A composition for treating and preventing novel coronavirus and preparation method thereof

Abstract

The present application relates to a composition for treating and preventing the novel coronavirus, wherein the active ingredients of the composition and their weight portions are: 55.7-223 parts of magnolol, 10.7-43 parts of pseudoephedrine, 5.2-20.6 parts of sennoside A, 4.3-17.2 parts of quercetin, 2.5-10.3 parts of benzoylpeonidin, 0.43-1.72 parts of isoliquiritigenin, and 1.3-5.2 parts of astragaloside IV. The composition has the effect of treating or preventing the novel coronavirus.

Description

[0001] Technical field: This application belongs to the field of traditional Chinese medicine, and in particular to the field of treating new coronavirus. Background technology:

[0002] The novel coronavirus is a highly contagious enveloped positive RNA virus that can cause respiratory diseases, fever, and pneumonia. The development of traditional Chinese medicine compound formulas (TCMFs) has been strongly supported and widely used for the prevention and treatment of COVID-19. Huashi Baidu Fang is a famous traditional Chinese medicine compound for COVID-19, which consists of 14 Chinese medicines, including monarch medicines: ephedra, patchouli, gypsum; ministerial medicines: bitter almond, pinellia, magnolia bark, atractylodes, tsaoko, tuckahoe; adjuvant medicines: astragalus, southern scutellaria, rhubarb; and guiding medicine: liquorice. Clinical studies have shown that Huashi Baidu Fang can promote the clearance of SARS-CoV-2 RNA, absorption of lung turbidity, reduce inflammation, improve flu-like symptoms, and has potential antiviral and anti-inflammatory activities. Previous studies have preliminarily identified 217 chemical components in Huashi Baidu Fang. In order to further study the active ingredients in the Hua Shi Baidu prescription, the active ingredients were identified through preliminary chemical and pharmacological studies, and in vivo and in vitro pharmacodynamic experiments were conducted to determine the composition of the active ingredients for the treatment and prevention of the new coronavirus.

[0003] Invention content: In order to obtain the active ingredient composition in the Hua Shi Baidu prescription, the present application conducted a large number of chemical and pharmacological related studies and finally screened out a composition for treating and preventing the new coronavirus.

[0004] The active ingredients of the composition for treating and preventing the new coronavirus involved in the present application and their weight portions are: 55.7-223 parts of magnolol, 10.7-43 parts of pseudoephedrine, 5.2-20.6 parts of sennoside A, 4.3-17.2 parts of quercetin, 2.5-10.3 parts of benzoylpeonidin, 0.43-1.72 parts of isoliquiritigenin, 1.3-5.2 parts of astragaloside IV; or 100-180 parts of magnolol, 20-35 parts of pseudoephedrine, 8.4-15.8 parts of sennoside A, 7.5-15.2 parts of quercetin, 4.5-9.6 parts of benzoylpeonidin, 0.56-1.35 parts of isoliquiritigenin, 2.1-4.5 parts of astragaloside IV; or 100 parts of magnolol, 20 parts of pseudoephedrine, 4.5-9.6 parts of sennoside A, 0.56-1.35 parts of isoliquiritigenin, 2.1-4.5 parts of astragaloside IV 8.4 parts, 7.5 parts of quercetin, 4.5 parts of benzoylpeonidin, 0.56 parts of isoliquiritigenin, and 2.1 parts of astragaloside IV; or 180 parts of magnolol, 35 parts of pseudoephedrine, 15.8 parts of sennoside A, 15.2 parts of quercetin, 9.6 parts of benzoylpeonidin, 1.35 parts of isoliquiritigenin, and 4.5 parts of astragaloside IV; or 111.5 parts of magnolol, 21.5 parts of pseudoephedrine, 10.3 parts of sennoside A, 8.6 parts of quercetin, 5.15 parts of benzoylpeonidin, 0.86 parts of isoliquiritigenin, and 2.6 parts of astragaloside IV.

[0005] The composition described in the present application can be prepared into a drug in the form of granules, tablets, capsules, oral solutions or injections.

[0006] The drug for treating and preventing the novel coronavirus described in the present application is prepared by combining the above-mentioned composition with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical preparation;

[0007] The use of the composition or medicine described in the present application in the preparation of a medicine for treating or preventing the new coronavirus.

[0008] The composition described in the present application has the effect of preventing or treating the new coronavirus.

[0009] The magnolol, pseudoephedrine, sennoside A, quercetin, benzoylpeonidin, isoliquiritigenin and astragaloside IV described in the present application can be prepared by extracting from traditional Chinese medicines using extraction methods in the art, or can be directly purchased from commercial products.

[0010] The technical solution of the present application is described below through specific embodiments. Description of the drawings:

[0011] Figure 1 Effect of the composition on the body weight of human ACE2 (hACE2) transgenic mice infected with SARS-CoV-2

[0012] Figure 2 Effect of the composition on the viral load of SARS-CoV-2 infected human ACE2 (hACE2) transgenic mice

[0013] Figure 3 Effect of the composition on chemokine MCP-1 in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0014] Figure 4 Effect of the combination on chemokine MIP-1α in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0015] Figure 5 Effect of the composition on chemokine IP-10 in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0016] Figure 6 Effect of the composition on inflammatory factor TNF-α in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0017] Figure 7 Effect of the composition on inflammatory factor IL-6 in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0018] Figure 8Effect of the composition on inflammatory factor IFN-γ in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0019] Fig. 9 Effect of the composition on inflammatory factor IL-17 in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0020] Fig.10 Effect of the composition on inflammatory factor IL-10 in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0021] Fig.11 Effect of the composition on inflammatory factor IL-12 in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0022] Fig.12 Effect of the composition on inflammatory factor IL-1α in lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2

[0023] Fig.13 HE staining results of lung tissue pathology in the control group of SARS-CoV-2 infected human ACE2 transgenic mouse model, showing moderate interstitial pneumonia (200×)

[0024] Fig.14 The results of HE staining of lung tissue of human ACE2 transgenic mice infected with SARS-CoV-2 showed mild interstitial pneumonia (200×) Specific implementation method:

[0025] Embodiment 1:

[0026] A composition, wherein the raw materials are: 111.5 mg of magnolol, 21.5 mg of pseudoephedrine, 10.3 mg of sennoside A, 8.6 mg of quercetin, 5.15 mg of benzoylpeonidin, 0.86 mg of isoliquiritigenin, and 2.6 mg of astragaloside IV. The preparation method of the composition into an oral liquid comprises: weighing the above active ingredients in sequence, mixing them thoroughly, and adding a pharmaceutically acceptable carrier to prepare the oral liquid.

[0027] Embodiment 2:

[0028] A composition, wherein the raw materials are: 180 mg of magnolol, 35 mg of pseudoephedrine, 15.8 mg of sennoside A, 15.2 mg of quercetin, 9.6 mg of benzoylpeonidin, 1.35 mg of isoliquiritigenin, and 4.5 mg of astragaloside IV. The preparation method of the composition into granules comprises: weighing the above active ingredients in sequence, mixing them thoroughly, and adding a pharmaceutically acceptable carrier to prepare the granules.

[0029] Embodiment 3:

[0030] A composition, wherein the raw materials are: 100 mg of magnolol, 20 mg of pseudoephedrine, 8.4 mg of sennoside A, 7.5 mg of quercetin, 4.5 mg of benzoylpeonidin, 0.56 mg of isoliquiritigenin, and 2.1 mg of astragaloside IV. The preparation method of the composition into capsules comprises: weighing the above active ingredients in sequence, mixing them thoroughly, and adding a pharmaceutically acceptable carrier to prepare the capsules.

[0031] Embodiment 4:

[0032] A composition, wherein the raw materials are: 55.7 mg of magnolol, 10.7 mg of pseudoephedrine, 5.2 mg of sennoside A, 4.3 mg of quercetin, 2.5 mg of benzoylpeonidin, 0.43 mg of isoliquiritigenin, and 1.3 mg of astragaloside IV. The preparation method of the composition into tablets comprises: weighing the above active ingredients in sequence, mixing them thoroughly, and adding a pharmaceutically acceptable carrier to prepare tablets.

[0033] Embodiment 5:

[0034] A composition, the raw materials of which are: 223 mg of magnolol, 43 mg of pseudoephedrine, 20.6 mg of sennoside A, 17.2 mg of quercetin, 10.3 mg of benzoylpeonidin, 1.72 mg of isoliquiritigenin, and 5.2 mg of astragaloside IV. The preparation method of the composition into an injection comprises: weighing the above active ingredients in sequence, mixing them thoroughly, and adding a pharmaceutically acceptable carrier to prepare the injection.

[0035] Pharmacodynamics experiment 1: Effect of the composition on novel coronavirus pneumonia

[0036] 2.1 Materials and methods

[0037] Ethics statement All infectious operations on mice were performed in a biosafety level 3 laboratory, and all experimental procedures were approved by the Animal Care and Use Committee of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences & Center for Comparative Medicine, Peking Union Medical College Hospital (BLL21008).

[0038] Virus: SARS-CoV-2 / human / CHN / Delta-2 / 2021 (Genbank: OM061695) was provided by the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences.

[0039] 2.2 Animal experiments

[0040] 6-8 week old SPF hACE2 mice were provided by the Institute of Medical Laboratory Animal Science, Chinese Academy of Medical Sciences. The mice were randomly divided into two groups (n=6), namely, a blank solvent group (the blank solvent gavage group was set as the model group) and a combination group (dose of 37.4 mg / kg) (i.e., the oral solution prepared in Example 1 of the present application), which were gavaged for 5 consecutive days; the combination group was gavaged with the corresponding drug, and the blank solvent group was given the solvent, which was gavaged for 5 consecutive days. After anesthesia, the mice were nasally instilled with 1×10 SARS-CoV-2 virus 5 TCID 50 / ml, and the infected mice were observed every day, and their body weight, clinical symptoms, and responses to external stimuli were recorded. The mice were killed 5 days after infection, and the lungs were taken for viral load analysis, cytokine and chemokine detection, and pathological examination.

[0041] 2.3 Detection

[0042] Viral load analysis by real-time fluorescence quantitative PCR

[0043] Total RNA was extracted from tissues using the RNeasy kit (Qiagen) and reverse transcribed. qRT-PCR was performed using the following cycle protocol and primers: 50°C for 2 min, 95°C for 2 min, 95°C for 15 s, 60°C for 30 s, 40 cycles, followed by a melting curve program of 95°C for 15 s, 60°C for 1 min, and 95°C for 45 s. The primers used to detect SARS-CoV-2 were as follows: forward primer, 5′-TCGTTTCGGAAGAGACAGGT-3′; reverse primer, 5′-GCGCAGTAAGGATGGCTAGT-3′.

[0044] Cytokine and chemokine detection

[0045] Cytokines and chemokines were analyzed using the LEGEND plexTM Mouse Inflammatory Factor Kit (13-plex). 25 μL of lung tissue homogenate was taken and incubated with the capture microspheres. After washing away the unbound antibodies and samples, biotin-labeled detection antibodies were added, and finally SA-PE was added to bind to biotin to emit fluorescence. The fluorescence intensity of PE on each microsphere was analyzed by flow cytometry to quantitatively analyze the soluble protein content in the sample, and the LEGENDplexTM data analysis software was used for data acquisition and analysis.

[0046] 2.4 Statistical analysis

[0047] All data were analyzed using GraphPad Prism 8.0 software (GraphPad software, Inc). Two-tailed unpaired t-test was used for comparison between groups. *p < 0.05, **p < 0.01 were considered statistically significant.

[0048] 2.5 Results:

[0049] The average weight loss rates of the model control group and the treatment group 5 days post infection (dpi) were: the model control group lost weight after infection, and the average weight loss rate on 5 dpi was 4.86%; the average weight loss rate on 5 dpi in the combination group was 2.69%. (See Appendix Figure 1 ).

[0050] The average viral load in lung tissue of the model control group was 10 6.84 The average viral load in lung tissue of the combination group was 10 6.35 copies / ml, compared with the model control group, the value decreased by 0.491g. (See attached Figure 2 ).

[0051] Five days after infection, the chemokines MCP-1, MIP-1α, IP-10 and inflammatory factors TNF-α, IL-6, IFN-γ, IL-12, IL-17, IL-10 and IL-1α in the lung tissue of the combination group were decreased compared with the model group. (See Appendix Figure 3-12 ).

[0052] In terms of lung pathological changes, the model control group showed moderate interstitial pneumonia, while the administration of the composition significantly improved lung inflammation (see Appendix Fig.14 ). Six cases (6 / 6) in the model control group showed moderate interstitial pneumonia in their lung tissues (see Appendix Fig.13 ); In the combination group, 4 cases (4 / 6) had mild interstitial pneumonia changes in lung tissue, and 2 cases (2 / 6) had moderate interstitial pneumonia changes in lung tissue.

[0053] Pharmacodynamics experiment 2: Effect of the composition on human coronavirus pneumonia

[0054] Test site: Biosafety Laboratory of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (ABSL-2 Laboratory)

[0055] 1. Test materials

[0056] 1.1 Drugs under investigation

[0057] The oral solution of the composition prepared in Example 1 of the present application.

[0058] 1.2 Experimental animals

[0059] SPF-grade ICR mice, half male and half female, weighing 13-15 g, were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd., certificate number: 110011211113827874 / 110011211113827747, license number: SCXK(Beijing)2021-0006. Breeding conditions: ABSL-2 laboratory, -20Pa, temperature 22±1℃, humidity 40-50%.

[0060] 1.3 Virus strains and cells

[0061] 1.3.1 Virus strain: Human coronavirus (HCoV-229E), purchased from ATCC, USA, propagated in this laboratory, and stored in a -80°C refrigerator.

[0062] 1.3.2 Cell line: Human embryonic lung epithelial cells (MRC-5) were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, passaged in our laboratory, and stored in liquid nitrogen.

[0063] 1.4 Experimental reagents

[0064]

[0065] 1.5 Test equipment

[0066] Instrument Name model Manufacturer Instrument use Type A2 Biological Safety Cabinet Thermo MSC1.8 Thermo Corporation Animal infection and autopsy Electronic balance YP1002 MAX100g Shanghai Yueping Scientific Instrument Co., Ltd. Weigh the mice Intelligent artificial climate chamber Ningbo Jiangnan Instrument Factory RXZ-380B Animal Modeling Electronic analytical balance AR1140 MAX110g Ohaus Corporation Weighing drugs and reagents Electronic balance AL204 MAX210g Mettler-Toledo Instruments Weighing tissue IVC mouse cage ZJ-4 Suzhou Fengshi Co., Ltd. Feeding mice Real-Time PCR Instrument Applied biosystems QuantStudio 5 Nucleic acid amplification testing Eight-row tube mini centrifuge Kylin-Bell Lab LX-300 Nucleic acid amplification testing Mini Vortex Mixer Haimen Qilin Bell Instrument Co., Ltd. OL-901 Nucleic acid amplification testing Multifunctional ELISA reader PerkinElmer, Germany Enspire type Inflammatory factor detection Flow cytometry BD Company of America <![CDATA[FACSAria TM llu type]]> T and B lymphocyte detection Centrifuge Eppendorf Company, Germany Centrifuge 5430 Centrifuge cells Low temperature high speed centrifuge Eppendorf Company, Germany Eppendorf-5810R Centrifugation of serum and lung tissue

[0067] 2. Test methods

[0068] 2.1 Dosage design and drug preparation

[0069] 2.1.1 Test drug: According to the recommended dosage regimen, after converting the dosage to the dosage for mice, 74.8 mg / kg, 37.4 mg / kg, 18.7 mg / kg and 9.35 mg / kg were used. 0.2 ml / 10 g body weight / time oral administration, once a day, for 3 consecutive days.

[0070] 2.2 Virus Passaging

[0071] Take a 25cm tube that has grown into a monolayer of MRC-5 cells 2 The culture flask was poured out of the culture medium, the cell surface was rinsed with cell maintenance medium for 3 times, and then 200 μl of HCoV-229E virus solution was added. The cells were cultured in a 37°C 5% CO2 incubator, and the cell pathological changes were observed under an inverted microscope every day for 72-96 hours. After 80% of the cells showed obvious pathological changes (CPE), the cell culture flask was placed in a -80°C low-temperature refrigerator for freezing. The virus solution was repeatedly frozen and thawed 3 times before being used to detect the virulence of the virus.

[0072] 2.3 Virus titer determination

[0073] Take the culture plate with MRC-5 cells grown into a monolayer, pour out the culture medium, rinse the cell surface with cell maintenance medium three times, and then inoculate HCoV-229E virus liquid of different titers at 10-fold dilution. -1 ~10 -8 There are 8 dilutions in total, 100 μl / well, 4 replicates for each concentration, and a normal cell control. The cells were cultured in a 37°C 5% CO2 incubator, and the cell lesions were observed under an inverted microscope every day for 72 to 96 hours. The cell lesions in each well were recorded. The 50% cell lesion concentration (TCID 50 ).

[0074] 2.4 Grouping and Detection

[0075] 80 ICR mice were randomly divided into normal control group, simple 229E infection control group, simple cold-dampness stimulation control group (hereinafter referred to as cold-dampness syndrome model group), cold-dampness epidemic toxin attack lung disease syndrome combined model group (hereinafter referred to as epidemic toxin attack lung model group), combination 74.8 mg / kg group, combination 37.4 mg / kg group, combination 18.7 mg / kg group, combination 9.35 mg / kg group according to weight level, 10 mice in each group, half male and half female. Except for the normal control group and 229E infection group, the remaining mice were placed in an artificial climate box with a relative humidity of 90±3%, no wind, and a temperature of 4±2°C every day, and taken out after each 4-hour stimulation for 7 consecutive days.

[0076] Except for the normal control group and the cold-dampness syndrome model group, the remaining mice were lightly anesthetized with isoflurane and then injected with 100TCID 50 HCOV-229E virus droplet infection, 50μl / mouse; the normal control group and cold-dampness syndrome model group were nasally infected with virus-free culture solution. Each group began to be administrated on the day of infection, with a volume of 0.2ml / 10g body weight, once a day for 3 consecutive days; the normal control group, cold-dampness syndrome model group, 229E infection group, and epidemic lung attack model group were gavaged with distilled water under the same conditions. After weighing on the 4th day of infection, the rats were killed and the lungs were weighed and observed and tested for the following indicators:

[0077] 2.4.1 Lung index and inhibition rate: Mice were killed and lungs were weighed. The lung index and lung index inhibition rate were calculated according to the following formula

[0078] Lung index = [lung wet weight (g) / body weight (g)] × 100

[0079]

[0080] 2.4.2 Nucleic acid detection in lung tissue (RT-PCR method): After the mouse is dissected, the lung tissue is divided into lysis medium and stored in a -80℃ low-temperature refrigerator. When testing, it is taken out from the -80℃ low-temperature refrigerator and 1ml TRIzol is added to each tube. Reagent, place on a shaking lyser for 20-25s, incubate at room temperature for 10min, and centrifuge at 4℃, 12000rpm for 10min; transfer the clarified supernatant to a new 1.5ml centrifuge tube; add 0.2ml chloroform, cover the tube tightly, shake the centrifuge tube vigorously for 15s, and incubate at room temperature for 2-3min until the liquid is separated; centrifuge at 4℃, 12000rpm for 15min; carefully transfer the transparent supernatant to a new 1.5ml centrifuge tube, add 0.5ml isopropanol, mix well, and incubate at room temperature for 30min; centrifuge at 4℃, 12000rpm for 10min; discard the supernatant, gently wash the precipitate with 1ml75% ethanol (so that the white precipitate floats lightly); centrifuge at 4℃, 7500rpm for 5min; aspirate the supernatant, briefly dry the RNA precipitate for 5-10min; dissolve the precipitate with 30μl DEPC water and store in a -80℃ low-temperature refrigerator.

[0081] Nucleic acid treatment of control substances: DEPC-H2O was used as negative control. Positive control substances were diluted 10, 100, and 1000 times.

[0082] Reagent preparation: Take n×18μl HCoV-229E nucleic acid fluorescence PCR detection mixture, n×1μl internal control, and n×1μl RT-PCR enzyme (n is the number of reaction tubes), shake and mix for a few seconds, and centrifuge at 3000rpm for a few seconds.

[0083] Add sample: Take 20μl of the above mixed solution and place it in a PCR tube, then add 5μl of sample nucleic acid extract, DEPC-H2O, and positive control into the PCR tube respectively, cover the tube tightly, centrifuge for a few seconds to allow all the liquid to settle at the bottom, and immediately carry out PCR amplification reaction.

[0084] The cycle parameters were set as follows: 45°C×10 min; 95°C×15 min; then 95°C×15 sec→60°C×60 sec, cycled 40 times; single-point fluorescence detection at 60°C, reaction system was 25 μl.

[0085] 2.4.3 Detection of gastrin (GAS), motilin (MTL) and inflammatory factors in mouse serum and lung tissue (ELISA method): Serum samples: Blood was collected from mouse eyeballs, placed at room temperature for 30 minutes, centrifuged at 3000 rpm for 10 minutes, and the supernatant was aspirated into a new EP tube and stored at -20°C. The test was performed according to the instructions of the kit, and each index was detected by the absorbance of 450nm using an ELISA reader.

[0086] Lung tissue homogenate sample: After weighing the lung tissue of mice, collect the mouse lung tissue in aliquots and store at -4°C. Weigh 50 mg of lung tissue and add 500 μL of saline, use an ultrasonic cell disruptor to homogenize the tissue, and use a low-temperature high-speed centrifuge to centrifuge at 4°C 1000rpm for 10 minutes. After absorbing the supernatant, aliquot and store it in a -80°C refrigerator for future use. During the test, follow the instructions of the kit and use an enzyme marker to detect each indicator at 450nm absorbance.

[0087] 2.4.4 Flow cytometry of T lymphocyte subsets and B lymphocyte ratio in mouse peripheral blood: Eyeballs of mice were removed to collect blood, and 3 drops of blood (about 150 μl) were added to a 15 ml centrifuge tube containing 10 ml 1×PBS, and centrifuged at 1600 rpm for 5 min at room temperature; the supernatant was carefully discarded with a pipette, and 1 ml of red blood cell lysis buffer was added to each tube to resuspend the cell pellet, and the cell pellet was lysed at room temperature for about 5-10 min until the liquid changed from turbid to clear, and 10 ml of PBS was added to terminate the lysis, and the cell pellet was centrifuged at 2000 rpm for 5 min at 4°C, and the supernatant was discarded. The cell pellet was resuspended with 10 ml PBS, centrifuged at 2000 rpm for 5 min at 4°C, and the supernatant was discarded. The cell pellet was resuspended with 200 μl of blocking buffer (PBS containing 5% FBS), and the cell suspension was transferred to a 1.5 ml ep tube and blocked at 4°C for 30 min. Prepare the flow cytometry antibodies in blocking solution in a dark place as follows: FITC-labeled anti-mouse CD3e, PE-labeled anti-mouse CD19, PerCP-Cy5.5-labeled anti-mouse CD4, APC-labeled anti-mouse CD8a. The preparation volume for each tube of cells is: 0.3 μl of each antibody and 50 μl of blocking solution.

[0088] Centrifuge the cell suspension at 4°C, 2000rpm for 5min, and discard the supernatant. Add 50μl of flow cytometry antibody to each tube, incubate at 4°C in the dark for 30min, add 1ml PBS, centrifuge at 4°C, 2000rpm for 5min, and discard the supernatant. Resuspend the cells with 200μl PBS containing 2% FBS, transfer to the flow cytometry tube, and detect on the machine.

[0089] 2.4.5 Pathological examination of mouse lung tissue

[0090] After the lungs of mice were removed, the lung tissues were fixed in 10% formalin solution, taken out after 48 hours, rinsed with running water, dehydrated with gradient ethanol, transparentized with xylene, embedded in wax, sliced, stained with HE, sealed with neutral gum, observed under a microscope, and photographed. The pathological morphological changes of the mouse lungs were observed under an optical microscope, and the groups were compared.

[0091] Microscopic observation standards:

[0092] “-”: There was no congestion or edema in the mouse lung interstitium, no inflammatory exudation around the bronchioles in the lungs, and the tissue structure was normal.

[0093] “+”: No exudation was observed in the lung interstitium of mice, no enlargement of alveoli, scattered congestion in the lung interstitium, a small amount of exudation, no cell proliferation; no obvious inflammatory exudation was observed in the bronchioles in the lungs. The structure is normal.

[0094] “++”: There was no significant enlargement of the alveoli in the lung tissue of mice, scattered red blood cell infiltration in the pulmonary interstitium, and small patches of inflammatory exudates in the interstitium; there were small patches of inflammatory exudates around the bronchioles, mainly lymphocytes, with a small number of eosinophils.

[0095] "+++": There are large patches of inflammatory exudates in the interstitial lungs of mice, mainly histiocytes, lymphocytes, and eosinophils, congestion in small blood vessels, degeneration of vascular membranes, displacement of intimal cells, and disordered arrangement. Inflammatory exudates around the bronchioles in the lungs.

[0096] "++++": The mice had extensive hemorrhage and inflammatory exudation in the lung interstitium, cell proliferation and hypertrophy, and a significant increase in lymphocytes and eosinophils. The small blood vessels became eosinophilic, the endothelial cells were displaced and arranged in disorder, the cell size was uneven, and the lesions were severe and diffuse. There was obvious inflammatory exudation around the bronchioles in the lungs, mainly lymphocytes.

[0097] 2.5 Statistical methods: One-way analysis of variance for comparison between groups.

[0098] 3. Results

[0099] 3.1 Effect of the composition on lung index of mice with human coronavirus pneumonia and cold-dampness toxin attacking the lungs

[0100] The results are shown in Table 1: After the mice were double stimulated by cold and damp environment and human coronavirus 229E infection, the lung index increased significantly, which was significantly different from the normal group (p < 0.01). After the mice were administered the drug, each dose group of the composition was able to significantly reduce the lung index of the mice, which was significantly different from the model group (p < 0.01), indicating that the composition has an obvious therapeutic effect on the mouse disease-syndrome combined model of human coronavirus pneumonia cold and dampness epidemic toxin attacking the lungs.

[0101] Table 1 Effect of the composition on lung index of mice with human coronavirus pneumonia and cold-dampness toxin attacking lung

[0102]

[0103] Note: Compared with the normal control group ## p<0.01; compared with the epidemic virus attack lung model group ** p<0.01.

[0104] 3.2 Effect of the composition on the lung viral load of mice with human coronavirus pneumonia and cold-dampness epidemic toxin attacking the lungs

[0105] The results in Table 2 show that there is almost no expression of viral nucleic acid in the lungs of mice in the normal group and the simple cold-dampness stimulation group, while the viral nucleic acid is highly expressed in the lung tissues of mice in the 229E infection group and the cold-dampness epidemic lung attack group. After administration, each dose group of the composition can reduce the viral load in the lung tissues of mice to varying degrees, and there is a significant difference compared with the model group (p < 0.01).

[0106] Table 2 Effect of the composition on the viral load of lung tissue in mice with human coronavirus pneumonia and cold-dampness epidemic toxin attacking the lung

[0107]

[0108] Note: Compared with the normal control group ## p<0.01; compared with the epidemic virus attack lung model group ** p<0.01.

[0109] 3.3 Effect of the composition on inflammatory factors in lung tissue of mice with human coronavirus pneumonia and cold-dampness toxin attack

[0110] The results in Table 3 show that after the mice were subjected to dual stimulation of cold and dampness + human coronavirus 229E, the levels of IL-6, IL-10, TNF-α, and IFN-γ in the body were significantly increased, which was significantly different from those in the normal group (p < 0.01). The inflammatory factors in each dose group of the combination were significantly reduced, which was significantly different from those in the model group (p < 0.01).

[0111] Table 3 Effects of the composition on inflammatory factors in the lungs of mice with human coronavirus pneumonia and cold-dampness toxin attacking the lungs

[0112]

[0113] Note: Compared with the normal control group ## p<0.01; compared with the epidemic virus attack lung model group ** p<0.01, * p<0.05.

[0114] 3.4 Effect of the combination on syndrome factors in the serum of mice with human coronavirus pneumonia and cold-dampness toxin attacking the lungs

[0115] The results in Table 4 show that the dual stimulation of cold and dampness and human coronavirus 229E significantly increased the motilin content and significantly decreased the gastrin content in mice. There was a significant difference compared with the normal group (p < 0.01). After administration to mice, each dose group of the composition was able to effectively improve the gastrointestinal reaction caused by cold and dampness toxin invasion, and effectively improve the disorder of motilin and gastrin, which was significantly different from the model group (p < 0.01).

[0116] Table 4 Effects of the combination on syndrome factors in the serum of mice with human coronavirus pneumonia and cold-dampness toxin attacking the lungs

[0117]

[0118] Note: Compared with the normal control group ## p<0.01; compared with the epidemic virus attack lung model group ** p<0.01.

[0119] 3.5 Effect of the composition on peripheral blood lymphocytes in mice with human coronavirus pneumonia and cold-dampness toxin attacking the lungs

[0120] The results in Table 5 show that the combination of human coronavirus cold-dampness epidemic toxin attacking the lung syndrome and mouse peripheral blood CD8 + The proportion of T cells was significantly reduced, with significant differences compared with the normal group (p < 0.01). After drug intervention, the composition 74.8 mg / kg group was able to significantly alleviate the reduction, with significant differences compared with the model group (p < 0.05, p < 0.01).

[0121] Table 5 Effects of the composition on peripheral blood lymphocytes in mice with human coronavirus pneumonia and cold-dampness toxin attacking the lungs

[0122]

[0123] Note: Compared with the normal control group ## p<0.01; compared with the epidemic virus attack lung model group * p<0.05.

[0124] 3.6 Effect of the composition on lung tissue and bronchial lesions in mice with human coronavirus infection and cold-dampness epidemic toxin attacking the lungs

[0125] The results in Table 6 show that: under pathological microscopic observation, the combination of human coronavirus cold-dampness epidemic toxin attacking the lung syndrome in mice showed large patches of inflammatory exudation in the lung interstitium, blood stasis in small blood vessels, vascular membrane degeneration, endothelial cell displacement, disordered arrangement, obvious inflammatory exudation around the bronchioles in the lungs, and the lesion grade was significantly different from that in the normal control group (p < 0.01). After administration to mice, each dose group of the composition was able to reduce the bronchial inflammatory exudation, the redness and swelling were relatively subsided, and the alveolar and pulmonary interstitial exudation inflammation and red blood cell exudation caused by viral infection were alleviated, and the blood stasis lesions were relatively alleviated, and the lesion grade was significantly different from that in the model group (p < 0.05, p < 0.01).

[0126] Table 6-1 Effects of the composition on the bronchi of mice with human coronavirus pneumonia and cold-dampness toxin attacking the lungs

[0127]

[0128] Table 6-2 Effect of the composition on lung tissue of mice with human coronavirus pneumonia and cold-dampness toxin attack

[0129]

Claims

1. A composition for treating and preventing the new coronavirus, wherein the active ingredients and their weight portions are: 55.7-223 parts of magnolol, 10.7-43 parts of pseudoephedrine, 5.2-20.6 parts of sennoside A, 4.3-17.2 parts of quercetin, 2.5-10.3 parts of benzoylpeonidin, 0.43-1.72 parts of isoliquiritigenin, and 1.3-5.2 parts of astragaloside IV.

2. The composition for treating and preventing the new coronavirus as claimed in claim 1, wherein the active ingredients and their weight portions are: 100-180 parts of magnolol, 20-35 parts of pseudoephedrine, 8.4-15.8 parts of sennoside A, 7.5-15.2 parts of quercetin, 4.5-9.6 parts of benzoylpeonidin, 0.56-1.35 parts of isoliquiritigenin, and 2.1-4.5 parts of astragaloside IV.

3. The composition for treating and preventing the new coronavirus as claimed in claim 2, wherein the active ingredients and their weight portions are: 100 parts of magnolol, 20 parts of pseudoephedrine, 8.4 parts of sennoside A, 7.5 parts of quercetin, 4.5 parts of benzoylpeonidin, 0.56 parts of isoliquiritigenin, and 2.1 parts of astragaloside IV.

4. The composition for treating and preventing the new coronavirus as claimed in claim 2, wherein the active ingredients and their weight portions are: 180 parts of magnolol, 35 parts of pseudoephedrine, 15.8 parts of sennoside A, 15.2 parts of quercetin, 9.6 parts of benzoylpeonidin, 1.35 parts of isoliquiritigenin, and 4.5 parts of astragaloside IV.

5. The composition for treating and preventing the new coronavirus as claimed in claim 2, wherein the active ingredients and their weight portions are: 111.5 parts of magnolol, 21.5 parts of pseudoephedrine, 10.3 parts of sennoside A, 8.6 parts of quercetin, 5.15 parts of benzoylpeonidin, 0.86 parts of isoliquiritigenin, and 2.6 parts of astragaloside IV.

6. A composition for treating and preventing the novel coronavirus as described in any one of claims 1 to 5, wherein the composition is prepared into a pharmaceutical dosage form of granules, tablets, capsules, oral liquid or injection.

7. A drug for treating and preventing the novel coronavirus, characterized in that: The composition for treating and preventing the novel coronavirus according to any one of claims 1 to 5 is prepared into a pharmaceutical preparation with a pharmaceutically acceptable carrier or excipient.

8. Use of the composition described in any one of claims 1 to 6 or the drug described in claim 7 in the preparation of a drug for treating or preventing the new coronavirus.

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