CN111297969A

CN111297969A - Application of Chaihingzhi preparation in preparing anti-coronavirus medicine and its preparation method

Info

Publication number: CN111297969A
Application number: CN202010215794.9A
Authority: CN
Inventors: 张贵民; 张子成; 关永霞; 程国良
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2020-03-25
Filing date: 2020-03-25
Publication date: 2020-06-19
Also published as: WO2021189743A1

Abstract

The invention discloses an application of a Chaihingzhiji in preparing an anti-coronavirus medicament and a preparation method thereof, belonging to the field of medicines. The Chaiyin preparation is prepared from eleven raw material medicinal materials of radix bupleuri, honeysuckle, scutellaria baicalensis, radix puerariae, schizonepeta, sweet wormwood herb, fructus forsythiae, platycodon grandiflorum, bitter apricot seed, mint and houttuynia cordata, and has the effects of clearing heat, relieving exterior syndrome, ventilating and smoothing lung, discharging heat, reducing phlegm, relieving sore throat and relieving cough. The medicine can obviously reduce the lung index of the lung disease caused by the fact that the human coronavirus pneumonia virus attacks the lung, obviously increase the percentages of CD4+ T lymphocytes, CD8+ T lymphocytes and total B lymphocytes in the peripheral blood of mice caused by the fact that the human coronavirus pneumonia virus attacks the lung, and has obvious treatment effect on the coronaviruses such as the human coronavirus through experiments.

Description

Application of Chaihingzhi preparation in preparing anti-coronavirus medicine and its preparation method

Technical Field

The invention relates to application of a Chaihin preparation and a preparation method thereof, in particular to application of a Chaihin preparation in preparation of anti-human coronavirus medicines and a preparation method thereof, and belongs to the field of medicines.

Background

Seven human coronaviruses (human coronaviruses, HCoV) have been found to cause human respiratory infections and other systemic organ damage, including HCoV-229E and HCoV-NL63 of α, HCoV-OC43 and HCoV-HKU1 of β A lineage, SARS-CoV and MERS-CoV of β C lineage of β B lineage, and COVID-19 (a 2019 novel coronavirus) which is abusing worldwide, HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoV-HKU1 all cause upper respiratory infections, wherein HCoV-229E and HCoV-OC43 are earlier found human coronaviruses which may cause upper respiratory infections of people of various ages, but also may cause lower respiratory infections including pneumonia in immunodeficient patients, and cause severe respiratory infections of CoVID-19 and worldwide deaths of CoVID, and severe respiratory infections of CoVID-19 have caused widespread consequences for human respiratory infections.

The outbreak of SARS, MERS and COVID-19 suggests that our coronavirus threat to human health is severe and persistent. Because the high occurrence of human coronavirus gene mutation greatly increases the risk of generating superstrong pathogenic virus and infecting human or generating mutation favorable for interpersonal transmission, it is necessary to monitor and research the variation or prevalence of human coronavirus and animal-carried coronavirus, and develop and discover more anti-human coronavirus drugs to better cope with the possible future prevalence of new human coronavirus.

According to TCM, the etiology of pneumonia caused by coronavirus is the pestilence qi which is the basic attribute of dampness, and from the onset season and nature of disease, it belongs to the pestilence category which is mainly pathogenic dampness, and it is called as "damp-toxin epidemic" and "cold-dampness syndrome". The coronavirus pneumonia is fundamentally caused by epidemic pathogens, but is characterized by dampness as an important characteristic, and from the development and evolution processes, the general pathogenesis of the coronavirus pneumonia can be summarized as dampness, toxicity, stasis and deficiency, the disease is located in the lung, and the course of the disease is lingering, so that the treatment needs to be spread around the dampness. The lung is the delicate organ, and it is advisable to remove dampness in the early stage of human coronavirus infection to prevent damp pathogen from being stagnated and then transforming into heat, which enters into yangming, and obstruction of fu-organs will aggravate the stagnation of lung qi. Since yangming pertains to the stomach and intestine, the lung and large intestine are exterior and interior, and lung qi is not dispersed and fu-organs are not lowered to form a vicious circle. The excess of yangming fu-organs, pathogenic qi adhering to it, damp-heat transforming into toxin, stagnant blood blocking the lung collaterals and pericardium, resulting in cough and dyspnea and hemoptysis. Without active treatment, it is easy to damp toxin, stasis and block qi movement, block qi movement and clear orifices, and the qi movement can not reach and enter into shock state with deep heat, resulting in Multiple Organ Dysfunction Syndrome (MODS).

The Chaihingzhiji oral liquid, Chaihingling granule and other oral medicinal preparation are prepared with ten kinds of Chinese medicinal materials including bupleurum root, honeysuckle, skullcap root, kudzu vine root, schizonepeta, sweet wormwood, forsythia, balloonflower root, bitter apricot seed, mint and cordate houttuynia, and have the functions of clearing away heat, relieving exterior syndrome, dispersing lung qi, clearing away heat, eliminating phlegm, relieving sore throat and stopping cough, and may be used in treating wind-heat exterior syndrome of pathogenic wind attacking lung and defending lung, such as fever, aversion to cold, headache, sore throat, red tongue edge and floating pulse. In the prescription, the bupleurum root is used as a monarch drug for relieving the heat retention and dispersing the dispersing function of the lung and dispersing the exterior heat; honeysuckle, scutellaria, forsythia, kudzu root, sweet wormwood and schizonepeta, and the six medicines have the effects of clearing heat, relieving exterior syndrome, ventilating lung, discharging heat, ventilating muscle and relieving fever and are used as ministerial medicines; mint, houttuynia cordata and bitter apricot kernel, which are used as adjuvant drugs for relieving sore throat, quenching thirst, clearing lung-heat, eliminating heat, reducing phlegm and relieving cough; the root of balloonflower is specialized in entering lung meridian, dispersing lung qi and resolving phlegm, relieving sore throat and stopping cough, and all the herbs in the carrier formula directly enter lung system to reach the effect of dispersing pathogenic factors and act as guiding drugs. The whole formula takes the pungent and cool dispersing powder as the main component and is matched with the medicinal flavors of sweet cold, bitter cold and pungent warm, thereby not only enhancing the effect of dispersing and clearing, but also having no disadvantage of bitter cold over-lingering the evil into the interior; the combination of pungent-cool, bitter-cold and pungent-warm herbs in the recipe strengthens the actions of ventilating the lung, dispersing pathogenic factors and relieving exterior syndrome, and can correct the bias of bitter-cold, sweet-cold and over-cold without the disadvantage of warm-dryness.

Tests show that the Bupleurum-silver preparation is an effective formula for resisting human coronavirus, and the symptoms of the lung coronavirus are obviously improved after the Bupleurum-silver particles are used for treating the positive lung coronavirus nucleic acid detection, the inflammatory reaction index is obviously increased, the lung tissues are obviously changed in pathology and the lung diseases are attacked by epidemic toxin. Has the characteristics of obvious effect, exact effect and low toxic and side effect, and has wide market popularization and application prospect.

Disclosure of Invention

The invention aims to provide an application of a Chaihingye preparation in preparing an anti-coronavirus medicine. The traditional Chinese medicine composition related to the patent is patented at 22.06.2005 (No. CN1207033C), and a series of Chaiyin preparations including Chaiyin granules, Chaiyin oral liquid and the like which are on the market are prepared by relying on the patent technology, and the preparation products are prepared from radix bupleuri, honeysuckle, scutellaria baicalensis, radix puerariae, herba schizonepetae, sweet wormwood herb, fructus forsythiae, platycodon grandiflorum, bitter almond, mint and herba houttuyniae.

The application of the Chaihin preparation in preparing the anti-coronavirus medicine is that the Chaihin preparation is prepared by the following traditional Chinese medicine components:

more preferably:

more preferably:

preferably, the coronavirus is a human coronavirus.

Preferably, the human coronavirus includes, but is not limited to, any one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and CoVID-19.

Further preferably, the application of the Chaihingzhiji in preparing anti-HcoV-229E medicines.

The traditional Chinese medicine raw material is mainly pungent and cool in nature and is matched with medicinal flavors of sweet, cold, bitter and warm, so that the effect of dispersing and clearing is enhanced, and the disadvantage that the bitter and cold are too lingering over to the interior is avoided; the combination of pungent-cool, bitter-cold and pungent-warm herbs in the recipe strengthens the actions of ventilating the lung, dispersing pathogenic factors and relieving exterior syndrome, and can correct the bias of bitter-cold, sweet-cold and over-cold without the disadvantage of warm-dryness. The medicines are used together to clear heat and relieve exterior syndrome, ventilate lung and release heat, resolve phlegm and relieve sore throat and relieve cough, and are used for treating fever and chills, headache and cough, sore throat, red tongue tip and red tongue and floating and rapid pulse of wind-heat exterior syndrome caused by pathogenic factors attacking lung and defensive qi due to wind-heat invasion.

Test results show that the lung index of the model mouse is obviously reduced in the high and low dose groups of the Bupleurum and argentum granules, and the lung index inhibition rates of the high and low dose groups are 61.02% and 55.45% respectively; the high and low dose groups of the Chaihin granules obviously increase the percentage of peripheral blood CD4+ T lymphocytes, CD8+ T lymphocytes and total B lymphocytes of the model mice, and have definite effect of resisting human coronavirus.

The invention also aims to provide a preparation method of the Chaihei preparation, which is characterized in that the traditional Chinese medicine is taken as a raw material, different auxiliary materials are added, and the medicine can be prepared into different oral medicinal preparations.

Preferably, the oral pharmaceutical preparation is granules, capsules, tablets, oral liquid, pills, pellets and microcapsules.

The preparation method of the Chailian oral pharmaceutical preparation comprises the following steps:

A. crushing proper amount of bitter almond into coarse particles, soaking seven kinds of materials including radix bupleuri, honeysuckle, sweet wormwood, forsythia, schizonepeta, mint, houttuynia and the like in water for 1 hour, distilling and extracting for 6-8 hours, collecting volatile oil, and collecting distillation residues and liquid medicine for later use;

B. taking the volatile oil in the step A, and adding cyclodextrin to prepare an inclusion compound for later use;

C. decocting the residue in the step A, the scutellaria baicalensis, the radix puerariae and the platycodon grandiflorum in water for 3 times, and each time for 2 hours, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.11-1.14(50 ℃), adding an alkalized ethanol solution to ensure that the alcohol content reaches 50% -70%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an alcohol precipitation solution I and an alcohol precipitation precipitate;

D. dissolving the alcohol precipitation with 50-70% ethanol, refrigerating, and filtering to obtain alcohol precipitation solution II; mixing the ethanol precipitation solutions I and II to obtain a total ethanol precipitation solution;

E. and D, taking the total alcohol precipitation liquid in the step D, concentrating under reduced pressure and recovering ethanol to obtain thick paste, and preparing the thick paste into an oral medicinal preparation directly through a conventional process or adding a pharmaceutically acceptable excipient.

Preferably, the alkalized ethanol solution added in step C is a 70% -95% ethanol solution with pH adjusted to 8-13 with 10% -30% sodium hydroxide solution.

Further preferably, the alkalized ethanol solution added in step C is an 80% ethanol solution adjusted to pH 9 with 20% sodium hydroxide solution.

The preferable preparation formulation of the invention is granules, and the preparation method of the bupleurum-silver granules comprises the following steps:

A. crushing proper amount of bitter almond into coarse particles, soaking seven kinds of materials including radix bupleuri, honeysuckle, sweet wormwood, forsythia, schizonepeta, mint, houttuynia and the like in water for 1 hour, distilling and extracting for 6 hours, collecting volatile oil, and reserving distilled dregs and liquid medicine for later use;

B. adding gamma-cyclodextrin into the volatile oil obtained in the step A to prepare an inclusion compound for later use;

C. decocting the residue in the step A, the scutellaria baicalensis, the radix puerariae and the platycodon grandiflorum in water for 3 times, and each time for 2 hours, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to an extract with the relative density of 1.13 at 50 ℃, adding an ethanol solution with the pH of 9 to ensure that the ethanol content reaches 80%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an ethanol precipitation solution I and an ethanol precipitation precipitate;

D. dissolving the precipitate with 60% ethanol, refrigerating, and filtering to obtain ethanol precipitation solution II; mixing the ethanol precipitation solutions I and II to obtain a total ethanol precipitation solution;

E. and D, taking the total alcohol precipitation liquid in the step D, concentrating under reduced pressure, recovering ethanol to obtain thick paste, and preparing the thick paste into granules directly through the conventional procedures or adding pharmaceutically acceptable excipients.

Another preferred dosage form of the present invention is a pellet formulation, and the preparation method of the pellet formulation comprises the following steps:

A. crushing bitter almond into coarse particles, soaking seven kinds of materials including radix bupleuri, honeysuckle, sweet wormwood, forsythia, schizonepeta, mint, houttuynia and the like in water for 1 hour, distilling and extracting for 6 hours, collecting volatile oil, and collecting distillation residues and liquid medicine for later use;

B. adding β -cyclodextrin into the volatile oil obtained in the step A to prepare an inclusion compound;

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, 2 hours each time, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.13(50 ℃), adding 80% ethanol with the pH value being adjusted to 9 by 20% sodium hydroxide solution to ensure that the fermentation content reaches 60%, uniformly stirring, refrigerating for 24 hours, filtering, and obtaining an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

E. d, collecting the total alcohol precipitation solution in the step D, concentrating under reduced pressure, recovering ethanol to obtain thick paste, belt-type vacuum drying at the vacuum degree of-0.08 MPa to-0.10 MPa and the drying temperature of 90 ℃, and crushing into fine powder;

F. mixing the fine powder obtained in step E and the volatile oil inclusion compound obtained in step B to obtain fine powder of the Bupleurum and silver extract for later use;

G. respectively weighing the fine powder of the firewood and silver extract, microcrystalline cellulose, cellulose acetate, superfine silica gel powder and lactose in the step F according to the formula amount, fully and uniformly mixing, adding 48 wt% of ethanol solution with the concentration of 35% of the formula amount, kneading to prepare soft materials, and extruding the soft materials into strips through a sieve plate with the aperture of 1.1mm of an extruder;

H. opening the spheronizer, selecting the rotation speed of 1250rpm, placing the strip-shaped objects in the step G into the spheronizer, spheronizing for 5.5min to prepare a semi-finished pellet, taking out the semi-finished pellet, drying at 85 ℃, and screening to obtain the pellet with 20-30 meshes.

In order to verify the effect of the inventive Chaihin preparation against human coronavirus, the inventors developed corresponding animal test studies. It should be noted that the animal tests are selected from the representative formulations and preparation methods of the present invention, and the other formulations and preparation methods of the present invention relate to the tests and results thereof, which are not exhaustive due to space limitations.

Test example 1 therapeutic Effect of drugs on coronavirus pneumonia and epidemic virus infected Lung-infected mouse disease binding model

In order to research the treatment effect of the Chailian preparation on the combined model of the disease of the mice infected with the coronavirus pneumonia and epidemic viruses, the inventor carries out animal experiment research, 48 mice are tested in total, and the number of the mice is half that of the mice, and the animal experiment research method and the result are as follows.

1 test Material

1.1 test drugs

1.2 Experimental animals

1.3 strains and cells

1.3.1 viral strains: human coronavirus 229E (HCoV-229E), provided by institute of medical and biotechnology, academy of Chinese medical sciences, was passaged in the laboratory and stored in a refrigerator at-80 ℃ for future use.

1.3.2 cell lines: human lung cancer cell A549, purchased from Beijing Beinan Chuanglian union of Industrial science, laboratory passage, and stored in liquid nitrogen for later use.

1.4 test reagents

1.5 test apparatus

2 test method

2.1 dosage design and drug formulation

2.1.1 dose design

Firewood and silver particles: the clinical dosage of human is 24g/60kg/d, and the dosage is converted into the dosage of mouse is 4.4 g/kg/d; the mouse dosage is set to be 8.8g/kg/d and 4.4g/kg/d during the test, namely the dosage is respectively equal to 2 times and equal times of the clinical dosage.

2.1.2 pharmaceutical formulation (in vivo test)

2.2 passage of Virus

Taking 25cm of A549, MRC-5 or MDCK cells which have grown into a monolayer²And (3) pouring out the culture solution from the culture bottle, flushing the cell surface for 3 times by using a cell maintenance solution, adding 200 mu l of HCoV-229E virus solution, culturing in a 37 ℃ 5% CO2 culture box, observing the pathological change condition of the cells under an inverted microscope every day for 72-96h until 80% of the cells have obvious pathological Change (CPE), placing the cell culture bottle in a-80 ℃ low-temperature refrigerator for freezing and storing, and repeatedly freezing and thawing the virus solution for 3 times for detecting the virus virulence.

2.3 Virus titre assay

Collecting culture plate with single layer of A549, MRC-5 or MDCK cells, pouring out culture solution, washing cell surface with cell maintenance solution for 3 times, diluting by 10 times, inoculating HCoV-229E virus solution with different titer, and 10 times diluting^-1-10^-8A total of 8 dilutions, 100. mu.l/well, 4 replicates per concentration, were included along with normal cell controls. Placing at 37 ℃ with 5% CO₂Culturing in an incubator, observing cytopathic condition under an inverted microscope every day, and recording the cytopathic condition of each well for 72-96 h. 50% cytopathic concentration (TCID) calculated as Reed-Muench₅₀)

2.4 modeling and administration of BALB/c mouse human coronavirus pneumonia and epidemic virus attacking lung syndrome

BALB/c mice 48, male and female, randomly divided into 2 batches, and normally raised 16 mice (male and female), and other 32 mice were modeled for cold-dampness syndrome, and the method was: the temperature is 4 +/-2 ℃, the relative humidity is 90% +/-3%, 4h every day, and 7d continuously. On the 5 th day of model building, 32 mice of the model of cold-dampness syndrome are randomly divided into a cold-dampness control group, a cold-dampness infection group (model group), a high-dose group and a low-dose group of the Chaihuang granules according to sex and weight, wherein each group comprises 8 mice and each half of male and female; the 16 normally bred mice are randomly divided into a normal control group and an infected control group according to sex and weight, each group comprises 8 mice, and the sex is half of the male and the female. After grouping, the mice in the control group, the cold-dampness infection group (model group) and the high and low dose group of the Chaihin granules are lightly anesthetized by ether and 100TCID₅₀HCoV-229E was administered in nasal drops 1 time daily for 2 days. On the first infection day, the high and low dose groups of radix bupleuri and argentum granules are administered by intragastric administration at 8.8 and 2.2g/kg/d, respectively, and the normal control group, infection control group, cold-dampness control group, and cold-dampness infection group(s) ((Model group) were each gazed with 20ml/kg/d distilled water 1 time a day for 3 consecutive days. Day of last administration 18: 00 per group basis 100g feed, 200ml water, 8 on day 2: 00 weighing the mass of each group of residual feed, measuring the volume of the residual drinking water of each group, and calculating the average food intake and the average water intake; weighing, collecting blood (anticoagulation or non-anticoagulation) from orbit, dissecting and weighing lung, and storing at-80 deg.C.

2.5 detection of nucleic acids in Lung tissues (real-time fluorescent quantitative RT-PCR method)

2.5.1 nucleic acid cleavage treatment

After the mouse is dissected, the lung tissue is subpackaged and stored in a low-temperature refrigerator at minus 80 ℃; the mouse lung tissue was removed from a-80 ℃ cryo-refrigerator, placed in a clean mortar, poured with a small amount of liquid nitrogen and ground to a powder using a pestle, the powder was collected in a 1.5ml centrifuge tube and immediately added to 1ml trizol Reagent, flicked off the bottom of the tube, and the sample was mixed as soon as possible to resuspension; placing the centrifuge tube at room temperature, and incubating for 20 min; centrifuging at 12000rpm at 4 deg.C for 10 min; transferring the clarified supernatant into a new 1.5ml centrifuge tube; adding 0.2ml chloroform, covering the tube cap tightly, shaking the centrifuge tube for 15s, and incubating at room temperature for 2-3min until liquid is layered; centrifuging at 12000rpm at 4 deg.C for 15 min; carefully transferring the transparent supernatant into a new 1.5ml centrifuge tube, adding 0.5ml isopropanol, mixing uniformly, and incubating at room temperature for 30 min; centrifuging at 12000rpm at 4 deg.C for 10 min; the supernatant was discarded and the pellet was washed gently with 1ml of 75% ethanol (the white pellet was floated gently); centrifuging at 7500rpm at 4 deg.C for 5 min; sucking up the supernatant, and drying the RNA precipitate for 5-10 min; dissolving the precipitate with 20 μ l DEPC water, and storing at-80 deg.C in refrigerator.

2.5.2 nucleic acid assays

And (3) treating the nucleic acid of the reference substance: DEPC-H₂O as a negative control. The positive control was diluted 10, 100, 1000 fold gradient.

Preparing a reagent: taking n x 18. mu.l HCoV-229E nucleic acid fluorescence PCR detection mixed solution, n x 1. mu.l internal control, and n x 1. mu.l RT-PCR enzyme (n is the number of reaction tubes), shaking and mixing for several seconds, and centrifuging at 3000rpm for several seconds.

Sample adding: placing 20 μ l of the above mixture in a PCR tube, and mixing with the nucleic acid extractive solution and DEPC-H₂O, positive control each 5. mu.lAdd to the PCR tube, modify the tube cap, centrifuge for several seconds to allow all the liquid to settle to the bottom, and immediately perform the PCR amplification reaction.

And (3) PCR amplification: the reaction tube is arranged on a quantitative fluorescent PCR instrument, and the circulation parameters are set as follows: multiplying at 45 ℃ for 10 min; multiplying at 95 ℃ for 15 min; circulating for 40 times according to 95 ℃ multiplied by 15sec → 60 ℃ multiplied by 60 sec; single-point fluorescence detection was performed at 60 ℃ in a 25. mu.l reaction system.

Fluorescence channel detection selection: FAM and HEX/VIC/JOE channels are selected.

Remarking: using ABI series PCR instrument, please choose "none" at both passive reference and query.

2.5.3 calculation method

Numbering	Channel	Ct value	Result judgment
				1	FAM	UNDET or 40	The sample is below the limit of detection and is reported as negative
2	FAM	≦38	Report as positive
				3	FAM	38～40	Rechecking once, if the recheck is still 38-40, the report is negative

2.6 detection of inflammatory factors in mouse Lung tissues (Elisa method)

2.6.1 sample Collection and storage

Tissue homogenate sample: after weighing lung tissues, the mice were collected from each group of mice No. 3, 4, 5, 6, 7, 8 and stored at-4 ℃. After 50mg of lung tissue was weighed and 500. mu.L of physiological saline was added, the tissue was homogenized using an ultrasonic cell disruptor and centrifuged at 1000x g ℃ for 10 minutes at a low temperature and high speed. Sucking supernatant, packaging, and storing in-80 deg.C refrigerator. Repeated freeze thawing is avoided.

2.6.2 sample testing

Sample preparation work: the sample was diluted 2-fold with diluent, i.e., 50. mu.L serum + 50. mu.L diluent.

And (3) detection: and (3) taking out the microporous plate from the sealed bag which is balanced to room temperature, and adding the standard substance, the experimental sample or the quality control substance with different concentrations into corresponding holes with the volume of 100 mu L per hole respectively. The reaction wells were sealed with a sealing plate of gummed paper and incubated at room temperature for 2 hours. The plate was washed with the washing solution and the operation was repeated 4 times. After the last washing, inverting the plate, and patting all residual liquid on absorbent paper; add 100. mu.L of enzyme-labeled detection antibody to each well. The reaction wells were sealed with a sealing plate of gummed paper and incubated at room temperature for 2 hours. The plate washing procedure of step 4 was repeated, and 100. mu.L of the chromogenic substrate was added to each well and incubated at room temperature for 30 minutes. Care was taken to avoid light. Absorbance at 450nm was measured using a microplate reader within 30 minutes after adding 100 μ L of stop solution to each well. And calculating a result.

2.7 mouse peripheral blood T lymphocyte and B lymphocyte ratio (flow cytometry detection)

Precooling at 4 ℃ by using a centrifuge. Blood is collected from mouse eyeball, 3 drops of blood (about 150 μ l) are added into a 15ml centrifuge tube filled with 10ml1 × PBS, 1600rpm is carried out for 5min, and centrifugation is carried out at room temperature; carefully discard the supernatant with a pipette, add 1ml of erythrocyte lysate per tube to resuspend the cell pellet, lyse at room temperature for about 5-10min until the fluid becomes clear from turbidity, add 10ml of PBS to stop lysis, centrifuge at 2000rpm for 5min, 4 ℃, discard the supernatant (step 4 can be repeated if more erythrocytes remain). The cell pellet was resuspended in 10ml PBS, centrifuged at 2000rpm for 5min at 4 deg.C, the supernatant was discarded, resuspended in 200. mu.l of blocking solution (PBS containing 5% FBS), and the cell suspension was transferred to a 1.5ml ep tube and blocked at 4 deg.C for 30 min. The flow antibody was prepared in blocking solution in the dark as follows: FITC-labeled anti-mouse CD3e, PE-labeled anti-mouse CD19, PerCP-Cy5.5-labeled anti-mouse CD4, APC-labeled anti-mouse CD8a, and the volume of each tube of cells was: each antibody was 0.3. mu.l, and the blocking solution was 50. mu.l.

The cell suspension was centrifuged at 2000rpm for 5min at 4 ℃ and the supernatant was discarded. Adding flow antibody, staining 50 μ l per tube at 4 deg.C in dark for 30min, adding 1ml PBS, centrifuging at 2000rpm for 5min at 4 deg.C, and discarding the supernatant. The cells were resuspended in 200. mu.l of PBS containing 2% FBS, transferred to a flow tube, and tested on the machine (if the machine test could not be performed on time, 4% paraformaldehyde-fixed solution was diluted to 2% with PBS, and the cells were resuspended at 200. mu.l per tube and stored overnight in the dark at 4 ℃).

2.8 mouse serum Motilin (MTL), Gastrin (GAS) assay (Elisa method)

Balancing the microporous plate to room temperature, adding standard substances, serum and quality control substances with different concentrations into corresponding holes, incubating for 2h at room temperature, wherein each hole has a volume of 100 mu L; washing the plate, repeating for 4 times, and sucking out residual liquid; adding 100 mu L of antibody into each hole, and incubating for 2h at room temperature; washing the plate, repeating for 4 times, and sucking out residual liquid; adding 100 μ L of chromogenic substrate into each well, and incubating for 30min at room temperature in the dark; 100 mu L of stop solution is added into each hole, and the absorbance at 450nm is detected by an enzyme-labeling instrument.

2.9 pathological examination of Lung tissue

Taking lung tissues of mice, dehydrating by gradient ethanol, transparentizing by xylene, embedding by wax immersion, slicing, HE dyeing, sealing by neutral gum, observing under a microscope, and taking pictures. The presence or absence of pathological morphological changes in the mouse lungs was observed under an optical microscope for comparison between groups.

2.10 statistical analysis

Statistical analysis is carried out by using SPSS17.0 software, differences among groups are subjected to one-way analysis of variance (ANOVA), LSD (least squares discriminant) test is carried out when the variances are uniform, Dunnett T3 test is carried out when the variances are irregular, and P <0.05 has statistical significance.

3 results of the experiment

3.1 general Observation of body signs

The normal control group mice had normal body weight, normal food intake and water intake, sensitive activity, smooth and glossy fur, and normal stool; the constitution of mice in the cold-dampness infection group (model group) is lightened, the food intake and the drinking water are reduced, the mice are intolerant of cold and do not move, the hair color is dark and dull, loose and rough, dysphoria, bite and fight, the stool is soft, the color is yellow, the limbs are weak, the reaction is slow, and the spirit is listened; the symptoms of the mice in the high and low dose groups of the bupleurum-silver particles are obviously improved.

3.2 Effect of Chaihin granule on the pulmonary index and inhibition ratio of BALB/c mice with syndrome of human coronavirus pneumonia and epidemic virus attacking lung

The method comprises the following steps of weighing a mouse body weight, then taking blood, dissecting lung tissue, weighing lung weight, and calculating the lung index and the inhibition rate of the mouse, wherein the specific calculation formula is as follows (lung index ═ lung wet weight (g)/body weight (g) ] × 100):

table 1, figure 1 results show: compared with a normal control group, the lung index of mice in an infected control group and a cold-dampness infected group (a model group) is obviously increased (P is less than 0.01), and the cold-dampness control group has no obvious change; compared with a cold-dampness infection group (a model group), the lung index of mice in the high and low dose groups of the Chaihuang granules is obviously reduced (P < 0.01); the lung index inhibition rates of mice in the high and low dose groups of the Chaihin granules are respectively 61.01% and 55.45%.

TABLE 1 therapeutic action of Chaiyin granules on combined model of human coronavirus pneumonia and epidemic virus attacking lung syndrome mouse

Note: comparing with normal control group^##P<0.01; comparison with model group^**P<0.01。

3.2 Effect of Chaihin granules on the percentage of BALB/c mice peripheral blood lymphocytes in the pattern of human coronavirus pneumonitis attacking the lungs

The results of table 2, fig. 3, fig. 4, fig. 5 show: compared with normal control group, infected control group, cold-dampness control group, and cold-dampness infected group (model group) mice peripheral blood CD4⁺T lymphocytes, CD8⁺Significant reduction in the percentage of T lymphocytes (P)<0.05，P<0.01), the percentage of total B lymphocytes in the peripheral blood of mice in the cold-dampness infection group (model group) is remarkably reduced (P)<0.05，P<0.01); in comparison with the cold-dampness infection group (model group), the mice in the high and low dose group of Chaihuang granules had peripheral blood CD4⁺T lymphocytes, CD8⁺The percentage of T lymphocytes and total B lymphocytes is remarkably increased (P)<0.01)。

TABLE 2 therapeutic action of Chaiyin granules on combined model of human coronavirus epidemic-virus lung attacking syndrome mouse

Note: comparing with normal control group^#P<0.05,^##P<0.01; comparison with model group^*P<0.05，^**P<0.01.

3.3 Effect of Chaihin granule on lung pathogen attack of human coronavirus pneumonia virus BALB/c mouse lung tissue pathology

Figure 6 the results show: the lung interstitium of the normal control group mouse has no inflammation and blood stasis, and the structure is normal; infected control mice have lung blood stasis, cells are hypertrophied and aggregated, and the shapes are inconsistent; the lung blood stasis of the mice in the cold-dampness control group causes cell hypertrophy and aggregation, and the shapes are inconsistent; the lung of the mice in the cold-dampness infection group (model group) is stained with flaky red, and inflammatory exudation is obvious; compared with the cold-dampness infection group (model group), the mice in the Chaihuang granule group have the advantages of reduced inflammatory exudation around the bronchus and interstitial substance in the lung and reduced inflammation.

4 small knot

In the experiment, general physical sign observation shows that the model mouse loses weight before infection, reduces food intake and drinking water, is intolerant of cold and does not move, has dark and dull hair color, is loose and rough, is dysphoria, is bitten and fights, has soft stool and yellow color, and is similar to the light clinical expression of the novel coronavirus pneumonia; after infection, the model mouse has obvious limb weakness, less motion, slow reaction, listlessness, positive lung virus nucleic acid detection, obviously raised inflammatory reaction index, obvious pathological change of lung tissue and typical epidemic virus attacking lung disease symptom. After the administration of the Chaihin granules, the symptoms of the above diseases are obviously improved.

The detection result shows that the lung index of the model mouse is obviously reduced in the high and low dose groups of the Bupleurum and argentum particles, and the lung index inhibition rates of the high and low dose groups are 61.02% and 55.45% respectively; the high and low dose groups of the Chaihin granules significantly increase the CD4 in the peripheral blood of the model mice⁺T lymphocytes, CD8⁺T lymphocytes and total B lymphocyte percentage. Experiments prove that the diesel-silver preparation has definite effect of resisting human coronavirus.

Drawings

FIG. 1: the influence of Bupleurum argenteum granule on BALB/c mouse lung index due to human coronavirus pneumonia and epidemic virus attacking lung, compared with normal control group^#P<0.05,^##P<0.01; comparison with model group^**P<0.01,n＝6.

FIG. 2: BALB/c mouse peripheral blood CD4 of lung disease infected by human coronavirus pneumonia virus⁺Influence of T percentage, compared with the Normal control group^#P<0.05,^##P<0.01; comparison with model group^**P<0.01,n＝6.

FIG. 3: BALB/c mouse peripheral blood CD8 of lung disease infected by human coronavirus pneumonia virus⁺Influence of T percentage, compared with the Normal control group^#P<0.05,^##P<0.01; comparison with model group^**P<0.01,n＝6.

FIG. 4: the influence of Chaihingn granule on BALB/c mouse peripheral blood B lymphocyte percentage of human coronavirus pneumonia virus attacking lung syndrome is compared with normal control group^#P<0.05,^##P<0.01; comparison with model group^**P<0.01,n＝6.

FIG. 5: content detection of T cells and B cells

FIG. 6: influence of Chaihingn granules on lung tissue pathology of BALB/c mouse with syndrome of lung being attacked by human coronavirus pneumonia and epidemic viruses

Note: the Chaihingsan particle group is a clinical equivalent dose group, namely a low dose group, and 2 observation visual fields are selected.

Detailed Description

The invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention in any way, as will be appreciated by those skilled in the art.

EXAMPLE 1 preparation of Chaihuyin capsules

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, 2 hours each time, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.12(50 ℃), adding 80% ethanol with the pH value being adjusted to 10 by 30% sodium hydroxide solution to ensure that the fermentation content reaches 60%, uniformly stirring, refrigerating for 24 hours, filtering, and obtaining an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

E. d, collecting the total alcohol precipitation solution in the step D, concentrating under reduced pressure, recovering ethanol to obtain thick paste, belt-type vacuum drying at a vacuum degree of-0.08 MPa to-0.10 MPa and a drying temperature of 95 ℃, and crushing into fine powder;

F. and D, uniformly mixing the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B, adding starch and microcrystalline cellulose (the weight ratio is 9: 1) according to the formula amount, uniformly mixing, granulating, drying, grading, filling, polishing in a polishing machine, and removing damaged capsules to obtain the capsule.

EXAMPLE 2 preparation of Chaiharge tablets

A. Crushing bitter almond into coarse particles, soaking seven kinds of materials including radix bupleuri, honeysuckle, sweet wormwood, forsythia, schizonepeta, mint, houttuynia and the like in water for 1 hour, distilling and extracting for 8 hours, collecting volatile oil, and collecting distillation residues and liquid medicine for later use;

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, 2 hours each time, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.14(50 ℃), adding 95% ethanol with the pH value being adjusted to 12 by 30% sodium hydroxide solution to ensure that the fermentation content reaches 70%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

D. dissolving the precipitate with 70% ethanol, refrigerating, and filtering to obtain ethanol precipitation solution II; mixing the ethanol precipitation solutions I and II to obtain a total ethanol precipitation solution;

F. and D, mixing the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B, adding microcrystalline cellulose and hydroxypropyl fiber (the weight ratio is 5: 3) in a formula amount to enable the weight to be 1000g, mixing uniformly, preparing into coarse granules, drying, crushing, sieving, preparing granules, drying at low temperature, finishing granules, adding 0.5% of magnesium stearate, mixing uniformly, pressing into 1000 tablets, and coating with a film coat to obtain the tablet.

EXAMPLE 3 preparation of Chaiyun pellets

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, each time lasting for 2 hours, mixing the decoction with the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.11(50 ℃), adding 70% ethanol with the pH value being adjusted to 8 by 10% sodium hydroxide solution to ensure that the fermentation content reaches 50%, uniformly stirring, refrigerating for 24 hours, filtering, and obtaining ethanol precipitation liquid I and ethanol precipitation sediment for later use;

D. dissolving the precipitate with 50% ethanol, refrigerating, and filtering to obtain ethanol precipitation solution II; mixing the ethanol precipitation solutions I and II to obtain a total ethanol precipitation solution;

E. d, collecting the total alcohol precipitation solution in the step D, concentrating under reduced pressure, recovering ethanol to obtain thick paste, belt-type vacuum drying at a vacuum degree of-0.08 MPa to-0.10 MPa and a drying temperature of 80 ℃, and crushing into fine powder;

F. and D, mixing the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B, adding sucrose powder and dextrin (weight ratio is 5: 1) in a formula amount to enable the weight to be 1000g, mixing uniformly, drying below 70 ℃, crushing into fine powder, adding 2.5% of low-substituted hydroxypropyl cellulose, mixing uniformly, preparing into 1000 pills by using water, drying below 70 ℃, polishing and coating to obtain the finished product.

EXAMPLE 4 preparation of Chaihingoral liquid

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, 2 hours each time, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.14(50 ℃), adding 80% ethanol with the pH value being adjusted to 10 by 30% sodium hydroxide solution to ensure that the fermentation content reaches 55%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

D. dissolving the precipitate with 55% ethanol, refrigerating, and filtering to obtain ethanol precipitation solution II; mixing the ethanol precipitation solutions I and II to obtain a total ethanol precipitation solution;

E. and D, collecting the total alcohol precipitation solution in the step D, concentrating under reduced pressure to recover ethanol, concentrating to obtain paste with the relative density of 1.14(50 ℃), adding β -cyclodextrin to prepare an inclusion compound, 3g of sodium benzoate and 200g of cane sugar, adding water to 1000ml, uniformly stirring, refrigerating for 24 hours, filtering, adjusting the pH value to 7.1 by using a sodium hydroxide solution, encapsulating, and sterilizing to obtain the traditional Chinese medicine composition.

EXAMPLE 5 preparation of Chaihsilver granules

B. adding β -cyclodextrin into the volatile oil obtained in step A to prepare an inclusion compound for later use;

E. d, collecting the total alcohol precipitation solution in the step D, concentrating under reduced pressure, recovering ethanol to obtain thick paste, and crushing into fine powder at the drying temperature of 90 ℃ and the vacuum degree of-0.08 MPa-0.10 MPa;

F. and D, uniformly mixing the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B, adding sucrose powder, hydroxypropyl starch and mannitol (in a weight ratio of 2: 2: 1) in a formula amount to enable the weight to be 1000g, uniformly mixing, granulating, drying, and finishing to prepare 1000 g.

EXAMPLE 6 preparation of silver microcapsules

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, 2 hours each time, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.13(50 ℃), adding 85% ethanol with the pH value being 12 regulated by 20% sodium hydroxide solution to ensure that the fermentation content reaches 65%, uniformly stirring, refrigerating for 24 hours, filtering, and obtaining ethanol precipitation liquid I and ethanol precipitation sediment for later use;

D. dissolving the precipitate with 65% ethanol, refrigerating, and filtering to obtain ethanol precipitation solution II; mixing the ethanol precipitation solutions I and II to obtain a total ethanol precipitation solution;

E. d, collecting the total alcohol precipitation solution in the step D, concentrating under reduced pressure, recovering ethanol to obtain thick paste, belt-type vacuum drying at a vacuum degree of-0.08 MPa to-0.10 MPa and a drying temperature of 85 ℃, and crushing into fine powder;

F. mixing the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B uniformly to obtain 282g of fine powder of the bupleurum-silver extract for later use;

G. 394g of sodium caseinate, 262g of starch syrup dry powder, 12g of octadecanol, 6g of titanium dioxide and 44g of propylene glycol are added into purified water, the mixture is heated and stirred at 52 ℃ to be dissolved, capsule wall material solution with the mass fraction of 36% is prepared, the solution is cooled to room temperature, 3.6g of the extract fine powder obtained in the step F, 3.6g of soybean lecithin and 5.4g of sucrose fatty acid ester are added under the stirring state, homogenized and emulsified to obtain emulsion, spray drying is carried out under the conditions of the air inlet temperature of 172 ℃, the spray pressure of 0.39MPa and the feed speed of 22.5ml/min, microcapsules are collected and cooled, and 865g of microcapsules are obtained.

EXAMPLE 7 preparation of silver particles

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, 2 hours each time, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.12(50 ℃), adding 90% ethanol with the pH value being adjusted to 8 by 10% sodium hydroxide solution to ensure that the fermentation content reaches 70%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

F. and D, uniformly mixing the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B, adding sucrose powder and dextrin (weight ratio is 3: 2) according to the formula amount, uniformly mixing, granulating, drying, and finishing to obtain 1000 g.

EXAMPLE 8 preparation of Chaiyin pellet formulation

F. uniformly mixing the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B to obtain 290g of fine powder of the bupleurum-silver extract for later use;

G. respectively weighing 282g of the faggot extract fine powder, 525g of microcrystalline cellulose, 105g of cellulose acetate, 50g of superfine silica gel powder and 38g of lactose in the step F according to the formula amount, fully and uniformly mixing, adding 48% (by weight) of 35% ethanol solution in the formula amount, kneading to prepare a soft material, and extruding the soft material into strips through a sieve plate with the aperture of 1.1mm of an extruder;

H. opening the spheronizer, selecting the rotation speed of 1250rpm, placing the strip-shaped objects in the step G into the spheronizer, spheronizing for 5.5min to prepare a pellet semi-finished product, taking out the pellet semi-finished product, drying at 85 ℃, and screening to obtain 932.5G of pellets with 20-30 meshes.

EXAMPLE 9 preparation of Chaiharge tablets

C. decocting the residues in the step A, the scutellaria baicalensis, the radix puerariae and the platycodon grandiflorum in water for 3 times, each time lasting for 2 hours, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.13(50 ℃), adding 75% ethanol with the pH value being adjusted to 13 by 25% sodium hydroxide solution to ensure that the fermentation content reaches 55%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

F. and D, adding the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B into microcrystalline fiber and hydroxypropyl fiber (the weight ratio is 5: 3) in a formula ratio, uniformly mixing, preparing coarse granules, drying, crushing, sieving, preparing granules, drying at a low temperature, finishing granules, adding 0.5% of magnesium stearate, uniformly mixing, pressing into 1000 tablets, and coating with a film coating to obtain the tablet.

EXAMPLE 10 preparation of Chaiharge Capsule

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, 2 hours each time, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.11(50 ℃), adding 90% ethanol with the pH value being adjusted to 9 by 20% sodium hydroxide solution to ensure that the fermentation content reaches 60%, uniformly stirring, refrigerating for 24 hours, filtering, and obtaining an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

F. and D, uniformly mixing the fine powder obtained in the step E and the volatile oil inclusion compound obtained in the step B, adding the starch, the superfine silica gel powder and the low-substituted hydroxypropyl cellulose (the weight ratio is 3: 1: 1) in the formula amount, uniformly mixing, granulating, drying, grading, filling, polishing in a polishing machine, and removing damaged capsules to obtain the capsule.

EXAMPLE 11 preparation of Chaihver micropellets

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, 2 hours each time, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.12(50 ℃), adding 95% ethanol with the pH value being adjusted to 11 by 30% sodium hydroxide solution to ensure that the fermentation content reaches 70%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

F. mixing the fine powder obtained in step E and the volatile oil inclusion compound obtained in step B uniformly to obtain 296g of fine powder of the Bupleurum and silver extract for later use;

G. weighing 296g of the fine powder of the firewood and silver extract, 200g of cellulose acetate, 418g of microcrystalline cellulose, 46g of sodium carboxymethyl starch, 25g of lactose and 15g of superfine silica gel powder in the step F according to the prescription amount, fully and uniformly mixing, adding 48 percent (by weight) of ethanol solution with the concentration of 20 percent in the prescription, kneading to prepare soft materials, and extruding the soft materials into strips through a sieve plate with the aperture of 1.0mm of an extruder;

H. opening the spheronizer, selecting the rotation speed of 1250rpm, placing the strip-shaped objects in the step G into the spheronizer, spheronizing for 6.5min to prepare a pellet semi-finished product, taking out the pellet semi-finished product, drying at 80 ℃, and screening to obtain 921.7G of pellets with 20-30 meshes.

Claims

1. Application of Chaihingzhi in preparing medicine for resisting coronavirus is disclosed.

2. The use according to claim 1, wherein the Bupleurum-silver preparation is an oral pharmaceutical preparation, preferably the oral pharmaceutical preparation is one of granules, capsules, tablets, oral liquids, pills, pellets and microcapsules.

3. The use of claim 2, wherein the oral pharmaceutical formulation is prepared from the following Chinese medicinal components:

more preferably:

more preferably:

4. use according to claim 1, wherein the coronavirus is a human coronavirus.

5. The use of claim 4, wherein said human coronavirus includes, but is not limited to, any one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and CoVID-19.

6. The use according to claim 2 or 3, wherein the process for the preparation of the oral pharmaceutical formulation comprises the following steps:

C. decocting the residue in the step A, the scutellaria baicalensis, the radix puerariae and the platycodon grandiflorum in water for 3 times, and each time for 2 hours, mixing the decoction and the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.11-1.14 at 50 ℃, adding an alkalized ethanol solution to ensure that the alcohol content reaches 50% -70%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an alcohol precipitation solution I and an alcohol precipitation precipitate;

7. The use according to claim 6, wherein the alkalized ethanol solution added in step C is a 70% -95% ethanol solution with pH 8-13 adjusted with 10% -30% sodium hydroxide solution; more preferably, the alkalized ethanol solution added in step C is an 80% ethanol solution with pH adjusted to 9 with 20% sodium hydroxide solution.

8. Use according to claim 2 or 3, wherein the granules are prepared by a process comprising the steps of:

9. Use according to claim 2 or 3, wherein the process for the preparation of the pellet formulation comprises the steps of:

C. decocting the residues in the step A, the scutellaria baicalensis, the kudzuvine root and the platycodon grandiflorum in water for 3 times, each time lasting for 2 hours, mixing the decoction with the liquid medicine distilled in the step A, filtering, concentrating the filtrate to obtain clear paste with the relative density of 1.13 at 50 ℃, adding 80% ethanol with the pH value being adjusted to 9 by 20% sodium hydroxide solution to ensure that the fermentation content reaches 60%, uniformly stirring, refrigerating for 24 hours, and filtering to obtain an ethanol precipitation solution I and an ethanol precipitation precipitate for later use;

G. respectively weighing the fine powder of the firewood and silver extract, microcrystalline cellulose, cellulose acetate, superfine silica gel powder and lactose in the step F according to the prescription amount, fully and uniformly mixing, adding 48% of ethanol solution with the concentration of 35% of the prescription amount, kneading to prepare soft material, and extruding the soft material into strips through a sieve plate with the aperture of 1.1mm of an extruder;

10. Use according to any one of claims 1 to 3, wherein the medicament is for the manufacture of an anti-HcoV-229E medicament.