Use of a traditional Chinese medicine composition in the preparation of a medicament for treating respiratory viral infection

Abstract

The application relates to an application of a traditional Chinese medicine composition in preparation of a medicine for treating respiratory virus infection, and belongs to the technical field of medicines. The technical problems to be solved are: to solve the problems that existing anti-virus medicines are prone to drug resistance, lack of broad-spectrum inhibiting effect on respiratory viruses or poor selectivity, and only relieve symptoms such as fever, headache, sore throat, cough and the like caused by cold. The technical solution points are: the application provides a new use of a traditional Chinese medicine composition (made of the following raw materials: dog liverwort, mountain sesame, indigo leaf, kudzu root, east wind orange, white and green, artemisia and windproof) in treating respiratory virus infection; the respiratory virus is one or more of the following: influenza A virus, influenza B virus, human rhinovirus, parainfluenza virus, respiratory syncytial virus and adenovirus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and provides an application of a traditional Chinese medicine composition in the preparation of a drug for treating respiratory viral infections. Background Technology

[0002] Respiratory viral infections are common human diseases, mostly caused by viruses, bacteria, and mycoplasma. Common pathogens causing respiratory infections include influenza virus, Mycoplasma pneumoniae, coronavirus, respiratory syncytial virus, human rhinovirus, adenovirus, human metapneumovirus, parainfluenza virus, hemolytic streptococci, Haemophilus influenzae, and Streptococcus pneumoniae. During peak seasons for respiratory infections, multiple pathogens can co-infect or alternate in epidemics. Among them, influenza virus is highly contagious and mutates rapidly, and can periodically cause global pandemics, posing a serious threat to public health.

[0003] Currently, the main drugs used clinically to treat respiratory viral infections include neuraminidase inhibitors (such as oseltamivir), M2 ion channel blockers (such as amantadine), and broad-spectrum antiviral drugs (such as ribavirin). However, existing antiviral drugs have the following limitations: (1) they have a single target and are prone to drug resistance; (2) they have a narrow therapeutic window and many adverse reactions; (3) they lack broad-spectrum inhibitory effects against various respiratory viruses; and (4) some drugs can only relieve symptoms and cannot directly eliminate the virus. Therefore, developing safe, effective drugs with unique mechanisms of action and broad-spectrum antiviral activity still has important clinical value.

[0004] Traditional Chinese medicine has a long history and unique advantages in the prevention and treatment of respiratory diseases. The Chinese Pharmacopoeia and the drug standards of the National Health and Family Planning Commission include a variety of traditional Chinese medicine preparations for treating colds. These medicines are usually guided by traditional Chinese medicine theories such as "dispelling wind and clearing heat", "relieving exterior syndromes and dispersing cold", and "stopping cough and resolving phlegm", and are used to relieve symptoms such as fever, headache, sore throat and cough caused by colds.

[0005] Existing traditional Chinese medicines for treating influenza (such as Lianhua Qingwen Capsules, Shufeng Jiedu Capsules, and Jinhua Qinggan Granules) are mostly composed of heat-clearing and detoxifying herbs, with "heat-clearing and detoxifying" as their main function. They rarely use astringent and tissue-regenerating herbs or herbs that regulate qi and resolve phlegm. They are mostly used for respiratory infection symptoms such as fever, headache, sore throat, cough, and excessive phlegm caused by influenza. It has not been disclosed whether they have a direct inhibitory effect on respiratory viruses, let alone revealed their antiviral activity against specific respiratory viruses such as influenza A virus, influenza B virus, human rhinovirus, parainfluenza virus, respiratory syncytial virus, and adenovirus, as well as their efficacy in reducing viral load in the lungs, improving lung inflammation, and increasing survival rate after viral infection.

[0006] Therefore, there is an urgent need in this field to develop new medicinal uses for existing antiviral traditional Chinese medicines for influenza, clarify their antiviral effects and efficacy, and provide a scientific basis for expanding their clinical applications. Summary of the Invention

[0007] The purpose of this invention is to provide: The application of a traditional Chinese medicine composition in the preparation of drugs for treating respiratory viral infections addresses the problems of existing antiviral drugs, such as easy development of drug resistance, lack of broad-spectrum inhibitory effect on respiratory viruses or poor selectivity, and only relieving symptoms such as fever, headache, sore throat and cough caused by colds. It expands the types of antiviral drugs for respiratory viruses and provides new treatment ideas for respiratory viral infections.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definition of the standard chemical term can be found in the reference "Pharmacopoeia of the People's Republic of China, 2025 Edition, China Medical Publishing House, October 2025".

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as filtration, concentration, and drying, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0014] The term "IC" 50 "Half-Maximal Inhibitory Concentration" refers to the concentration of a drug or inhibitor required to inhibit a specified biological process (such as enzyme activity, receptor binding, cell proliferation, viral replication, etc.) to a 50% level. It is a core quantitative indicator in pharmacology and biochemistry for characterizing the antagonistic ability of inhibitors.

[0015] The term "EC" 50 "Half Maximal Effective Concentration" refers to the concentration of a drug, antibody, or toxin that, after a specific period of exposure, achieves 50% of the maximum biological effect, reflecting the drug's ability to produce an effect.

[0016] The term "100 TCID" 50 "(50% Tissue Culture Infective Dose)" refers to the half-maximal infectious dose of a tissue culture, which is the viral dilution that can cause 50% of cultured cells to become infected / pathogenic. It is used to indicate the infectivity / virulence of a virus.

[0017] The term "LD" 50 "Median Lethal Dose" (LD50) is a standard indicator in toxicology used to describe the toxicity of a toxin / virus. It refers to the dose of virus / toxin required to kill half of the test animals under specified experimental conditions. The unit is usually adjusted according to the experimental scenario, and in virus experiments, it generally refers to the amount of virus used. 50 The smaller the value, the more virulent the virus. "2LD" 50 That's twice the median lethal dose.

[0018] This invention is achieved through the following technical solution: The application of a traditional Chinese medicine composition in the preparation of a drug for treating respiratory viral infections, said composition being made from the following raw materials: *Gnaphalium affine*, *Sesame aralia*, *Isatis tinctoria* leaves, *Pueraria lobata* root, *Citrus aurantium*, *Bletilla striata*, *Artemisia annua*, and *Saposhnikovia divaricata*. The respiratory viruses mentioned are one or more of the following: influenza A virus, influenza B virus, human rhinovirus, parainfluenza virus, respiratory syncytial virus, and adenovirus.

[0019] In the herbal composition of this invention, *Gnaphalium affine*, *Sesame arvense*, and *Isatis indigotica* are all heat-clearing and detoxifying herbs, serving as the principal herbs to work together to clear heat and detoxify, targeting the heat-toxin pathogenesis of respiratory viral infections. *Pueraria lobata* is the assistant herb, relieving muscle tension and reducing fever, assisting the principal herb in clearing heat and relieving exterior symptoms. *Citrus aurantium* regulates qi and resolves phlegm, *Bletilla striata* astringes and promotes tissue regeneration, and *Artemisia annua* clears heat and relieves summer heat; these three herbs assist the principal and assistant herbs to enhance efficacy. *Saposhnikovia divaricata* dispels wind and releases the exterior, guiding the medicine upwards. The entire formula embodies the characteristics of "using heat-clearing and detoxifying with dispelling wind and releasing the exterior, and addressing phlegm and cough with astringing and promoting tissue regeneration," directly inhibiting viral replication, regulating the body's immune response, reducing inflammatory damage, and protecting and repairing the respiratory mucosa.

[0020] Preferably, the respiratory virus is one or more of influenza A virus, influenza B virus, human rhinovirus, and adenovirus; the traditional Chinese medicine composition of the present invention has better therapeutic effects on the aforementioned four viral infections.

[0021] More preferably, the respiratory virus is influenza A virus and / or influenza B virus.

[0022] In this invention, the traditional Chinese medicine composition treats respiratory viral infections by reducing the lung index, reducing viral load in the lungs, improving lung inflammation, and increasing survival rate after viral infection.

[0023] Preferably, the composition is made from the following raw materials in parts by weight: 45-55 parts of dog liver vegetable, 26-30 parts of mountain sesame, 45-55 parts of big green leaf, 45-55 parts of kudzu root, 26-30 parts of Dongfeng orange, 13-15 parts of Bletilla striata, 8-12 parts of Artemisia annua and 4-6 parts of Saposhnikovia divaricata.

[0024] More preferably, the composition is made from the following raw materials in parts by weight: 50 parts of dog liver vegetable, 28 parts of mountain sesame, 50 parts of indigo leaf, 50 parts of kudzu root, 28 parts of Dongfeng orange, 14 parts of Bletilla striata, 10 parts of Artemisia annua and 5 parts of Saposhnikovia divaricata.

[0025] More preferably, the preparation method of the traditional Chinese medicine composition is as follows: (1) Take dog liver vegetable, mountain sesame, big indigo leaf, Dongfeng orange and kudzu root, add water and decoct, filter and concentrate to obtain thick paste; (2) Take Bletilla striata, Artemisia annua and Saposhnikovia divaricata, crush and sieve them to obtain medicinal powder; (3) Mix the medicinal powder with the thick paste, dry and pulverize to obtain the mixed medicinal powder; (4) The mixed powder is made into pills and coated to obtain the final product.

[0026] In step (1), the decoction is performed 1-2 times, with 8-12 times the amount of water added each time and decocted for 1-2 hours.

[0027] The present invention also provides the application of a traditional Chinese medicine composition in the preparation of a drug for treating pneumonia caused by respiratory viral infection, the composition being made from the following raw materials: dog liver vegetable, mountain sesame, indigo leaf, kudzu root, tangerine peel, bletilla rhizome, artemisia annua, and saposhnikovia root.

[0028] Furthermore, the pneumonia caused by the respiratory virus infection is pneumonia caused by influenza A virus and / or pneumonia caused by influenza B virus.

[0029] Compared with the prior art, the present invention has the following beneficial effects: The existing drug Qinggan Wan (a traditional Chinese medicine composition prepared from *Gnaphalium affine*, *Sesame arvense*, *Isatis tinctoria*, *Pueraria lobata*, *Citrus aurantium*, *Bletilla striata*, *Artemisia annua*, and *Saposhnikovia divaricata*) is only used for "respiratory infections caused by colds, such as fever, headache, sore throat, cough, and excessive phlegm," which is a traditional use for relieving cold symptoms. This invention reveals for the first time that it has a direct inhibitory effect on respiratory viruses. It has been found that it can effectively reduce the viral load in the lungs of rats infected with one or more of the following respiratory viruses: influenza A virus, influenza B virus, human rhinovirus, parainfluenza virus, respiratory syncytial virus, and adenovirus; improve lung inflammation; and increase survival rate after viral infection. This provides a scientific basis for its use in treating influenza, human rhinovirus infection, and respiratory syncytial virus infection, breaking through the traditional cognitive limitation of "for relieving cold symptoms" and realizing a completely new application of this known traditional Chinese medicine composition in the field of antiviral therapy.

[0030] Furthermore, the pharmaceutical composition of this invention differs from the "broad-spectrum antiviral" properties claimed by existing antiviral traditional Chinese medicines. Instead, it exhibits strong selectivity against respiratory viruses, particularly showing significant inhibitory effects against six respiratory viruses: influenza A virus, influenza B virus, human rhinovirus, parainfluenza virus, respiratory syncytial virus, and adenovirus (ECV). 50 The effective concentration range was 52.41-94.97 μg / mL, but it showed no significant inhibitory effect on human coronavirus 229E and enterovirus 71 within the same concentration range (EC). 50 (Value >200 μg / mL). This selective antiviral result suggests that the antiviral effect of the traditional Chinese medicine composition of the present invention is virus-specific, rather than a broad-spectrum cytotoxic effect, nor a simple superposition of the antiviral effects of single herbs, but rather a synergistic effect produced by specific compatibility. Attached Figure Description

[0031] Figure 1 The results of the effects of the traditional Chinese medicine composition of the present invention on the pathological effects of mouse lung tissue of influenza A virus (HE staining, 100×). Figure 2 The results show the effect of the traditional Chinese medicine composition of the present invention on the survival curve of a mouse model of influenza A virus; wherein, each group is compared with the model control group. * expressp ≤0.05, ** express p ≤0.01; Figure 3 The results show the effects of the traditional Chinese medicine composition of the present invention on the pathological effects of influenza B virus on the lung tissue of mice (HE staining, 100×). Detailed Implementation

[0032] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0033] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0034] Example 1 I. Preparation of Traditional Chinese Medicine Compositions: The prescription for the Chinese herbal combination is as follows: 1250g of dog liver vegetable, 700g of mountain sesame, 1250g of Daqingye (Isatis tinctoria leaf), 1250g of kudzu root, 700g of Dongfengju (Citrus aurantium), 350g of Bletilla striata, 250g of Artemisia annua, and 125g of Saposhnikovia divaricata.

[0035] The preparation method is as follows: (1) Take the prescribed amount of dog liver vegetable, mountain sesame, big indigo leaf, Dongfeng orange and kudzu root, add water and decoct twice, each time add 10 times the amount of water, decoct for 1 hour, filter, combine the filtrates, and concentrate under reduced pressure to a thick paste with a relative density of 1.20 to 1.30 (measured at 60℃); (2) Take the prescribed amount of Bletilla striata, Artemisia annua and Saposhnikovia divaricata, grind them separately, pass them through a 60-mesh sieve, mix them evenly, and obtain fine powder; (3) Mix the fine powder obtained in step (2) with the thick paste obtained in step (1) evenly, dry under vacuum at 75°C, pulverize, and pass through a 100-mesh sieve to obtain the powder of the traditional Chinese medicine composition.

[0036] Appearance: This product is a brownish-brown powder; it has a slightly bitter taste.

[0037] II. Preparation of the Formulation 1. Pills: Take the powder of the above-mentioned Chinese herbal medicine composition, and for every 100g of powder, use 45g of refined honey and an appropriate amount of water to form pills. Coat the pills with activated carbon, dry them, and polish them to obtain the pills.

[0038] 2. Granules Take 100g of the above-mentioned Chinese herbal medicine powder, add 80g of sucrose powder and 50g of dextrin, mix evenly, granulate with 85% ethanol, dry at 60℃, and granulate to obtain granules.

[0039] 3. Tablets Take 100g of the above-mentioned Chinese herbal medicine powder, add 50g of starch and 30g of microcrystalline cellulose, mix evenly, granulate with 85% ethanol, dry at 60℃, granulate, add 1g of magnesium stearate, mix evenly, compress into tablets to obtain tablets.

[0040] 4. Capsules Take 100g of the above-mentioned Chinese herbal medicine powder, add 30g of starch, mix well, sieve, and fill into capsules to obtain capsules.

[0041] Experimental Example 1: In vitro antiviral activity experiment 1. Experimental Materials 1.1 Test Drug Test drug: The powder of the traditional Chinese medicine composition prepared in Example 1 was prepared into drug solutions with different concentration gradients using DMEM culture medium, and then filtered through a 0.22 μm filter membrane for sterilization and set aside.

[0042] Positive control drug: Oseltamivir acid, used as a positive control for influenza A and influenza B viruses. Batch number: 341605, expiry date: October 15, 2024, purchased from MCE.

[0043] Ribavirin is used as a positive control for human rhinovirus, respiratory syncytial virus, parainfluenza virus, enterovirus, coronavirus, and adenovirus. Batch number: C01024, specification: 1g / vial, expiration date: July 8, 2026, manufacturer: Shanghai Yuanye Biotechnology Co., Ltd.

[0044] 1.2 Virus strains (see Table 1 below) Table 1

[0045] 1.3 Virus culture conditions and corresponding hosts (see Table 2 below) Table 2

[0046] 1.4 Main Reagents DMEM medium (Gibco), fetal bovine serum (Gibco), 0.25% trypsin-EDTA trypsin (Gibco), CCK8 (Gibco), PBS (Gibco), virus maintenance solution (DMEM medium with 2% FBS, influenza virus requires additional addition of 2μg / mL TPCK Treated Trypsin, mix well, store at 2-8℃ for later use).

[0047] 2. Experimental Methods 2.1 Cytotoxicity test of traditional Chinese medicine composition (1) Experimental groups: Blank control group: only serum-free DMEM medium; Cell control group: only cells and serum-free DMEM medium; Traditional Chinese medicine combination group and positive control drug group: the test drug and positive control drug were serially diluted to 6 concentrations in serum-free DMEM medium; 3 replicates were set for each concentration in each group. The specific concentrations are shown in Table 3 below.

[0048] Table 3 Concentration settings for cytotoxicity assays

[0049] (2) Cell plating: Cells are prepared into plates with a density of 5×10⁻⁶. 4 Single-cell suspensions of 100 μL / well were seeded into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0050] (3) Drug addition detection: Discard the culture medium and wash the cells twice with 1×PBS. Add 100 μL of culture medium containing different concentrations of drugs to each well for each experimental group. Incubate at 37℃ and 5% CO2 for 48 h. Add 10 μL of CCK-8 to each well and incubate for 1-4 h. Measure the OD 450nm value on a microplate reader and calculate the cell inhibition rate and the half-maximal inhibitory concentration (IC50) of the test drug on the cells. 50 The results are shown in Table 5.

[0051] Cell inhibition rate (%) = [(OD450nm value of cell control group - OD450nm value of blank control group) - (OD450nm value of drug group - background OD450nm value of drug group - OD450nm value of blank control group)] / (OD450nm value of cell control group - OD450nm value of blank control group) × 100%.

[0052] 2.2 In vitro antiviral test of the test subject (1) Experimental grouping: The concentration settings were determined based on the results of the cytotoxicity test and the literature reports on the antiviral effects of the positive control drug, as shown in Table 4 below. The virus + drug group consisted of the test substance group and the positive control drug group (diluted to 5 concentrations using virus maintenance solution), the virus control group (with virus but no drug), and the cell control group (with only virus maintenance solution). Each group had 4 replicates for each concentration.

[0053] Table 4. In vitro antiviral drug concentration settings

[0054] (2) Cell plating: Cells are prepared into plates with a density of 5×10⁻⁶. 4 Single-cell suspensions of 100 μL / well were seeded into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0055] (3) Observation and record of drug addition: Discard the culture medium, wash twice with 1×PBS, and add 100 TCID. 50 50 µL of virus solution was added to each well, and the mixture was allowed to adsorb for 2 h. The virus solution was then discarded, and the wells were washed twice with 1×PBS. Then, 100 μL of culture medium containing different concentrations of the drug was added to each well according to the above group settings, and the wells were cultured for another period of time.

[0056] Cytopathic effect (CPE) was observed, and culture was terminated when the viral control group showed approximately 75% CPE. The degree of CPE was expressed as the proportion of cells exhibiting CPE, judged according to the following 5-level criteria: (-) Normal cell growth, no lesions; (+) Less than 25% of the entire cell monolayer showed lesions; (++) 25%–50% of cells showed lesions; (+++) 50%–75% of cells showed lesions; (++++) 75%–100% of cells showed lesions. The inhibition rate was calculated using the Reed-Muench method based on the degree of CPE to determine the half-maximal effective concentration (EC50) of the drug. 50 The results are shown in Table 6.

[0057] 2.3 Calculation of Treatment Index Therapeutic Index (TI) = IC 50 / EC 50 When the therapeutic index (TI) is greater than 2, it indicates that the drug has low toxicity and high activity; when TI is less than 1, it indicates ineffectiveness. The results are shown in Table 6.

[0058] 3. Experimental Results 3.1 Cytotoxicity Results Table 5. Cytotoxicity IC50 of test drug / positive control drug 50 Summary of results (n=3)

[0059] The table above shows that the IC50 of the traditional Chinese medicine composition on the cytotoxicity of MDCK, Hep-2, LLC-MK2, and HeLa cells is [not specified]. 50 The values ​​were 222.78, 189.10, 167.60, and 321.60 μg / mL, respectively. Oseltamivir's IC50 for MDCK cytotoxicity was... 50 Value >100 μg / mL. The cytotoxic IC50 of ribavirin against MDCK, Hep-2, LLC-MK2, and HeLa cells. 50 The values ​​were 3365, 1410, 5975, and 222.30 μg / mL, respectively.

[0060] 3.2 In vitro antiviral results Table 6. In vitro antiviral EC of traditional Chinese medicine composition / positive control drug 50 Summary of results (n=4)

[0061] As can be seen from the table above, the traditional Chinese medicine composition of this invention has a certain inhibitory effect on virus-induced cytopathic effects. The traditional Chinese medicine composition also exhibits inhibitory effects on the EC50 of IAV, IBV, RhV, PIV, RSV, and ADV viruses. 50 The values ​​were 73.71, 76.91, 59.96, 52.41, 94.97, and 93.57 μg / mL, respectively. The therapeutic indices (TIs) of the traditional Chinese medicine composition against IAV, IBV, RhV, PIV, RSV, and ADV viruses were 3.02, 2.90, 5.68, 3.20, 1.99, and 3.44, respectively. The tested traditional Chinese medicine composition did not show significant inhibitory effects against HCoV-229E and EV71 viruses.

[0062] In summary, it can be determined that the traditional Chinese medicine composition powder of the present invention has a significant inhibitory effect on six respiratory viruses, namely IAV, IBV, RhV, PIV, RSV, and ADV. 50 The concentrations ranged from 52.41 to 94.97 μg / mL, and the therapeutic index (TI) ranged from 1.99 to 5.68. No significant inhibitory effect was shown against HCoV-229E and EV71 viruses.

[0063] Experiment Example 2: In vivo efficacy experiment against influenza virus (influenza A virus) 1. Experimental Materials Test drug: Take the powder of the traditional Chinese medicine composition prepared in Example 1 and prepare a suspension of the required concentration with distilled water before use.

[0064] Positive control drug: Oseltamivir phosphate tablets, Roche Pharmaceuticals Shanghai Co., Ltd., batch number: M1174.

[0065] 2. Experimental Methods 2.1 Animal modeling and grouping Quarantined Balb / c mice were randomly divided into a normal control group, a model control group, a positive control group, and low-, medium-, and high-dose groups of the traditional Chinese medicine composition, with 10 animals in each group. Freshly prepared influenza A virus strain H1N1 / PR8 was added to PBS solution and kept on ice. Two hours before the first administration, mice in each group were mildly anesthetized with isoflurane to induce anesthesia. Except for the normal control group, mice in the other groups received 2 LD of the traditional Chinese medicine composition via nasal instillation. 50 Influenza virus solution, 50 μL / animal, and normal control group, 50 μL PBS solution was administered via nasal drops.

[0066] Administration and observation: Treatment was administered on the day of viral infection and 2 hours after infection, followed by continuous treatment for 6 days, for a total of 7 days. During this period, the mice's condition was closely monitored and indicators were measured.

[0067] 2.2. Detection Indicators (1) Lung index detection: After dissecting the mice, the lungs were weighed and the lung index was calculated. The results are shown in Table 7. Then, the right lung was cryopreserved at -80℃ and the left lung was fixed in paraformaldehyde for HE staining and pathological observation. The results are shown in Table 7. Figure 1 .

[0068] (2) Detection of viral load in the lungs: Frozen right lung tissue was taken for detection of viral load in the lungs. The results are shown in Table 8.

[0069] 3. Statistical methods One-way ANOVA was used for comparisons between groups, and the results are expressed as mean ± standard deviation. The significance level was [value missing]. p ≤0.05 indicates that the difference is statistically significant.

[0070] 4. Experimental Results (1) Effects of traditional Chinese medicine composition on lung index in mice Table 7. Results of the effect of traditional Chinese medicine composition on the lung index of influenza A virus in mice ( )

[0071] Note: The normal control group was compared with the model control group. # express p ≤0.05, ## express p ≤0.01; Compared with the treatment group, the model control group, * express p ≤0.05, ** express p ≤0.01.

[0072] As can be seen from the table above, compared with the normal control group, the lung index of the model control group animals showed a significant increase ( p ≤0.01). Compared with the model control group, the lung index of the positive control drug oseltamivir group was significantly decreased ( p ≤0.01). The lung index decreased significantly in both the medium and high-dose groups of the traditional Chinese medicine composition. p ≤0.01). A decreasing trend was observed at low doses, but no significant difference was observed ( ). p >0.05).

[0073] (2) Effect of traditional Chinese medicine composition on viral load in mouse lungs Table 8. Results of the effect of traditional Chinese medicine composition on viral load in the lungs of mice with influenza A virus ( )

[0074] Note: The normal control group was compared with the model control group. # express p ≤0.05, ## express p ≤0.01; Compared with the treatment group, the model control group, * express p ≤0.05, ** express p ≤0.01.

[0075] As can be seen from the table above, compared with the normal group, the viral load in the lungs of the model control group animals increased significantly after infection. p ≤ 0.01). Compared with the model control group, the viral load in the lungs of the positive control drug oseltamivir group was significantly reduced ( p ≤0.01). The low, medium, and high dose groups of the traditional Chinese medicine composition all showed a significant decrease in viral load in the lungs after infection. p ≤0.01 or p ≤0.05).

[0076] (3) Effects of traditional Chinese medicine composition on the pathological results of the lungs of mice with influenza A virus like Figure 1 As shown, the normal control group had intact lung structure, regular alveolar arrangement, unobstructed cavities, normal airway and alveolar epithelium morphology without damage, uniform narrowing of the lung interstitium without congestion, edema, inflammatory cell infiltration, and no abnormal lesions such as exudation, hemorrhage, necrosis, hyaline membrane and fibrosis, which are consistent with normal physiological structural characteristics.

[0077] Compared with the normal control group, the model control group showed extensive degeneration and necrosis of airway epithelial cells, with epithelial cell shedding in some areas; diffuse widening of the pulmonary interstitium with significant congestion, and diffuse infiltration of inflammatory cells, mainly mononuclear cells and lymphocytes, within the interstitium; extensive collapse of alveolar spaces, with diffuse hemorrhage visible within the spaces, destruction of alveolar wall structure in some areas, and focal coagulative necrosis. The necrotic areas showed disintegration of lung tissue structure and homogeneous red staining, manifesting as severe pneumonia. The lung tissue lesions affected approximately 85% of the mice.

[0078] Compared with the model control group, the positive control group showed mild degeneration, necrosis, and epithelial cell shedding in local airway epithelial cells; diffuse widening and slight congestion of the pulmonary interstitium, with a small amount of inflammatory cell infiltration mainly composed of mononuclear cells and lymphocytes in the interstitium; a small number of alveolar cavities collapsed, with a small amount of hemorrhage within the cavities, and destruction of the collapsed alveolar wall structure. A small amount of focal necrotic lung tissue was also observed, presenting as mild pneumonia. The lung tissue lesions affected approximately 30% of the mice.

[0079] In the low-dose group of the traditional Chinese medicine composition, mild degeneration and necrosis of airway epithelial cells and partial epithelial cell shedding were observed. Diffuse widening of the pulmonary interstitium was observed, accompanied by mild congestion, with diffuse infiltration of inflammatory cells, mainly monocytes and lymphocytes, within the interstitium. Extensive collapse of alveolar spaces was observed, with diffuse, minor hemorrhage within the spaces. Destruction of the alveolar wall structure was observed in some areas, along with mild, focal coagulative necrosis. The necrotic areas showed structural disintegration of the lung tissue and homogeneous red staining, resembling severe pneumonia. The lung tissue lesions affected approximately 60% of the mouse lung tissue.

[0080] In the medium-dose group of the traditional Chinese medicine composition, a small amount of airway epithelial cells showed degeneration and necrosis, with some areas exhibiting epithelial cell shedding; the pulmonary interstitium showed localized mild widening with mild congestion, and diffuse infiltration of inflammatory cells, mainly mononuclear cells and lymphocytes, within the interstitium; the alveolar spaces were extensively collapsed, with diffuse small amounts of hemorrhage within the spaces, presenting as mild pneumonia. The lesions affected approximately 40% of the lung tissue in mice.

[0081] In the high-dose group of the traditional Chinese medicine composition, a small amount of airway epithelial cells showed degeneration and necrosis, with some areas exhibiting epithelial cell shedding; localized mild widening of the pulmonary interstitium, accompanied by mild congestion, with diffuse infiltration of inflammatory cells, mainly mononuclear cells and lymphocytes, within the interstitium; widespread collapse of alveolar spaces, with diffuse small amounts of hemorrhage within the spaces, presenting as mild pneumonia. The lesions affected approximately 20% of the lung tissue in mice.

[0082] All dosage groups of the traditional Chinese medicine composition improved lung tissue damage caused by influenza A virus to varying degrees, manifested as reduced airway epithelial damage, suppression of interstitial pulmonary inflammation, and relief of alveolar collapse and hyaline membrane formation, exhibiting a clear dose-dependent effect: the low-dose group showed a certain protective effect, while the improvement effects of the medium- and high-dose groups were close to those of the oseltamivir control group. Therefore, the traditional Chinese medicine composition of this invention has a significant effect on improving lung inflammation in mice infected with influenza A virus, and can also downregulate the viral load in the lungs of viral pneumonia, indicating that the traditional Chinese medicine composition has a protective effect against influenza A virus-induced pneumonia in mice.

[0083] Experimental Example 3: In vivo test of traditional Chinese medicine composition against influenza A virus (mortality protection) 1. Experimental Materials Test drug: Take the powder of the traditional Chinese medicine composition prepared in Example 1 and prepare a suspension of the required concentration with distilled water before use.

[0084] Positive control drug: Oseltamivir phosphate tablets, Roche Pharmaceuticals Shanghai Co., Ltd., batch number: M1174.

[0085] 2. Experimental Methods 2.1 Animal modeling and grouping Quarantined Balb / c mice were randomly divided into a normal control group, a model control group, a positive control group, and low-, medium-, and high-dose groups of the traditional Chinese medicine composition, with 10 animals in each group. Freshly prepared influenza A virus strain H1N1 / PR8 was added to PBS solution and kept on ice. Two hours before the first administration, mice in each group were mildly anesthetized with isoflurane. Except for the normal control group, mice in other groups received 50 μL of the influenza virus solution with an LD50 via nasal instillation, while the normal control group received 50 μL of PBS solution via nasal instillation.

[0086] Administration and observation: Treatment was administered on the day of viral infection and 2 hours after infection, followed by continuous treatment for 13 days, for a total of 14 days. During this period, the mice's condition was closely monitored and indicators were measured.

[0087] 2.2 Detection Indicators Record the mortality rate of mice in each group, and calculate the mortality protection rate and life extension rate. The calculation formulas are as follows: Mortality protection rate = (mortality rate in the model control group - mortality rate in the drug treatment group) / mortality rate in the model control group × 100%; Life extension rate = (mean survival days in the treatment group - mean survival days in the model control group) / mean survival days in the model control group × 100%; See results Figure 2 and Table 9 below.

[0088] 3. Experimental Results Table 9. Effects of Traditional Chinese Medicine Compositions on Mortality Protection in Mouse Models of Influenza A Virus

[0089] From Table 9 and Figure 2 It can be seen that the survival of mice in the oseltamivir control group (positive control drug) was significantly improved, with the mortality rate reduced to 20%, the mortality protection rate reaching 70%, and the life extension rate reaching 14.77%. The survival of mice in the medium and high dose groups of the traditional Chinese medicine composition was also significantly improved. In the medium dose group, the mortality rate was reduced to 50%, the mortality protection rate reached 40%, and the life extension rate reached 30.68%. In the high dose group, the mortality rate was reduced to 40%, the mortality protection rate reached 50%, and the life extension rate reached 33.36%. In the low dose group of the traditional Chinese medicine composition, the mortality rate was 70%, the mortality protection rate reached 20%, and the life extension rate reached 14.77%. Therefore, the traditional Chinese medicine composition of this invention has a significant protective effect against mortality in mice infected with influenza A virus.

[0090] Experiment Example 4: In vivo efficacy experiment against influenza virus (influenza B virus) 1. Experimental Materials Test drug: Take the powder of the traditional Chinese medicine composition prepared in Example 1 and prepare a suspension of the required concentration with distilled water before use.

[0091] Positive control drug: Oseltamivir phosphate tablets, Roche Pharmaceuticals Shanghai Co., Ltd., batch number: M1174.

[0092] 2. Experimental Methods 2.1 Animal modeling and grouping Quarantined Balb / c mice were randomly divided into a normal control group, a model control group, a positive control group, and low-, medium-, and high-dose groups of the traditional Chinese medicine composition, with 10 animals in each group. Freshly prepared Florida influenza B virus strain was added to PBS solution and kept on ice. Two hours before the first administration, mice in each group were mildly anesthetized with isoflurane. Except for the normal control group, mice in other groups received 50 μL of the influenza virus solution with an LD50 concentration via nasal instillation, while the normal control group received 50 μL of PBS solution via nasal instillation.

[0093] Administration and observation: Treatment was administered on the day of viral infection and 2 hours after infection, followed by continuous treatment for 6 days, for a total of 7 days. During this period, the mice's condition was closely monitored and indicators were measured.

[0094] 2.2 Detection Indicators (1) Lung index detection: After dissecting the mice, the lungs were weighed and the lung index was calculated. The results are shown in Table 10. Then, the right lung was cryopreserved at -80℃ and the left lung was fixed in paraformaldehyde for HE staining and pathological observation. The results are shown in Table 10. Figure 3 As shown.

[0095] (2) Detection of viral load in the lungs: Frozen right lung tissue was taken for detection of viral load in the lungs. The results are shown in Table 11.

[0096] 3. Statistical methods One-way ANOVA was used for comparisons between groups, and the results are expressed as mean ± standard deviation. The significance level was [value missing]. p ≤0.05 indicates that the difference is statistically significant.

[0097] 4. Experimental Results (1) Effects of traditional Chinese medicine composition on lung index in mice Table 10 Results of the effect of traditional Chinese medicine composition on the lung index of influenza B virus in mice ( )

[0098] Note: The normal control group was compared with the model control group. # express p ≤0.05, ## express p ≤0.01; Compared with the treatment group, the model control group, * express p ≤0.05, ** express p ≤0.01.

[0099] As can be seen from the table above, compared with the normal control group, the lung index of the model control group animals showed a significant increase ( p ≤0.01). Compared with the model control group, the lung index of the positive control drug oseltamivir group was significantly decreased ( p ≤0.01). Compared with the model control group, the lung index of the low, medium and high dose groups of the traditional Chinese medicine composition all decreased significantly ( p ≤0.01).

[0100] (2) Effect of traditional Chinese medicine composition on viral load in mouse lungs Table 11 Results of the effect of traditional Chinese medicine composition on viral load in the lungs of mice with influenza B virus ( )

[0101] Note: The normal control group was compared with the model control group. # express p ≤0.05, ## express p ≤0.01; Compared with the treatment group, the model control group, * express p ≤0.05, ** expressp ≤0.01.

[0102] As can be seen from the table above, compared with the normal group, the viral load in the lungs of the model control group animals increased significantly after infection. p ≤0.01). Compared with the model control group, the viral load in the lungs of the positive control drug oseltamivir group was significantly reduced ( p ≤0.01). Both the medium and high-dose groups of the traditional Chinese medicine composition showed a significant decrease in viral load in the lungs after infection. p ≤0.05).

[0103] (3) Effects of traditional Chinese medicine composition on the pathological results of the lungs of mice with influenza B virus like Figure 3 As shown, the normal control group had intact lung structure, regular alveolar arrangement, unobstructed cavities, normal airway and alveolar epithelium morphology without damage, uniform narrowing of the lung interstitium without congestion, edema, inflammatory cell infiltration, and no abnormal lesions such as exudation, hemorrhage, necrosis, hyaline membrane and fibrosis, which are consistent with normal physiological structural characteristics.

[0104] Compared with the normal control group, the model control group showed mild degeneration, necrosis, and epithelial cell shedding in local airway epithelial cells; diffuse widening of the pulmonary interstitium with mild congestion, and diffuse infiltration of inflammatory cells, mainly mononuclear cells and lymphocytes, within the interstitium; widespread collapse of alveolar spaces with mild hemorrhage, destruction of alveolar wall structure in some areas, and focal necrosis and cell shedding of small amounts of lung tissue, presenting as severe pneumonia. The lung tissue lesions affected approximately 70% of the mice.

[0105] Compared with the model control group, the positive control group showed mild degeneration, necrosis, and epithelial cell shedding in the local airway epithelial cells; slight widening and mild congestion in the local lung interstitium, with mild infiltration of inflammatory cells, mainly mononuclear cells and lymphocytes, in the interstitium; a few alveolar cavities collapsed, with mild hemorrhage within the cavities, a small amount of collapsed alveolar walls, structural damage, and a small amount of focal necrotic lung tissue, presenting as mild pneumonia. The lung tissue lesions affected approximately 40% of the mice.

[0106] In the low-dose group of the traditional Chinese medicine composition, mild degeneration and necrosis of airway epithelial cells and partial epithelial cell shedding were observed; mild widening of the pulmonary interstitium with slight congestion and diffuse infiltration of inflammatory cells, mainly mononuclear cells and lymphocytes, were seen in the interstitium; alveolar cavity collapse and destruction of alveolar wall structure were observed in some areas, along with a small amount of focal necrotic lung tissue, presenting as mild pneumonia. The lung tissue lesions in mice affected approximately 35% of the lung tissue.

[0107] In the medium-dose group of the traditional Chinese medicine composition, the airway epithelial cells showed mild degeneration and necrosis, with some areas of epithelial cell shedding; the pulmonary interstitium showed diffuse widening with mild congestion, and diffuse infiltration of inflammatory cells, mainly mononuclear cells and lymphocytes, within the interstitium; the alveolar spaces collapsed extensively, with diffuse small amounts of hemorrhage within the spaces, and a small amount of focal necrotic lung tissue was also observed, presenting as mild pneumonia. The lung tissue lesions affected approximately 40% of the mice.

[0108] In the high-dose group of the traditional Chinese medicine composition, airway epithelial cells showed mild degeneration and necrosis, with a small amount of epithelial cell shedding in some areas; the pulmonary interstitium showed mild widening with slight congestion, and diffuse infiltration of inflammatory cells, mainly mononuclear cells and lymphocytes, within the interstitium; local alveolar cavities showed mild collapse with diffuse hemorrhage within the cavities, and a small amount of focal necrotic lung tissue was also observed, presenting as mild pneumonia. The lung tissue lesions affected approximately 35% of the mice.

[0109] It is evident that the various dosage groups of the traditional Chinese medicine composition of the present invention can improve lung tissue damage caused by influenza B virus to varying degrees, manifested as reduced airway epithelial damage, suppression of interstitial pulmonary inflammation, and relief of alveolar collapse and hyaline membrane formation. This indicates that the traditional Chinese medicine composition of the present invention has a significant ameliorative effect on lung inflammation in mice infected with influenza B virus, and can also downregulate the viral load in the lungs of viral pneumonia, demonstrating that the traditional Chinese medicine composition has a protective effect against influenza B virus-induced pneumonia in mice.

[0110] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for treating respiratory viral infections, characterized in that, The composition is made from the following raw materials: dog liver vegetable, mountain sesame, indigo leaf, kudzu root, tangerine peel, bletilla rhizome, artemisia annua, and saposhnikovia root; The respiratory viruses mentioned are one or more of the following: influenza A virus, influenza B virus, human rhinovirus, parainfluenza virus, respiratory syncytial virus, and adenovirus.

2. The application according to claim 1, characterized in that, The respiratory virus is one or more of the following: influenza A virus, influenza B virus, human rhinovirus, and adenovirus.

3. The application according to claim 2, characterized in that, The respiratory virus in question is influenza A virus and / or influenza B virus.

4. The application according to claim 1, characterized in that, The traditional Chinese medicine composition treats respiratory viral infections by reducing the lung index, reducing viral load in the lungs, improving lung inflammation, and / or increasing survival rate after viral infection.

5. The application according to claim 1, characterized in that, The composition, by weight, is made from the following raw materials: 45-55 parts of dog liver vegetable, 26-30 parts of mountain sesame, 45-55 parts of big green leaf, 45-55 parts of kudzu root, 26-30 parts of Dongfeng orange, 13-15 parts of Bletilla striata, 8-12 parts of Artemisia annua and 4-6 parts of Saposhnikovia divaricata.

6. The application according to claim 5, characterized in that, The composition, by weight, is made from the following raw materials: 50 parts of dog liver vegetable, 28 parts of mountain sesame, 50 parts of indigo leaf, 50 parts of kudzu root, 28 parts of Dongfeng orange, 14 parts of Bletilla striata, 10 parts of Artemisia annua, and 5 parts of Saposhnikovia divaricata.

7. The application according to any one of claims 1-6, characterized in that, The preparation method of the traditional Chinese medicine composition is as follows: (1) Take dog liver vegetable, mountain sesame, big indigo leaf, Dongfeng orange and kudzu root, add water and decoct, filter and concentrate to obtain thick paste; (2) Take Bletilla striata, Artemisia annua and Saposhnikovia divaricata, crush and sieve them to obtain medicinal powder; (3) Mix the medicinal powder with the thick paste, dry and pulverize to obtain the mixed medicinal powder; (4) The mixed powder is made into pills and coated to obtain the final product.

8. The application according to claim 7, characterized in that, In step (1), the decoction is performed 1-2 times, with 8-12 times the amount of water added each time and decocted for 1-2 hours.

9. The use of a traditional Chinese medicine composition in the preparation of a drug for treating pneumonia caused by respiratory viral infection, characterized in that, The composition is made from the following raw materials: dog liver vegetable, mountain sesame, indigo leaf, kudzu root, tangerine peel, bletilla rhizome, artemisia annua, and saposhnikovia root.

10. The application according to claim 9, characterized in that, The pneumonia caused by the respiratory virus infection is pneumonia caused by influenza A virus and / or pneumonia caused by influenza B virus.

Images