Use of baicalin in treating coronavirus infectious diseases

Abstract

The application discloses an application of baicalin in treating coronavirus infectious diseases. The new application of baicalin is verified through in-vitro cell tests and mouse tests infected with human coronavirus HCoV-229E, the technical scheme is novel, and a curative effect test of baicalin in treating pneumonia caused by HCoV-229E virus infection is carried out, and the results show that baicalin has a certain inhibitory effect on HCoV-229E infected cells in vitro; baicalin shows good drug efficacy in lung index and inhibition rate of pneumonia mice infected with HCoV-229E virus, can reduce the virus load of lung tissue of pneumonia mice infected with HCoV-229E virus, increase the proportion of peripheral blood lymphocytes of mice, and reduce the content of IL-2, IL-10, TNF-alpha and IFN-beta in lung tissue of mice.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical treatment, in particular to the application of baicalin in treating coronavirus infectious diseases. BACKGROUND

[0002] Coronaviruses are positive-sense single-stranded RNA viruses with an envelope. There are spines on the viral envelope, and the spines of different coronaviruses differ significantly, looking like a crown, hence the name "coronavirus". The virus particles are surrounded by a lipid membrane, and the membrane surface has three types of glycoproteins: spike glycoprotein (S, Spike Protein, which is the receptor binding site, cytolysis and main antigen site); envelope glycoprotein (E, Envelope Protein, which is a smaller protein combined with the envelope); and membrane glycoprotein (M, Membrane Protein, which is responsible for the transmembrane transport of nutrients, the budding release of new viruses and the formation of the viral envelope). A few species also have hemagglutinin glycoprotein (HE protein, Haemaglutinin-esterase). The nucleic acid of coronavirus is non-segmented single-stranded (+) RNA, which is 27-31 kd, the longest RNA nucleic acid chain among RNA viruses, and has important structural features unique to positive-strand RNA: a methylated "cap" at the 5' end of the RNA chain and a PolyA "tail" structure at the 3' end. This structure is very similar to eukaryotic mRNA, and is also an important structural basis for the RNA itself to function as a translation template, eliminating the RNA-DNA-RNA transcription process. The recombination rate between coronaviruses is very high, and the virus is prone to variation, resulting in changes in antigenicity, which leads to the failure of the original vaccine.

[0003] There are seven types of coronaviruses, four of which cause common cold symptoms, namely 229E, NL63, OC43, and HKU1, and the remaining two are the dreaded SARS (atypical pneumonia) and MERS-CoV (Middle East Respiratory Syndrome Coronavirus), which have been selected as one of the most terrible viruses in human history. On January 12, 2020, the World Health Organization officially named it "2019 Novel Coronavirus" (2019-nCoV), which is the 7th human coronavirus (HCoV) identified in human history.

[0004] In addition, the coronavirus genome sequence also has homology and can be divided into four genera, alpha, beta, gamma, and delta. The gamma and delta coronavirus genera only infect animals, the alpha coronavirus genus includes human coronaviruses 229E and NL63, and the beta coronavirus genus includes four families, HKU1 and OC43 belong to the A family; the B family includes SARS, which is associated with severe pneumonia; and MERS belongs to the beta coronavirus C family.

[0005] Human coronavirus HCoV-229E, an alpha group positive strand RNA virus coronavirus, is a pathogen causing upper respiratory tract infection in human, and often causes common cold in human. In addition to causing respiratory tract infection, it can also cause gastrointestinal diseases and nervous system symptoms, such as pneumonia. Studies have shown that when HCoV-229E infects the lungs, it releases pro-inflammatory cytokines, including IL-2, IL-10, TNF-α, IFN-β, causing pneumonia.

[0006] Therefore, it is urgent to develop a drug for preventing and treating human coronavirus. SUMMARY

[0007] Baicalin is a flavonoid compound extracted and separated from the dried roots of Scutellaria baicalensis Georgi of the dicotyledonous Labiatae family, and is a yellow needle-like crystal. It is generally stable under dilute acid conditions, such as not hydrolyzing in a 2% aqueous sulfuric acid solution, but can be hydrolyzed to form glucuronic acid and baicalein when the acid concentration is increased and the temperature is increased to 110°C.

[0008] Clinical studies have found that baicalin has significant biological activity, such as bacteriostatic, diuretic, anti-inflammatory, anti-allergic, spasmolytic, and strong anticancer responses, and other physiological effects. Baicalin can also absorb ultraviolet rays, scavenge oxygen free radicals, and inhibit the production of melanin. However, there is no report discussing that baicalin can treat human coronavirus HCoV-229E.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0010] According to one aspect of the present application, the application of baicalin in the treatment of coronavirus infectious diseases is provided, the coronavirus is human coronavirus HCoV-229E, and the coronavirus infectious disease is a disease caused by human coronavirus HCoV-229E infection.

[0011] Optionally, the disease caused by human coronavirus HCoV-229E infection includes pneumonia, nephritis, and hepatitis.

[0012] Optionally, the administration mode of the disease caused by human coronavirus HCoV-229E infection is selected as nebulization inhalation administration.

[0013] Optionally, the single dose of the nebulization inhalation administration is 13.6 mg / 60 kg-54.4 mg / 60 kg. The administration dose is calculated according to the mouse coefficient 11 and the human body weight 60 kg based on the high, medium, and low three doses of 2 ml of nebulization inhalation administration in Example 2.

[0014] Optionally, the formulation form of the aerosol inhalation administration is an inhalation solution, wherein the concentration of baicalin is 1.25 mg / ml-5 mg / ml.

[0015] Optionally, the disease caused by the human coronavirus HCoV-229E infection is constructed by infecting human embryo lung fibroblast MRC-5 cells in vitro or a BALB / c mouse model.

[0016] In the process of applying baicalin to human coronavirus HCoV-229E infected human embryo lung fibroblast MRC-5 cells in vitro, first, the toxicity test of baicalin on MRC-5 cells in vitro is carried out to determine the maximum non-toxic concentration of baicalin. Within the range of the maximum non-toxic concentration, the baicalin raw liquid is diluted by 2 times to 8 concentrations for testing, wherein the 8 concentrations are arranged in descending order as 2.5 mg / ml, 1.25 mg / ml, 0.625 mg / ml, 0.31 mg / ml, 0.16 mg / ml, 0.08 mg / ml, 0.04 mg / ml, and 0.02 mg / ml. The inhibitory effect of baicalin on human coronavirus HCoV-229E in vitro cultured MRC-5 cells is verified.

[0017] Optionally, the application of baicalin in a BALB / c mouse model infected with human coronavirus HCoV-229E.

[0018] In the application of baicalin in a BALB / c mouse model infected with human coronavirus HCoV-229E, the baicalin raw liquid is diluted by 2 times to 3 concentrations for testing, wherein the high concentration is 5 mg / ml, the medium concentration is 2.5 mg / ml, and the low concentration is 1.25 mg / ml. The mice are administered by atomization, and a mouse pneumonia model infected with human coronavirus HCoV-229E is used to detect the effectiveness of the test drug baicalin in terms of mouse lung index and inhibition rate, lung tissue viral load, lung tissue inflammatory factor content, and peripheral blood lymphocyte percentage.

[0019] The technical scheme of the present application verifies the new use of baicalin through in vitro cell tests infected with human coronavirus HCoV-229E, and mouse tests infected with human coronavirus HCoV-229E, and the technical scheme has novelty, and the efficacy test of baicalin in treating pneumonia caused by HCoV-229E virus infection is carried out, and the test results show that baicalin has certain inhibitory effect on human coronavirus HCoV-229E infected cells in vitro; can inhibit the expression of TNF alpha, IL 6 and other cytokines induced by virus HCoV-229E, and shows good drug efficacy on lung index and inhibition rate of pneumonia mice infected with HCoV-229E virus, can reduce the virus load of lung tissue of pneumonia mice infected with HCoV-229E virus, increase the proportion of peripheral blood lymphocytes of mice, and reduce the content of IL-2, IL-10, TNF-alpha and IFN-beta in lung tissue of mice. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The result column chart of lung index and inhibition rate of pneumonia mice infected with HCoV-229E virus treated by baicalin is shown in the following figure;

[0021] Figure 2 The result column chart of lung tissue virus load of pneumonia mice infected with HCoV-229E virus treated by baicalin is shown in the following figure;

[0022] Figure 3 The result column chart of peripheral blood CD4 T lymphocyte percentage of pneumonia mice infected with HCoV-229E virus treated by baicalin is shown in the following figure; +4

[0023] Figure 4 The result column chart of peripheral blood CD8 T lymphocyte percentage of pneumonia mice infected with HCoV-229E virus treated by baicalin is shown in the following figure; +8

[0024] Figure 5 The result column chart of peripheral blood B lymphocyte percentage of pneumonia mice infected with HCoV-229E virus treated by baicalin is shown in the following figure;

[0025] Figure 6 The result column chart of lung tissue inflammatory factor IL-2 of pneumonia mice infected with HCoV-229E virus treated by baicalin is shown in the following figure;

[0026] Figure 7 The result column chart of lung tissue inflammatory factor IL-10 of pneumonia mice infected with HCoV-229E virus treated by baicalin is shown in the following figure;

[0027] Figure 8 ​​A bar chart of the results of treating lung tissue inflammatory factor TNF-α of the mice with pneumonia infected with HCoV-229E virus with baicalin;

[0028] Figure 9 A bar chart of the results of treating lung tissue inflammatory factor IFN-β of the mice with pneumonia infected with HCoV-229E virus with baicalin. DETAILED DESCRIPTION

[0029] In order to make the objects, technical solutions and advantages of the present application clearer, the following will further describe the embodiments of the present application with reference to the drawings.

[0030] Example 1

[0031] An in vitro cultured MRC-5 cell model is infected with human coronavirus HCoV-229E, and the TC0, TC50, IC50 and TI of the drug are observed to evaluate the inhibitory effect of baicalin on human coronavirus HCoV-229E in vitro.

[0032] I. Test materials

[0033] 1.1. The cell strain is human embryonic lung fibroblast MRC-5, which is purchased from Beijing Beinaiceli Biotechnology Research Institute, subcultured in the laboratory, and stored in liquid nitrogen for standby.

[0034] 1.2. The virus strain is human coronavirus HCoV-229E, which is provided by the Institute of Medical Biotechnology of the Chinese Academy of Medical Sciences, subcultured in the laboratory, and stored in a-80℃ refrigerator for standby.

[0035] 1.3. The test drug is baicalin, with a concentration of 5mg / ml, a molecular weight of 446, and a solvent of phosphate buffered saline (PBS).

[0036] 1.4. The experimental reagents are shown in Table 1. Among them, DMEM is a culture medium containing various amino acids and glucose.

[0037] Table 1:

[0038] Reagent name Manufacturer Batch number DMEM medium Corning 14019014 PBS solution Corning 14819005 Trypsin Corning 110519010 Fetal bovine serum Corning 35081006 Secondary antibody Gibco 1796440

[0039] 1.5. The test instruments are shown in Table 2.

[0040] Table 2:

[0041] Instrument name Manufacturer Model number Electronic analytical balance Ohaus Corp. Brock. NJ. USA AR1140 CO2 incubator Thermo Thermo-371 Biological safety cabinet Thermo Thermo MSC1.2 Clean bench airtech SW-CJ-2FD Centrifuge eppendorf Centrifuge 5430 Full-automatic cell counter countstar IC 1000 Inverted microscope olympus CKX41

[0042] II. Test method

[0043] 2.1. Preparation of the original liquid

[0044] Weigh 500 mg of baicalin into a 50 ml volumetric flask. Dose to volume with pH 7 phosphate buffer solution and shake well to obtain a 10 mg / ml solution. To improve solubility, dilute the prepared solution 2-fold to a 5 mg / ml stock solution for pharmacological experiments.

[0045] The 5 mg / ml baicalin stock solution was diluted 2-fold to 2.5 mg / ml, 1.25 mg / ml, 0.625 mg / ml, 0.31 mg / ml, 0.16 mg / ml, 0.08 mg / ml, 0.04 mg / ml, and 0.02 mg / ml, for a total of 8 concentrations.

[0046] 2.2 Toxicity test of baicalin on MRC-5 cells cultured in vitro

[0047] The eight prepared dilutions of the drug were added to MRC-5 cell culture plates that had grown into a monolayer, 100 μL / well, with four replicates for each dilution. A normal cell control was also set up. The culture plates were cultured at 37°C in a 5% CO2 incubator. The cytopathic effects were observed daily under an inverted microscope for 48 hours. The lowest dilution at which no obvious cytopathic effects were observed (maximum non-toxic concentration TC0) was determined, and the 50% cytotoxic concentration (TC0) was calculated using the Reed-Muench method. 50 ), see Tables 3 and 4.

[0048] Table 3:

[0049]

[0050] Table 4:

[0051]

[0052] As shown in Tables 3 and 4, the maximum non-toxic concentration TC0 of baicalin is 0.078 mg / ml, and the concentrations used in the following experiments were all carried out below the TC0 concentration.

[0053] 2.3. The inhibitory effect of baicalin on human coronavirus HCoV-229E in in vitro cultured MRC-5 cells.

[0054] Take the culture plate that has grown into a monolayer of cells, pour out the culture medium, and inoculate the HCoV-229E virus solution, 100TCID 50, 100 μL / well, adsorbed for 1 h in a 37℃, 5% CO2 incubator, discarded the virus liquid, washed the cell surface with cell maintenance liquid for 3 times, then added 6 dilutions of each drug liquid below the non-toxic concentration, 100 μL / well, 3 duplicate wells for each dilution, set normal cell control and virus control at the same time, placed in a 37℃, 5% CO2 incubator, cultured, observed the cytopathic effect under an inverted microscope every day, recorded the test results when the virus control group cytopathic effect was +++ ~ ~ ~ + + + at 48-72 h.

[0055] Among them, the cytopathic effect was judged according to 6-level standard:

[0056] -: normal cell growth, no lesions appeared;

[0057] ±: less than 10% of the entire monolayer cell lesions;

[0058] +: cell lesions accounted for less than 25% of the entire monolayer;

[0059] ++: cell lesions accounted for less than 50% of the entire monolayer;

[0060] +++: cell lesions accounted for less than 75% of the entire monolayer;

[0061] ++++: cell lesions accounted for more than 75% of the entire monolayer.

[0062] The 50% inhibition concentration (IC 50 ) and the therapeutic index (TI) were calculated according to Reed-Muench, TI = TC 50 / IC 50 , the results are shown in Table 5, Table 6.

[0063] Table 5:

[0064]

[0065] Table 6:

[0066]

[0067] The results of Table 5, 6 show that baicalin has a certain degree of inhibition on the cytopathic effect of human coronavirus HCoV-229E on MRC-5 cells in vitro, can inhibit the expression of TNF α, IL 6 and other cytokines induced by virus HCoV-229E, and the strength of the effect is related to the concentration of baicalin, the greater the concentration of baicalin, the stronger the inhibitory effect, the IC 50 is 56 ug / ml.

[0068] III. Experimental conclusion

[0069] Baicalin has a certain degree of inhibitory effect on HCoV-229E infection induced MRC-5 cell pathogenesis in vitro at non-toxic concentration, and the strength of the effect is related to the concentration of baicalin. The greater the concentration of baicalin, the stronger the inhibitory effect, and the therapeutic index TI = 2.

[0070] Example 2

[0071] The human coronavirus HCoV-229E infected mouse pneumonia model was used to evaluate the therapeutic effect of baicalin on the human coronavirus HCoV-229E mouse pneumonia model from four aspects of lung index and inhibition rate, viral nucleic acid expression in lung tissue, inflammatory factor content in lung tissue, and percentage of immune cells in peripheral blood.

[0072] I. Experimental materials

[0073] 1.1. The positive drug is chloroquine phosphate tablets, purchased from Sichuan Shenghe Pharmaceutical Co., Ltd., batch number 2002114, specification 0.25g / tablet, oral, 0.5g / 60kg / day.

[0074] 1.2. The test drug is baicalin with a concentration of 5mg / ml, a transparent and clear liquid, administered by inhalation.

[0075] 1.3. The experimental animals are BALB / c mice, 51 males and 51 females respectively, SPF level, weight 11±1g, certificate numbers NO.1100112011015252(male), NO.1100112011015253(female), license number SCXK(Jing)2016-0006, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0076] 1.4. The virus strain is human coronavirus HCoV-229E, provided by the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, and subcultured in our laboratory, stored in a-80℃ refrigerator for standby use.

[0077] 1.5. The experimental reagents are shown in Table 7

[0078] Table 7:

[0079]

[0080] 1.6. The experimental instruments are shown in Table 8

[0081] Table 8:

[0082]

[0083] II. Experimental methods

[0084] 2.1. Drug preparation

[0085] Table 9: Preparation of chloroquine phosphate tablets

[0086]

[0087] Table 10: Preparation of test drugs

[0088]

[0089] 2.2, Virus passage

[0090] Take 25 cm 2 culture bottle of MRC-5 cells which have grown into monolayer, discard the culture solution, rinse the cell surface with cell maintenance solution for 3 times, then add 5 ml of cell maintenance solution, and add 200 μl of HCoV-229E virus solution, place in a 37℃, 5% CO2 incubator for culture for 72-96 h, observe the cytopathic effect (CPE) under inverted microscope every day until 80% of the cells show obvious CPE, then place the cell culture bottle in a -80℃ low temperature freezer for storage, and the virus solution is repeatedly frozen and thawed for 3 times before use for virus titer determination.

[0091] Take 96-well plates of MRC-5 cells which have grown into monolayer, discard the culture solution, rinse the cells with cell maintenance solution for 3 times, then dilute and inoculate HCoV-229E virus solution of different titers by 10 times of dilution (10 -1 ~ 10 -8 ) dilution, 100 μl / well, 4 replicate wells for each dilution, and set normal cell control at the same time. Place the 96-well plates in a 37℃, 5% CO2 incubator for culture for 72-96 h, observe the cytopathic effect under inverted microscope every day, and record the cytopathic effect of each well. Calculate the 50% cytopathic concentration (TCID 50 ) according to Reed-Muench method.

[0092] 2.3, Construction of mouse model and administration

[0093] BALB / c mice 102, according to the body weight was randomly divided into normal control group, model control group, chloroquine control group, baicalin high, medium and low dose group. Among them, high, medium and low dose were the original drug solution, 1 / 2 times the original drug solution, 1 / 4 times the original drug solution. Normal control group, model control group, chloroquine control group, 10 in each group, half male and half female; Baicalin high, medium and low dose group, 8 in each group, half male and half female. After grouping, except for the normal control group, other groups of mice were lightly anesthetized with ether, and were infected with 100 TCID50 HCoV-229E by nasal drops, 50 μl per mouse, once every other day, a total of 2 times. On the day of the first infection, the chloroquine control group was given baicalin by gavage at 0.09 g / kg / d, and each dose group of mice was given 2 ml of drug solution by aerosol inhalation every 20 min, the normal control group, model control group were given distilled water by gavage under the same conditions, once a day, for 4 consecutive days. One day after the last administration, the body weight was measured, the orbital blood was collected (anticoagulation), the lung was dissected and weighed, and the lung was stored at-80℃.

[0094] 2.4, lung index and inhibition rate of mice

[0095] After weighing the mice, the blood was taken, the lung tissue was dissected and weighed, and the lung index and inhibition rate of mice were calculated, the specific calculation formula was as follows:

[0096] Lung index = wet lung weight (g) x 100 / body weight (g)

[0097]

[0098] 2.5, nucleic acid detection in lung tissue of mice (real-time fluorescent quantitative RT-PCR method)

[0099] 2.5.1, nucleic acid lysis treatment

[0100] The mouse lung tissue stored at-80℃ was placed in a clean mortar, a small amount of liquid nitrogen was poured into the mortar, and the lung tissue was ground into powder. The powder was placed in a 1.5 ml centrifuge tube, 1 ml of TRIzol reagent was added, the tube bottom was shaken, and the lung tissue powder was resuspended. The centrifuge tube was placed horizontally at room temperature, and incubated for 20 min. At 4℃, 12000 rpm, centrifugation was performed for 10 min. The supernatant was transferred to a new 1.5 ml centrifuge tube, 0.2 ml of chloroform was added, and the mixture was shaken for 15 s. Incubation was performed at room temperature for 3 min to separate the liquid layers. At 4℃, 12000 rpm, centrifugation was performed for 15 min. The supernatant was transferred to a new 1.5 ml centrifuge tube, 0.5 ml of isopropanol was added, and the mixture was mixed. Incubation was performed at room temperature for 30 min. At 4℃, 12000 rpm, centrifugation was performed for 10 min. The supernatant was discarded, and the precipitate was washed with 75% ethanol 1 ml. At 4℃, 7500 rpm, centrifugation was performed for 5 min. The supernatant was aspirated, and the RNA precipitate was briefly dried for 5-10 min. The precipitate was dissolved with 20 μl of DEPC water, and stored in a-80℃ low temperature refrigerator.

[0101] 2.5.2, Nucleic acid assay

[0102] Control nucleic acid treatment: DEPC-H2O as negative control. Positive control was diluted by 10, 100, 1000 times. Reagent preparation: take n x 18 μl HCoV-229E nucleic acid fluorescent PCR detection mixture, n x 1 μl internal control, n x 1 μl RT-PCR enzyme (n is the number of reaction tubes), shake well for several seconds, centrifuge at 3000 rpm for several seconds. Sample loading: take 20 μl of the above mixture and put it into a PCR tube, then add 5 μl of sample nucleic acid extraction solution, DEPC-H2O, and positive control into the PCR tube respectively, improve the tube cap, centrifuge for several seconds to make all the liquid at the bottom, and then immediately perform PCR amplification reaction. PCR amplification: reaction tube is placed on a quantitative fluorescent PCR instrument, and the cycle parameters are set as follows: 45°C x 10 min; 95°C x 15 min; then 95°C x 15 sec→60°C x 60 sec, cycle 40 times; single point fluorescence detection at 60°C, and the reaction system is 25 μl.

[0103] 2.5.3, Nucleic acid detection calculation method, see Table 11.

[0104] Table 11:

[0105] Ct value Result judgment UNDET or 40 Negative ≦38 Positive 38～40 Review once, if still 38-40, then negative

[0106] 2.6, Detection of inflammatory factors in mouse lung tissue (Elisa method)

[0107] 2.6.1, Sample collection and storage

[0108] Tissue homogenate sample: weigh 50 mg of lung tissue, add 500 μl of physiological saline, and then use an ultrasonic cell crusher to homogenize the tissue. Use a low-temperature high-speed centrifuge to centrifuge at 4°C, 10000 rpm for 10 minutes. After aspirating the supernatant, store it in a -80°C refrigerator for storage, and avoid repeated freezing and thawing.

[0109] 2.6.2, Sample detection

[0110] Sample preparation: the sample was diluted 2 times with diluent before detection. Detection: the microwell plate was taken out from the sealed bag which had been equilibrated to room temperature, and different concentrations of standard, sample or quality control were added into the corresponding wells, 100 μl per well. The reaction wells were sealed with sealing glue paper, and incubated at room temperature for 2 hours. The plate was washed with washing solution, and the operation was repeated 4 times. After the last washing, the plate was inverted and all residual liquid was wiped off with absorbent paper; 100 μl of enzyme-labeled detection antibody was added into each microwell. The reaction wells were sealed with sealing glue paper, and incubated at room temperature for 2 hours. The plate washing operation of step 4 was repeated, and 100 μl of color developing substrate was added into each microwell, and incubated at room temperature for 30 minutes. Note that the operation should be carried out in the dark. Within 30 minutes after 100 μl of termination solution was added into each microwell, the absorbance value at 450 nm was measured by using an enzyme-labeled instrument, and the result was calculated.

[0111] 2.7. Detection of the proportion of peripheral blood lymphocytes in mice (flow cytometry)

[0112] After the centrifuge was pre-cooled at 4°C, the mouse was taken out of the eye ball to collect blood, 3 drops of blood (about 150 μl) were added into a 15 ml centrifuge tube containing 10 ml of 1xPBS, and centrifuged at 1600 rpm for 5 min at room temperature; the supernatant was carefully removed by using a pipette, 1 ml of red blood cell lysis solution was added into each tube to resuspend the cell precipitate, and the lysis was carried out at room temperature for about 5-10 min until the liquid changed from turbid to clear, 10 ml of PBS was added to terminate the lysis, and centrifuged at 2000 rpm for 5 min at 4°C, and the supernatant was discarded (if there were still a lot of red blood cells, the step 4 could be repeated). The cell precipitate was resuspended with 10 ml of PBS, centrifuged at 2000 rpm for 5 min at 4°C, the supernatant was discarded, and the cell suspension was resuspended with 200 μl of blocking solution (5% FBS in PBS), and the cell suspension was transferred into a 1.5 ml ep tube, and blocked at 4°C for 30 min. The flow cytometry antibodies were prepared in the blocking solution in the dark as follows: FITC-labeled anti-mouse CD3e, PE-labeled anti-mouse CD19, PerCP-Cy5.5-labeled anti-mouse CD4, and APC-labeled anti-mouse CD8a, and the preparation volume for each tube of cells was as follows: 0.3 μl of each antibody, and 50 μl of blocking solution.

[0113] The cell suspension was centrifuged at 2000 rpm for 5 min at 4°C, and the supernatant was discarded. The flow cytometry antibodies were added into each tube, 50 μl per tube, and the staining was carried out in the dark at 4°C for 30 min, 1 ml of PBS was added, and centrifuged at 2000 rpm for 5 min at 4°C, and the supernatant was discarded. The cells were resuspended with 200 μl of PBS containing 2% FBS, and transferred into a flow cytometry tube for detection (if the detection could not be carried out in time, the 4% paraformaldehyde fixing solution could be diluted to 2% with PBS, the cells were resuspended, and the volume of each tube was 200 μl, and the cells were stored in the dark at 4°C overnight).

[0114] 2.8. Statistical analysis

[0115] Data were expressed as (MEAN ± SD), and the differences between groups were analyzed by T test. P < 0.05 was considered statistically significant.

[0116] III. Results

[0117] 3.1, Effects of Baicalin on lung index and inhibition rate of pneumonia mice infected with HCoV-229E virus

[0118] Table 12:

[0119]

[0120] Note: Compared with the normal control group, P < 0.01; compared with the model control group, P < 0.01

[0121] From Table 12 and Figure 1 , it can be seen that compared with the normal control group, the lung index of the model control group was significantly increased (P < 0.01); compared with the model control group, the lung index of the Baicalin high, medium and low dose groups was significantly reduced (P < 0.01), and the inhibition rates of the Baicalin high, medium and low dose groups were 73.61%, 44.32% and 56.17%, respectively. It can be seen that the Baicalin high, medium and low dose groups all showed good efficacy.

[0122] 3.2, Effects of Baicalin on lung tissue viral load of pneumonia mice infected with HCoV-229E virus

[0123] Table 13:

[0124]

[0125] Note: Compared with the normal control group, P < 0.01; compared with the model group, P < 0.01

[0126] From Table 13, Figure 2 , it can be seen that compared with the normal control group, the lung tissue viral load of the model control group was significantly increased (P < 0.01); compared with the model control group, the lung tissue viral load of the Baicalin high and medium dose groups was significantly reduced (P < 0.01).

[0127] 3.3, Effects of Baicalin on peripheral blood lymphocyte proportion of pneumonia mice infected with HCoV-229E virus

[0128] Table 14:

[0129]

[0130] Note: Compared with the normal control group, P < 0.01; compared with the model control group, P < 0.05

[0131] From Table 14, Figure 3 ,Figure 4 、 Figure 5 Results show that: compared with the normal control group, the model control group mice peripheral blood CD 4+ T lymphocytes, CD 8+ T lymphocyte percentage significantly decreased (P<0.01) ; compared with the model control group, baicalin high dose group mice peripheral blood CD 4+ T lymphocytes, CD 8+ T lymphocyte percentage significantly increased (P<0.05).

[0132] 3.4, the influence of baicalin on the inflammatory factors of HCoV-229E virus infected mice with pneumonia

[0133] Table 15:

[0134]

[0135] Note: compared with the normal control group P<0.05, P<0.01; compared with the model control group P<0.05, P<0.01

[0136] From table 15, Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 Results show that: compared with the normal control group, the model control group mice lung tissue IL-2, IL-10, TNF-α, IFN-β content significantly increased (P<0.05, P<0.01) ; compared with the model control group, baicalin high, medium and low dose group mice lung tissue IL-2, IL-10, TNF-α content significantly reduced (P<0.05, P<0.01), baicalin medium dose group mice lung tissue IFN-β content significantly reduced (P<0.01).

Claims

1. The use of baicalin in the preparation of a drug for treating a coronavirus infectious disease, characterized in that, The coronavirus is human coronavirus HCoV-229E, and the coronavirus infectious disease is a disease caused by human coronavirus HCoV-229E infection; The disease caused by human coronavirus HCoV-229E infection is constructed by infecting human embryo lung fibroblast MRC-5 cultured in vitro or a BALB / c mouse model with HCoV-229E virus; The disease caused by human coronavirus HCoV-229E infection includes pneumonia; The administration mode of the disease caused by human coronavirus HCoV-229E infection is selected from aerosol inhalation administration.

2. Use according to claim 1, wherein The single dose of the aerosol inhalation administration is 13.6 mg / 60 kg-54.4 mg / 60 kg.

3. Use according to claim 2, wherein the compound is ###0002### The preparation form of the aerosol inhalation administration is an inhalation solution, wherein the concentration of baicalin is 1.25 mg / ml-5 mg / ml.

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