1 50 =51.46 μg / mL, and the selectivity index for H3N2 is 52.66. It can be seen that the Chinese medicine composition of Example 1 exhibits a certain in vitro antiviral activity against H3N2 strains (Table 4).

[0140] In addition, as shown in Table 4, the EC of the Chinese medicine compositions of Comparative Examples 1 to 6 50 The values ​​are significantly higher than those of Examples 1 to 3, which indicates that the Chinese medicine composition of the present invention has significant advantages over Comparative Examples 1 to 6 in inhibiting the virus strain H3N2.

[0141] In addition, as shown in Table 4, the SI of the Chinese medicine compositions of Examples 1 to 3 are significantly higher than those of Comparative Examples 1 to 6, indicating that the Chinese medicine compositions of the present invention have higher safety and effectiveness in inhibiting the virus strain H3N2 than Comparative Examples 1 to 6.

[0142] Table 4 Selection index in MDCK cells infected with three tested virus strains

[0143]

[0144]

[0145] a The half-cytotoxic concentration (CC) of the Chinese medicine composition of Example 1 and oseltamivir acid 50 ); As an illustration, due to CC 50 is the half cytotoxic concentration of MDCK cells not infected by the tested virus strain, so CC corresponding to the examples and comparative examples in H1N1 / MDCK, H1N1-OC / MDCK and H3N2 / MDCK 50 The values ​​are the same;

[0146] b The 50% effective concentrations (EC) of the pharmaceutical composition of Example 1 and oseltamivir acid against the tested virus strains 50 );

[0147] c Selection Index (SI, CC 50 / EC 50 ).

[0148] Note: Compared with the Chinese medicine composition of Example 1, # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001; compared with the Chinese medicine composition of Example 2, *P<0.01, **P<0.01, ***P<0.001, ****P<0.0001; compared with the Chinese medicine composition of Example 3, ★ P<0.05, ★★ P<0.01, ★★★ P<0.001, ★★★★ P<0.0001.

[0149] Experiment 2 In vivo experiments

[0150] 1. Experimental Materials

[0151] 1 Drugs, reagents and animals

[0152] 1.1 Drugs and reagents

[0153] The Chinese medicine compositions prepared in the above embodiments and comparative examples.

[0154] Oseltamivir phosphate was purchased from Yichang East Sunshine Changjiang Pharmaceutical Co., Ltd., batch number: 6002211067.

[0155] IL-6, IL-1β and TNF-α ELISA detection kits were purchased from Shanghai Yuanju Biotechnology Center.

[0156] 1.2 Experimental animals and virus strains

[0157] SPF-grade ICR mice, weighing 11-14 g, half male and half female, a total of 100, were provided by the Center for Comparative Medicine of Yangzhou University, and the sales unit license number is: SCXK(Su)2022-0009.

[0158] The experimental animals were used by China Pharmaceutical University, and the license number is SYXK(Su)2018-0019.

[0159] SPF-grade experimental mouse pellet feed was provided by Qinglongshan Animal Breeding Farm in Jiangning District, Nanjing.

[0160] Mice were raised in the Experimental Animal Center of China Pharmaceutical University, with males and females in separate cages. After purchase, the animals were allowed free access to food and water and acclimatized normally for 3 days at room temperature of 23-26°C and humidity of 46-58%.

[0161] The influenza A virus strain A / FM / 1 / 47 (H1N1) was preserved in the Microbiology Laboratory of the School of Life Sciences and Technology, China Pharmaceutical University. The virus was amplified and cultured in nine-day-old SPF-grade chicken embryos and then packaged. The chicken embryo allantoic fluid was frozen in a -80℃ refrigerator in the laboratory.

[0162] 1.3 Drug preparation and animal grouping

[0163] Preparation of the Chinese medicine composition prepared in the above embodiments and comparative examples: Weigh 2.7g of the Chinese medicine composition and add it to 6mL of 0.5 wt% CMC-Na aqueous solution (dissolve 0.5g CMC-Na with physiological water and adjust the volume to 100ml), the final concentration is 0.9g / mL, and grind it into a uniform suspension using a mortar. Dilute it in equal proportions and prepare medium-dose (0.45g / mL) and low-dose (0.225g / mL) solutions for use. Give mice oral gavage treatment at a dosing volume of 100μL / 10g body weight, once / d, for 5 consecutive days.

[0164] Table 5 Grouping and dosing schedule of drug-treated animals

[0165]

[0166]

[0167] 2 Statistical methods

[0168] All data were analyzed using GraphPad Prism 6.0 statistical software package. One-way analysis of variance was used for comparison among multiple groups, and Student's t test was used for pairwise comparison among groups. p < 0.05 was considered statistically significant.

[0169] (II) Experimental methods and results

[0170] 1 Animal modeling and drug administration

[0171] SPF grade ICR mice weighing 11-14 g, half male and half female, were taken and anesthetized with ether. After induction of anesthesia, the mice were treated with 4×LD 50 The mice were infected by intranasal drops of titrated H1N1 virus dilution (80 μL / mouse). 2 hours after infection, mice were gavaged with the corresponding dose of drug suspension (dosing volume (V): 100 μL / 10g body weight) once a day for 5 consecutive days. The survival of some mice was observed every day, and the weight of the mice was weighed. The observation was continued for 14 days, and the weight change curve was drawn; on the third day of infection, blood was collected from the orbital venous plexus of other mice, and serum was collected by centrifugation. On the 7th day of infection, 6 mice were taken, blood was collected from the orbital venous plexus, centrifuged, and serum was collected. The whole lung tissue of the mice was dissected under sterile conditions, and the dry and wet weights were weighed. The inflammation of virus-infected mice and the degree of pathological damage of lung tissue were detected by ELISA / western blot / RT-qPCR and other methods, and the relief of the lesions of infected mice by the experimental drugs was evaluated.

[0172] 2. Detection indicators

[0173] (1) General indicators: Observe the fur, activity, diet and death of mice, and measure the weight of mice.

[0174] (2) Virus infection: Observe whether the mice lose weight, have reduced activity, have their hair erected, or curled up.

[0175] (3) Pathological changes in the lung tissue of infected mice, dry and wet weights of lung tissue, levels of serum inflammatory factors, and expression levels of inflammation-related proteins in lung tissue.

[0176] 3 Experimental results

[0177] 3.1 Treatment with the Chinese medicine composition of the present invention can improve the general condition of virus-infected mice

[0178] Before modeling, all mice were in good condition, with shiny fur and normal activities. After modeling, mice showed poor mental state, listlessness, and fatigue; their activities were significantly reduced; their food and water intake were reduced; their fur was messy and dull, and their weight was significantly reduced. In severe cases, mice showed symptoms such as slow movement, dragging, and death. Figure 1As shown, starting from the 8th day, the body weight of mice in the medium-dose group (4.5 g / kg) and the high-dose group (9.0 g / kg) of Example 1 gradually recovered, and by the 14th day, the body weight of mice basically recovered to a level close to that of the normal group. At the same time, drug treatment can alleviate the conditions of erect hair, slow activity and reduced food intake in mice. The results show that the Chinese medicine composition of Example 1 has a more obvious protective effect on the body weight of mice infected with influenza A virus A / FM / 1 / 47 (H1N1), and its effect at a high dose is comparable to that of oseltamivir phosphate.

[0179] 3.2 Treatment with the Chinese medicine composition of the present invention can improve the survival rate of virus-infected mice

[0180] like Figure 2 As shown, the results show that the low, medium and high dose groups of Example 1 have a certain protective effect on the death of mice infected with influenza A virus (H1N1), and can prolong the survival time of mice infected with influenza virus. In the low-dose (2.25g / kg) treatment group, the survival rate of mice increased by 40% compared with the model group, the survival rate of virus-infected mice in the medium-dose treatment group increased by 50%, and the survival rate of virus-infected mice in the high-dose (9.0 g / kg) treatment group increased by 70%. In addition, the Chinese medicine composition of the present invention can prolong the survival time of virus-infected mice in a dose-dependent manner, showing a good in vivo antiviral effect.

[0181] 3.3 Treatment with the Chinese medicine composition of the present invention reduces the lung index of mice

[0182] like Figure 3 As shown, different doses of the Chinese medicine composition of the present invention can reduce the ratio of lung wet weight to body weight and the ratio of lung dry weight to wet weight of mice to varying degrees. The above results show that the Chinese medicine composition of the present invention can reduce pulmonary edema and congestion in mice infected with influenza virus, and its effect at high doses is equivalent to or better than that of oseltamivir phosphate.

[0183] 3.4 Effect of the Chinese medicine composition of the present invention on the virus titer in the lung tissue of mice infected with H1N1

[0184] On the 7th day after administration, the mice were killed to separate the whole lungs, and the homogenate was prepared with a high-speed homogenizer on an ice bath. The homogenate was then centrifuged at low temperature and high speed, and the supernatant was taken and sterilized by filtration with a 0.22 μm sterile filter for standby use. The lung tissue homogenate prepared in each group was diluted 10 times by gradient and added to MDCK cells grown to a monolayer to determine the ability of the virus in the lung tissue homogenate to infect MDCK cells. The results showed that compared with the mouse lung tissue homogenate from the model group, the Chinese medicine composition treatment of the present invention can reduce the virus titer of the mouse lung tissue. In the high-dose drug treatment group, the reduction level of the virus titer of the mouse lung tissue is the most obvious, and the drug can also significantly reduce the virus titer of the H1N1 strain infected mouse lung tissue under low and medium dose administration conditions, proving that the Chinese medicine composition of the present invention can effectively inhibit the replication and proliferation of influenza A virus (H1N1) in vivo.

[0185] (1) The lung tissue homogenate was diluted in a gradient manner and infected with MDCK cells respectively. The pathological effects of the virus contained in the lung tissue homogenate at different dilutions on MDCK cells were observed, and the virus titer in the lung tissue of mice in different treatment groups was measured. Figure 4 As shown, the results show that the Chinese medicine composition of the present invention can significantly reduce the virus titer in the lung tissue of infected mice, and the high-dose treatment group has the best inhibitory effect on the virus titer, which is equivalent to oseltamivir phosphate.

[0186] (2) The M gene of the virus was detected by PCR, and the virus titer of the lung tissue of H1N1 infected mice was evaluated by the relative expression of the M gene. The results are shown in the following table.

[0187] Table 6 Effect of drug treatment on virus titer in lung tissue of H1N1 infected mice (x±s, n=6)

[0188]

[0189] Note: Compared with the model group, ◆ P<0.05, ◆◆ P<0.01, ◆◆◆ P<0.001; each experimental group was compared with the medium dose group of Example 1, # P<0.05, ## P<0.01, ### P<0.001.

[0190] The data in the table show that in terms of reducing the virus titer in the lung tissue of mice infected with the H1N1 virus, the oseltamivir phosphate group and each example and comparative example group all showed significant inhibitory effects compared with the model group. It is worth noting that the high-dose group of Example 1 was comparable to the oseltamivir phosphate group in terms of inhibiting the virus titer.

[0191] Further analysis revealed that the inhibitory effects of comparative examples 1 to 6 were significantly lower than those of the medium-dose group of Example 1 at the same dosage, which further confirmed that continuous oral gavage therapy with the Chinese medicine composition of the present invention had a more significant inhibitory effect on the viral titer in the lung tissue of mice infected with the H1N1 virus.

[0192] In summary, the Chinese medicine composition of the present invention exhibited an antiviral effect comparable to that of oseltamivir phosphate at a high dose, and also showed an inhibitory effect significantly superior to that of the control group at a medium dose, indicating that it has potential clinical application value in the treatment of H1N1 virus infection.

[0193] 3.5 Effect of the Chinese medicine composition of the present invention on the alleviation of lung tissue lesions in mice infected with H1N1

[0194] The Chinese medicine composition of the present invention was used for continuous treatment for 5 days. On the 7th day, the lung tissue of the mice was taken, fixed with 4% paraformaldehyde, embedded in paraffin, and stained with conventional H&E. The lung tissue damage of the mice in different treatment groups was pathologically scored according to the scoring criteria in the following table to determine whether the Chinese medicine composition of the present invention has an alleviating effect on the lung damage caused by H1N1 strain infection. The scoring criteria are shown in the following table.

[0195] Table 7 Underwood's histopathological scoring system for pulmonary inflammatory changes

[0196]

[0197] The results show that: Figure 5 As shown in the figure, intranasal infection with the H1N1 strain can cause lung tissue damage in mice, which is manifested as: a large number of eosinophils and lymphocytes infiltrating around the medium-sized blood vessels and bronchi of the lung tissue (blue arrows), a large number of bronchial mucosal epithelial cells necrotic and sloughed, and some structures disappeared, leaving only the tube wall (orange arrows), a large number of eosinophils and macrophages infiltrating in the alveolar cavity (yellow arrows), rare alveolar wall capillary congestion (black arrows), and occasionally eosinophilic flocs attached to the alveoli to form a transparent membrane (brown arrows).

[0198] Treatment with the Chinese medicine composition of the present invention can alleviate the lung tissue damage of mice caused by infection to varying degrees. After drug treatment, it can be seen that there is no obvious abnormality in the bronchi of the lung tissue, a small amount of free macrophage infiltration (blue arrow) can be seen in the alveolar cavity, occasional flocs in the alveolar cavity (purple arrow), occasional perivascular lymphocyte infiltration (yellow arrow), and a small amount of vascular congestion (green arrow). Among them, the degree of relief of lung tissue damage in mice in the medium and high dose treatment groups is more obvious.

[0199] The specific scores of mouse lung tissue edema and inflammatory cell infiltration are shown in the table below.

[0200] Table 8 Effects of drug treatment on lung tissue injury scores of H1N1 infected mice (x±s, n=6)

[0201]

[0202] Note: Compared with the dosage group in Example 1, # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001; compared with the Chinese medicine composition of Example 2, *P<0.01, **P<0.01, ***P<0.001, ****P<0.0001; compared with the Chinese medicine composition of Example 3, ★ P<0.05, ★★ P<0.01, ★★★ P<0.001, ★★★★ P<0.0001; compared with the model group, ◆ P<0.05, ◆◆ P<0.01, ◆◆◆ P < 0.001, ◆◆◆◆ P<0.0001.

[0203] The data in the table show that in terms of relieving lung tissue damage in mice, the oseltamivir phosphate group and each embodiment and comparative example group all showed significant relieving effects compared with the model group. Further analysis found that the medium and high dose groups of Example 1 and the oseltamivir phosphate group had comparable effects in relieving lung tissue damage in mice; the relieving effects of comparative examples 1 to 6 were lower than those of the embodiments, which confirmed that the use of the Chinese medicine composition of the present invention can better alleviate lung tissue damage in mice.

[0204] In summary, the Chinese medicine composition of the present invention can significantly alleviate the lung tissue damage of mice at different doses, especially the medium dose group and high dose group of Example 1 showed better relief effect. These results not only confirm the effectiveness of the Chinese medicine composition of the present invention, but also provide strong data support for future clinical applications.

[0205] 3.6 Mechanism of the protective effect of the Chinese medicine composition of the present invention on H1N1 infected mice

[0206] 3.6.1 The Chinese medicine composition of the present invention inhibits the production of pro-inflammatory factors in H1N1 infected mice

[0207] Blood was collected from the orbital venous plexus on the 3rd and 7th days after infection with the H1N1 strain, and serum was separated to measure the production of inflammatory factors in virus-infected mice under drug treatment. Figure 6As shown, the results show that: H1N1 infection can significantly increase the levels of IL-1β, TNF-α and IL-6 in mouse serum, and the treatment with the Chinese medicine composition of the present invention can reduce the excessive release of inflammatory factors induced by viral infection to a certain extent. On the 7th day, the effect of the Chinese medicine composition of the present invention at a high dose is equivalent to that of oseltamivir phosphate.

[0208] 3.6.2 The Chinese medicine composition of the present invention inhibits the activation of TLR-7-MyD88-NF-KB pathway in lung tissue of H1N1 infected mice

[0209] According to literature reports, influenza virus H1N1 infection can induce activation of the TLR-7-MyD88-NF-KB pathway in mice. Figure 7 As shown, the expression levels of MyD88, TLR7 and p-NF-KB-p65 in mouse lung tissue increased significantly after virus infection, indicating that the TLR-7-MyD88-NF-KB inflammatory pathway was activated in the lung tissue of mice infected with influenza A virus A / FM / 1 / 47 (H1N1). After 5 days of continuous treatment with the Chinese medicine composition of the present invention, the expression levels of MyD88, TLR7 and p-NF-KB-p65 proteins in the lung tissue of mice showed a downward trend, among which the medium and high doses of the Chinese medicine composition of the present invention significantly inhibited the expression level of p-NF-κB-p65, and were better than oseltamivir phosphate, indicating that the Chinese medicine composition treatment of the present invention can inhibit the activation of the TLR-7-MyD88-NF-κB pathway in the lung tissue of mice by virus infection, thereby reducing the damage to the lung tissue caused by the virus-induced inflammatory response.

[0210] 3.6.3 The Chinese medicine composition of the present invention inhibits the expression level of TLR-7 and MyD88 mRNA in the lung tissue of virus-infected mice

[0211] In addition to detecting the expression levels of inflammation-related proteins in the lung tissue of infected mice, the present invention also detected the expression levels of MyD88 and TLR-7 mRNA. Figure 8 As shown, it was also found that the Chinese medicine composition of the present invention can significantly inhibit the expression of MyD88 and TLR-7 mRNA, especially in the high-dose treatment group, the corresponding mRNA expression level decreased by about 4 times, and its inhibitory effect was equivalent to that of the positive control drug oseltamivir phosphate.

[0212] In summary, the present invention evaluates the inhibitory effect of the Chinese medicine composition of the present invention on influenza A virus strains A / FM / 1 / 47 (H1N1), A / Beijing / 32 / 92 (H3N2) and oseltamivir-resistant influenza A virus strains (H1N1-OC) on MDCK cells. The results show that the Chinese medicine composition of the present invention has a certain inhibitory effect on the above-mentioned tested virus strains, especially showing a good inhibitory effect on oseltamivir-resistant influenza virus H1N1. At the same time, the present invention determines the toxicity of the Chinese medicine composition of the present invention on MDCK cell lines, and the results show that the Chinese medicine composition of the present invention has low cytotoxicity, high safety, and has a high selection index for H3N2 and oseltamivir-resistant H1N1-OC.

[0213] In addition, in the influenza A virus A / FM / 1 / 47 (H1N1) infected mouse model, the weight, survival days and survival rate of the mice were observed, and it was found that the Chinese medicine composition of the present invention can improve the survival rate of virus-infected mice, prolong the survival time of animals, and reverse the weight loss of mice caused by virus infection. Further analysis shows that the Chinese medicine composition of the present invention can significantly reduce the virus titer in the lung tissue of infected mice, inhibit the activation of the TLR-7-My88-NF-KB signaling pathway in the lung tissue, and reduce the production of pro-inflammatory factors IL-6, IL-1β and TNF-a, thereby alleviating the symptoms of infectious pneumonia such as pulmonary edema, congestion and tissue damage in mice induced by virus infection.

[0214] In summary, the Chinese medicine composition of the present invention has a certain inhibitory effect on influenza A virus in vitro, and also shows a good anti-influenza A virus infection effect in virus-infected mice. The present invention can provide a basis for the clinical treatment of lung infection caused by influenza A virus with the Chinese medicine composition of the present invention.

[0215] The above are only several exemplary embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention is disclosed as above with preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to obtain equivalent or equivalent embodiments using the above disclosed technical content, which fall within the scope of the present invention.

Claims

1. A use of a Chinese medicine composition in preparing a medicament for preventing and / or treating influenza A and related diseases, wherein the Chinese medicine composition comprises the following components or is prepared from the following raw materials, by weight: 20-40 parts of gypsum, 10-30 parts of golden buckwheat, 10-30 parts of houttuynia cordata, 3-15 parts of roasted ephedra, 3-15 parts of stir-fried bitter almonds, 3-15 parts of Fritillaria cirrhosa, 5-25 parts of dwarf tea, 3-15 parts of loquat leaves, 3-15 parts of stir-fried perilla seeds and 3-15 parts of roasted liquorice.

2. The use according to claim 1, wherein The Chinese medicine composition is the only active ingredient in the medicine.

3. The use according to claim 1 or 2, wherein The traditional Chinese medicine composition comprises the following components or is prepared from the raw materials comprising the following components in parts by weight: 25-35 parts of gypsum, 15-25 parts of golden buckwheat, 15-25 parts of houttuynia cordata, 8-12 parts of roasted ephedra, 8-12 parts of stir-fried bitter almonds, 8-12 parts of Fritillaria cirrhosa, 10-20 parts of dwarf camellia, 8-12 parts of loquat leaves, 8-12 parts of stir-fried perilla seeds and 8-12 parts of roasted licorice.

4. The use according to any one of claims 1 to 3, wherein In parts by weight, the traditional Chinese medicine composition comprises the following components or is prepared from the raw materials comprising the following components: 30 parts of gypsum, 20 parts of golden buckwheat, 20 parts of houttuynia cordata, 9 parts of roasted ephedra, 9 parts of stir-fried bitter almonds, 9 parts of Fritillaria cirrhosa, 15 parts of dwarf camellia, 9 parts of loquat leaves, 9 parts of stir-fried perilla seeds and 9 parts of roasted licorice.

5. The use according to any one of claims 1 to 4, wherein The influenza A is caused by one or more influenza A virus strains selected from the group consisting of H1N1 subtype, H3N2 subtype and drug-resistant influenza A virus strains.

6. The use according to any one of claims 1 to 5, wherein The influenza A is caused by an H1N1 subtype influenza A virus strain and / or an oseltamivir-resistant influenza A virus strain.

7. The use according to any one of claims 1 to 6, wherein The H1N1 subtype influenza A virus strain is A / FM / 1 / 47; Preferably, the H3N2 subtype influenza A virus strain is A / Beijing / 32 / 92; Preferably, the oseltamivir-resistant influenza A virus strain is H1N1-OC.

8. The use according to any one of claims 1 to 7, wherein The influenza A-related disease is pneumonia.

9. The use according to any one of claims 1 to 8, wherein The Chinese medicine composition or the raw material drug further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutically acceptable excipient is selected from one or more of a diluent, an excipient, a binder, a preservative and a flavoring agent.

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