CN117338880A

CN117338880A - Application of pediatric wind-heat clearing mixture in preparation of anti-coronavirus drugs

Info

Publication number: CN117338880A
Application number: CN202311485184.0A
Authority: CN
Inventors: 杨子峰; 马钦海; 李润峰; 殷苗苗
Original assignee: Handan Pharmaceutical Co ltd
Current assignee: Handan Pharmaceutical Co ltd
Priority date: 2023-11-09
Filing date: 2023-11-09
Publication date: 2024-01-05

Abstract

The invention provides application of a pediatric wind-heat clearing agent in preparation of anti-coronavirus medicines, and relates to the technical field of anti-coronavirus medicines. The commercially available pediatric wind-heat clearing mixture is taken as an active ingredient to prepare the medicine for preventing or treating coronavirus infection. After the anti-coronavirus medicament provided by the scheme of the invention is externally used or internally used, the synergistic effect is achieved from multiple aspects such as inhibiting the replication of coronaviruses, inhibiting the over-expression of inflammatory factors, improving the survival quality of individuals after coronavirus infection, and the like, so that the anti-coronavirus effect is achieved, and the anti-coronavirus medicament has remarkable antiviral capability on new coronavirus wild strains and Omicron variant strains. Not only expands the application mode of the children wind-heat clearing mixture product and treats diseases, but also provides new raw materials and ideas for preparing the anti-coronavirus medicament.

Description

Application of pediatric wind-heat clearing mixture in preparation of anti-coronavirus drugs

Technical Field

The invention belongs to the technical field of anti-coronavirus medicines, and particularly relates to application of an infant wind-heat clearing agent in preparation of an anti-coronavirus medicine.

Background

Coronaviruses were first isolated from chickens in 1937, and the virus particles were 60-200 nm in diameter and 100nm in average diameter, spherical or oval in shape, and polymorphic. The virus has envelope, spinous processes exist on the envelope, the whole virus is like coronaries, and the spinous processes of different coronaviruses have obvious differences. Tubular inclusion bodies are sometimes seen in coronavirus infected cells. Coronavirus particles are irregularly shaped and have a diameter of about 60-220nm. The virus particles are coated with fat film, and three kinds of glycoprotein are arranged on the surface of the film: spike glycoprotein (S, spike Protein, a receptor binding site, cytolytic and primary antigenic site); small Envelope glycoproteins (E, envelope proteins, smaller, envelope-binding proteins); membrane glycoprotein (M, membrane Protein), responsible for transmembrane transport of nutrients, budding release of nascent virus and formation of viral outer envelope). There are also a few kinds of hemagglutinin glycoproteins (HE proteins, haemagglutinin-esterase). The coronavirus nucleic acid is non-segmented single-stranded (+) RNA, is 27-31kb long, is the longest RNA nucleic acid strand in RNA viruses, and has important structural characteristics specific to positive strand RNA: namely, the 5 '-end of the RNA strand has a methylation "cap" and the 3' -end has a PolyA "tail" structure. This structure is very similar to eukaryotic mRNA and is also an important structural basis for the fact that genomic RNA itself can serve as a translation template, eliminating the RNA-DNA-RNA transcription process.

Coronaviruses are currently classified into four groups, α, β, γ, and δ, based on genetic differences and serological properties, where the α and β groups primarily infect humans and other mammals and the γ and δ groups primarily infect birds. Among the a and β groups that can infect humans, seven coronaviruses are currently known to cause disease in humans, four of which mainly cause relatively mild symptoms of upper respiratory tract infection, while the other three cause severe and even life-threatening conditions, in particular:

SARS-CoV: infection of the human body can lead to Severe Acute Respiratory Syndrome (SARS);

MERS-CoV: infection of the human body may lead to Middle East Respiratory Syndrome (MERS);

SARS-CoV-2: infection of humans can result in novel coronavirus infection (covd-2019), including wild strains and Omicron variants;

HCoV-OC43, HCoV-229E, HCOV-NL63 and HCOV-HKU1: these four viruses mainly cause relatively mild upper respiratory tract infections.

The recombination rate between coronavirus RNA and RNA is very high, and it is this high recombination rate that the virus is mutated. After recombination, the RNA sequence changes, and thus the amino acid sequence encoded by the nucleic acid changes, and the protein composed of amino acids changes accordingly, so that the antigenicity changes. The result of the change in antigenicity is the failure of the original vaccine and the failure of immunity. Thus, effective agents for treating coronavirus infection have been explored as effective means for combating coronaviruses.

The pediatric wind-heat clearing mixture is a traditional Chinese medicine compound preparation, and is composed of honeysuckle, weeping forsythiae capsule, radix isatidis, peppermint, radix bupleuri, burdock, schizonepeta spike, gypsum, radix scutellariae, gardenia, platycodon grandiflorum, radix paeoniae rubra, reed rhizome, fried bitter apricot kernel, lophatherum gracile, fructus aurantii, fried medicated leaven, stiff silkworm, radix sileris and liquorice, and has the functions of clearing heat and detoxicating, relieving cough and relieving sore throat, and is clinically mainly used for pediatric wind-heat cold, fever, cough, expectoration, nasal obstruction and nasal discharge and throat redness and swelling pain.

However, the application of the pediatric wind-heat clearing agent in resisting coronaviruses is not disclosed in the prior art, and the potential effect and possible molecular regulation mechanism of the pediatric wind-heat clearing agent in resisting coronaviruses are not disclosed. Based on the above, the invention takes the pediatric wind-heat clearing agent as a research object, and explores the possible effect of the pediatric wind-heat clearing agent in the field of resisting coronaviruses.

Disclosure of Invention

The invention aims to provide an application of a pediatric wind-heat clearing mixture in preparing an anti-coronavirus medicament. The commercially available pediatric wind-heat clearing mixture is taken as an active ingredient to prepare the medicine for preventing or treating coronavirus infection. After the anti-coronavirus drug is externally or internally used, the anti-coronavirus drug has synergistic effects in multiple aspects of inhibiting coronavirus replication, inhibiting inflammatory factor over-expression, improving individual survival quality after coronavirus infection and the like, thereby achieving the effect of resisting coronavirus, and particularly has remarkable antiviral capability on new coronavirus wild strains and Omicron variant strains.

In a preferred embodiment, the coronavirus comprises one or more of OC43, wild-type strain SARS-CoV-2 and Omacron variant.

In a preferred embodiment, the active ingredient of the anti-coronavirus drug is a pediatric wind-heat clearing agent.

In a preferred embodiment, the anti-coronavirus drug is prepared by concentrating or further purifying a commercially available pediatric wind-heat clearing agent, and the concentration or purification method can be any method known to those skilled in the art, which is not limited thereto.

In a preferred embodiment, the anti-coronavirus drug comprises a pediatric wind-heat scavenger and/or a pharmaceutically acceptable carrier, more preferably the carrier comprises a pharmaceutically acceptable diluent, wetting agent, binder, flash-disintegrating agent, lubricant, color-flavor modulator, solvent, solubilizing agent, co-solvent, emulsifier, antioxidant, metal complexing agent, preservative, pH adjuster, surfactant, excipient, filler and synergist. Wherein the diluent comprises starch, sucrose, cellulose, inorganic salts, etc.; wetting agents include, for example, water, ethanol, and the like; binders include, for example, starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, and the like; disintegrants include, for example, starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium dicarboxymethyl cellulose, surfactants, and the like; lubricants include, for example, talc, calcium stearate, magnesium lauryl sulfate, polyethylene glycol, and the like; color, flavor, and taste flavoring agents include, for example, coloring agents, sweeteners, flavors, mucilages, and the like; solvents include, for example, water, glycerol, ethanol, and the like; the solubilizer includes, for example, tweens, sellers, sulfates, sulfonates, etc.; the cosolvent comprises organic acid (such as citric acid) and its salts, inorganic salts, polyethylene glycol, etc.; emulsifying agents include span, glycerol fatty acid ester, acacia, gelatin, agar, sodium alginate, etc.; antioxidants include, for example, sulfites, ascorbic acid, gallic acid, salts thereof, and the like; the metal complexing agent includes, for example, disodium edetate, polycarboxylic acid compounds, etc.; preservatives including, for example, parabens, quaternary ammonium compounds, chlorhexidine acetate, and the like; the pH regulator includes, for example, hydrochloric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, citrate, etc.

In a preferred embodiment, the anti-coronavirus agent is in a dosage form including one or more of powder, granule, tablet, capsule, suspension, emulsion, syrup, spray, external preparation, suppository, and sterile injectable solution. It will be appreciated that the anti-coronavirus drug of the present invention may be based on different excipients and different dosage forms, and accordingly, the mode of administration may also be varied.

In a preferred embodiment, the anti-coronavirus drug comprises: more preferably, the agent for preventing or treating coronavirus infection comprises: inhibiting coronavirus replication, inhibiting inflammatory factor overexpression, and improving survival quality of individuals after coronavirus infection.

In a preferred embodiment, vero E6 cells are used as the cells of the experimental model, the anti-coronavirus drug CC ₅₀ 40.14mg/mL; antiviral action IC of the anti-coronavirus drug on coronavirus wild strain SARS-CoV-2 ₅₀ Antiviral action against coronavirus Omicron variant IC at 3.491mg/mL ₅₀ 2.415mg/mL.

In a preferred embodiment, the anti-coronavirus drug has a coronavirus wild strain therapeutic index SI of 11.50; the therapeutic index SI of the pediatric wind-heat clearing agent on the coronavirus Omicron variant strain is 16.62.

In a preferred embodiment, the anti-coronavirus agent is capable of significantly inhibiting the overexpression of cytokines TNF- α, IL-6, MCP-1 and IP-10 induced by coronavirus infection at a concentration of 12.5-3.13 mg/mL.

In a preferred embodiment, the anti-coronavirus agent has a significant inhibitory effect on the expression of IFN-gamma, TNF-alpha and IP-10 in brain tissue; specifically, the anti-coronavirus drug has a significant inhibitory effect on the expression of IFN-gamma (2.9 mL/kg/d, p <0.01;11.6mL/kg/d, p < 0.001), TNF-alpha (2.9 mL/kg/d, p <0.01;11.6mL/kg/d, p < 0.01) and IP-10 (2.9 mL/kg/d, p <0.01;11.6mL/kg/d, p < 0.05) in brain tissue.

In a preferred embodiment, the anti-coronavirus agent is capable of significantly reducing the expression of TNF- α in lung tissue; in particular, the anti-coronavirus drug (2.9 mL/kg/d) can significantly reduce the expression of TNF-alpha in lung tissue (p < 0.05).

In a preferred embodiment, the anti-coronavirus agent significantly improves survival curves of the mice after coronavirus infection.

In a preferred embodiment, the anti-coronavirus agent is capable of significantly reducing the viral titer of the brain tissue of a milk mouse.

In a preferred embodiment, the anti-coronavirus agent provides a significant improvement in brain cell shrinkage and necrosis and neutrophil and lymphocyte infiltration caused by coronavirus infection.

Compared with the prior art, the technical scheme of the invention has the following advantages:

1. the invention takes the commercial product, namely the pediatric wind-heat clearing mixture, as an active ingredient, expands the application mode and treats the symptoms, and provides new raw materials and ideas for preparing the anti-coronavirus medicament.

2. In the present invention, it was confirmed by in vivo and in vitro experiments: the Chinese medicinal composition has antiviral effect on wild strain and Omicron variant strain of new coronavirus in vitro, and has inhibiting effect on replication of new coronavirus in cells, and therapeutic indexes SI are 11.50 (wild strain) and 16.62 (Omicron variant strain), respectively; in addition, the traditional Chinese medicine composition can obviously inhibit the over-expression of cell inflammatory factors TNF-alpha, IL-6, MCP-1 and IP-10 induced by virus infection at the concentration of 12.5-3.13mg/mL, and exert the anti-inflammatory effect, so that the anti-coronavirus medicine prepared by the pediatric wind-heat clearing agent can achieve the anti-coronavirus effect by the double effects of inhibiting virus replication and inhibiting the over-expression of inflammatory factors.

3. In the invention, the anti-coronavirus medicine prepared by the pediatric wind-heat clearing agent has good antiviral effect on common coronavirus infection, has good anti-inflammatory effect on encephalitis caused by coronavirus infection, and can improve the survival quality of infected individuals.

Drawings

These and/or other aspects and advantages of the present invention will become more apparent and more readily appreciated from the following detailed description of the embodiments of the invention, taken in conjunction with the accompanying drawings, wherein:

FIG. 1 effect of anti-coronavirus drug used in example 1 on survival of Vero E6 cells;

FIG. 2 antiviral effect of anti-coronavirus drug used in example 1 on wild-type strain of novel coronavirus;

FIG. 3 antiviral effect of anti-coronavirus drugs used in example 1 on novel coronavirus Omicron variant;

FIG. 4 shows the results of infection of cells with novel coronavirus Omacron variants with anti-coronavirus drugs used in example 1 (P <0.05, P <0.01, P <0.001 compared to the Virus group);

fig. 5 changes in body weight (a) (±sd, n=6) and survival (B) (n=8) of OC 43-infected rats with anti-coronavirus drug used in example 2 (wherein # # represents P <0.001,Ctrl vs Placebo; represents P <0.01; drug group vs Placebo; represents P <0.05; drug group vs Placebo);

FIG. 6 inhibition of viral titres of the anti-coronavirus drugs used in example 2 (LogTCID 50, + -SD, n=6) (wherein, P <0.001, drug group vs Placebo, P <0.01, drug group vs Placebo; P <0.05, drug group vs Placebo);

FIG. 7 inhibition of inflammatory mediator expression in brain tissue of rats (. + -. SD, n=5-6) with anti-coronavirus drugs used in example 2 (wherein # #, p <0.01; # #, p <0.001,Ctrl vs Placebo; p <0.05; p <0.01; p <0.001, drug group vs Placebo);

FIG. 8 inhibition of inflammatory mediator expression (. + -. SD, n=6) in lung tissue of rats using anti-coronavirus drug used in example 2 (wherein # #, p <0.01; # #, p <0.001,Ctrl vs Placebo; p <0.05; p <0.01; p <0.001, drug group vs Placebo);

FIG. 9 effect of anti-coronavirus drugs used in example 2 on improvement of pathological changes in brain and lung tissues of rats.

Detailed Description

For a better understanding of the present invention, those skilled in the art will now make further details with reference to the drawings and the detailed description, but it should be understood that the scope of the invention is not limited by the detailed description.

The technical scheme of the invention aims to solve the problems, and the general idea is as follows:

the in vitro experiment is used to evaluate the in vitro anti-novel coronavirus (SARS-CoV-2) effect of the Chinese medicinal composition, and provides basis for clinical study. In vitro experiments Vero E6 cytotoxicity test, anti-SARS-CoV-2 wild strain and Omicron variant virus efficacy test were performed. In vivo animal experiments develop the research of the protection effect of the traditional Chinese medicine composition on the common coronavirus OC43, and aim to clarify the antiviral and anti-inflammatory effects and the death protection effect of the traditional Chinese medicine composition on OC43 infection, thereby providing reference for the clinical application. The method specifically comprises the following steps:

experimental example 1

1. Experimental materials

1. Test drug: the anti-coronavirus medicine is commercially available infant wind-heat clearing and the volume is concentrated to 1/5 by a conventional method. Commercial infant Fengshui qing batch number 03222015, production date 2022, 8 and 18, and the property is tan liquid extract. Storing the test object: shading, sealing and storing at 4 ℃.

2. Cell and source: vero E6 cells were maintained by the respiratory disease national emphasis laboratory virus laboratory of the respiratory health institute, guangzhou.

3. Virus origin and quality attributes: the novel coronavirus wild strain and omacron variant strain were isolated from throat swabs of patients in eighth people hospital in Guangzhou, and stored in Guangzhou customs technical center P3 laboratory (respiratory disease national emphasis laboratory high pathogenic microorganism laboratory).

Preservation conditions: preserving in a refrigerator at the ultralow temperature of 80 ℃ below zero.

The culture method comprises the following steps: virus isolation culture was performed in Vero E6 cell vector with DMEM medium containing 2% fetal bovine serum.

Suitability evaluation before use: the coronavirus (SARS-CoV-2) strains of different subtypes can cause Vero E6 cells to have obvious lesions, and can obtain adaptive culture and amplification in the Vero E6 cells for later use.

2. Experimental reagent and instrument

1. Main experiment reagent

TABLE 1 list of main reagents

2. Main experimental instrument

Table 2 list of main instruments

3. Experimental method

1. Cytotoxicity test

Experiments were performed in BSL-2 laboratories. The cell control, the blank control (solvent control) and the test substance groups with different concentrations are set, and the administration doses are shown in Table 3. The cell density was set at 2X 10 ⁵ The cells/mL of VeroE6 cell suspension was plated in sterile 96 well plates at 100. Mu.L/well at 37℃with 5% CO ₂ Culturing for 24 hours in the environment; removing culture supernatant, washing monolayer cells with PBS for 1 time, adding 100 μl of 2-fold gradient diluted drug into each well, adding equal volume culture solution into normal cell group and blank control group, and culturing for 4 days; adding 20 mu L of MTT solution with the concentration of 5mg/mL into each hole, and continuously incubating for 4 hours; removing supernatant, adding 100 μl DMSO into each well, oscillating at low speed for 5min to melt the crystal sufficiently, and measuring at 490nm wavelength with enzyme-labeled instrumentAbsorbance (OD) was determined and inhibition was calculated. Inhibition = (average OD value of normal group-average OD value of drug group)/(average OD value of normal group-average OD value of blank group) ×100%. Drug median toxicity concentration (CC) was calculated using GraphPad prism5.0 software ₅₀ )。

TABLE 3 cytotoxicity test group design List

2. Antiviral test

Experiments were performed in BSL-3 laboratories. The cell density was set to 2X 10 by using the cell control, blank control (solvent control), virus control (negative control) and drug group as shown in Table 4 at the doses shown in Table 4 ⁵ The cells/mL of VeroE6 cell suspension was plated in sterile 96-well plates with 100. Mu.L of each well at 37℃with 5% CO ₂ Culturing for 24 hours in the environment; the culture supernatant is discarded, 100TCID50 virus solution (wild strain and Omicron variant strain) is added into the experimental group and the virus control group, 100 mu L/hole is added, and the conventional culture is carried out for adsorption for 2 hours; the culture supernatant was discarded, 100. Mu.L of drug at different concentrations was added per well, 100. Mu.L/well, 4 duplicate wells per concentration, and the culture was incubated for 3-4 days in conventional. Cytopathy (CPE) was observed under an optical microscope and the degree of cytopathy was recorded according to the following grade 6 standard: "-" is that the cells are free of lesions; "±" is less than 10% of cytopathic effect; "+" is about 25% of cytopathic; "++" is about 50% of cytopathic changes; "+". ++'s is a cell lesion about 75%: "+". ++'s is a cell lesions were over 75%. Half maximal inhibitory concentrations (IC 50) were calculated using GraphPad prism5.0 software. Antiviral drug efficacy outcome criteria: determining a drug efficacy criterion by using a selection index SI (si=cc50/IC 50), SI>1 indicates effectiveness, and the larger the index (SI value), the larger the safety margin.

TABLE 4 antiviral (wild strain and Omicron variant) test group design List

3. Anti-inflammatory test

The cells are then culturedDensity of 1X 10 ⁶ cell suspensions of cells/mL were plated in sterile 12-well plates, 500. Mu.L of each well was added, and incubated at 37℃in 5% CO2 for 24h; removing culture supernatant, washing monolayer cells with 500 μl PBS for 1-2 times, adding 500 μl diluted virus solution into each well, adding normal cell control group into equal volume of culture medium containing 2% foetal calf serum, and conventional culturing for 1 hr; discarding supernatant, adding the drug to be tested diluted by a multiple ratio, and adding an equal volume of culture medium containing 2% of fetal bovine serum into a normal cell control group; conventional culture and incubation for 48h, collecting supernatant, and preserving at-80deg.C for use; the cells were washed 1-2 times with PBS, and cellular RNA was extracted by the trizol method, reverse transcribed into cDNA using a reverse transcription kit, and transferred to BSL-2 laboratory for PCR detection.

TABLE 5 list of infection-induced inflammation test group designs against virus (wild strain and Omicron variant)

4. Experimental results

1. As shown in figures 1-3, the antiviral test results show that the traditional Chinese medicine composition shows CC of the traditional Chinese medicine composition for VeroE6 cytotoxicity results ₅₀ 40.14mg/mL; the antiviral test results show that the Chinese medicinal composition has antiviral effect IC on SARS-CoV-2 wild strain and Omicron variant strain ₅₀ 3.491mg/mL (wild strain) and 2.415mg/mL (Omicron variant strain), respectively.

2. As shown in FIG. 4, the detection results of inflammatory factors show that the traditional Chinese medicine composition has inhibition effect on the over-expression of inflammatory factors TNF-alpha, IL-6, MCP-1 and IP-10 induced by new coronavirus Omicron variant infected cells at the concentrations of 12.5, 6.25 and 3.13 mg/mL.

In summary, under the present study conditions, the Chinese medicinal composition has antiviral effect on wild strain and Omicron variant strain of new coronavirus in vitro, has inhibitory effect on the replication of new coronavirus in cells, and has therapeutic indexes SI of 11.50 (wild strain) and 16.62 (Omicron variant strain), respectively; in addition, the traditional Chinese medicine composition can obviously inhibit the over-expression of cell inflammatory factors TNF-alpha, IL-6, MCP-1 and IP-10 induced by Omicron variant strain infection at the concentration of 12.5-3.13mg/mL, plays an anti-inflammatory role, and is inferred to achieve the anti-new coronavirus role by inhibiting virus replication and inhibiting the over-expression of inflammatory factors.

Experimental example 2

1. Experimental materials

1. Test drug: the anti-coronavirus drug is commercially available infant wind-heat clearing, and the concentration of the anti-coronavirus drug is purified to be 5 times of that of a commercially available sample by a conventional method. Commercial infant Fenghui qing (batch number: 0322003; production date: 2022, 3, 5 days) is brown extract, and is stored at 4deg.C in dark place. When the test of the suckling mice is carried out, deionized water is used for preparing the children wind-heat clearing liquid medicine into the test concentration, and the test concentration is split charging and stored at 4 ℃ for standby.

2. Cells and viruses: human colon cancer tumor cells (HRT-18) and human coronavirus OC43 (HCoV-OC 43, OC 43) murine brain-adapted strain were maintained by the national focus laboratory virus laboratory for respiratory disease, guangzhou respiratory health institute.

The OC43 amplification method is as follows: 10 healthy C57BL/6 milk mice (weight 4-6 g) within 10 days old, injecting 30 mu L of 1000 LD50 OC43 into each of the cranium, taking brain tissue after 4 days of virus attack, homogenizing, centrifuging for 15min at 4 ℃ under 5000g/min, and collecting supernatant to obtain the newly amplified OC43 virus liquid.

The LD50 (median lethal dose) of OC43 on the milk mice was determined as follows: the newly amplified OC43 virus solution was taken and subjected to 10-fold gradient dilution with sterile PBS for a total of 7 dilution gradients. Healthy rats within 42 days of age 10 were randomly divided into 7 groups (10 ^-1 ～10 ^-7 Gradient dilution inoculation), 6 per group, each group was injected intracranially with 30 μl of virus gradient dilutions, respectively. Experimental observations were made for 7 days, the body weight and the number of deaths were recorded daily and LD50 was calculated using Reed-Muench. The LD50 of the virus used in this experiment was 10-5.45/30. Mu.L.

2. Test method

1. Research on in vivo efficacy of traditional Chinese medicine composition

Healthy C57BL/6 milk mice (body weight 4-6 g) within 10 days of age were randomly divided into 6 groups, namely a normal group (Ctrl), an OC43 group (Placebo), a Ruidexivir group (RDV, 25 mg/kg), a Chinese medicinal composition (XF) low, medium and high dose group (2.9 mL/kg, 5.8mL/kg and 11.6 mL/kg). Virosomes and pharmaceutical groups the rats received an intracranial injection of 30 μl of OC43 virus solution containing 1 LD50, and the normal group was given an equal volume of PBS. RDV was co-dissolved with 12% aqueous sulfobutyl ether- β -cyclodextrin and adjusted to ph=5.0 with NaOH/HCl. 2 hours after the toxin is removed, the XF group and the normal group are respectively infused with the corresponding dose of the medicament and the equal volume of deionized water, and the RDV group is injected subcutaneously. All groups were dosed continuously for 4 days, body weights were weighed daily, the rats were dissected and weighed on day 4 for lung and brain tissue, brain index (brain weight/body weight x 100) and lung index (lung weight/body weight x 100) were calculated, left brain and left lung were homogenized with 1mL of PBS (5000 g, centrifuged at 4 ℃ for 15 min), and supernatants were taken for virus titer (see 3. Virus titer assay) and inflammatory factor expression (see 4.Bio-Plex assay for inflammatory factor expression); after right brain and lung were fixed with neutral formalin, H & E sections were prepared (see 5. Pathological changes observations).

2. Research on death protection effect of traditional Chinese medicine composition

The experimental group and administration mode were almost the same as 1 (study of efficacy in the body of the Chinese medicinal composition), and the virus inoculation amount was adjusted to 1000 LD50 s. Each group of mice was weighed daily and recorded for 7 consecutive days.

3. Virus titer detection

Brain and lung tissue homogenates were 10-fold gradient diluted with 1640 medium containing 2% serum, different gradient dilutions were added to HRT-18 cells, 4 duplicate wells per gradient, and cultured for 5 days. Cytopathy was recorded on day 5 by supplementing 1640 medium with 2% serum on day 3. TCID50 was calculated using the Reed-Muench method.

Bio-Plex detection of inflammatory factor expression

The detection of inflammatory factor protein level is carried out according to the specification, and is specifically as follows:

(1) Standard preparation

And (3) sucking 250 mu L of standard substance diluent, adding the diluent into the freeze-dried powdery standard substance, vortexing for 15s, incubating on ice for 30min, and sequentially diluting the standard substance according to a 4-time gradient after the standard substance powder is completely dissolved.

(2) Preparation of antibody-precoated microspheres

The number of wells to be measured N was calculated, 50. Mu.L of 1X microspheres were required per well, and an additional 10% volume consumption was calculated, with the total 1X microsphere volume required being NX 50X 1.1. Mu.L.

(3) Washing liquid preparation

After the 10X concentrated washing solution was returned to room temperature, it was diluted to 1X with the detection buffer.

(4) Preparation of detection antibodies

The number of wells to be measured N was calculated, 25. Mu.L of detection antibody was required per well, and an additional 10% volume consumption was calculated, giving a total required detection volume of N.times.25.times.1.1. Mu.L.

(5) Preparation of SA-PE solutions

The number of wells N to be measured was calculated, 50. Mu.L of SA-PE solution was required for each well, and an additional 10% volume consumption was calculated, with the total required SA-PE solution being Nx25X1.1. Mu.L.

(6) Experimental procedure

Washing the thin bottom 96-well plate to be used with a 1X washing solution at 200. Mu.L per well at 300rpm/min for 5min at room temperature; 50 mu L of standard substance is added into the standard substance hole; 50. Mu.L of detection buffer was added to the background wells; adding 50 mu L of sample into the sample hole; 50 μl of 1×microsphere was added to all wells; incubating for 30min at normal temperature in a dark place, and setting the rotating speed to 850+/-50 rpm/min; washing 3 times by using an automatic plate washing machine; adding 25 mu L of detection antibody into each hole, incubating for 30min at room temperature in a dark place, and setting the rotating speed to 850+/-50 rpm/min; washing 3 times by using an automatic plate washing machine; adding 50 mu L of SA-PE solution into each hole, incubating for 15min at room temperature in a dark place, and setting the rotating speed to 850+/-50 rpm/min; washing 3 times by using an automatic plate washing machine; 125. Mu.L of detection buffer was added to each well and after sufficient re-suspension of the microspheres, the beads were subjected to the Bio-Plex 200 assay (50 microspheres/region). Finally, data quality control analysis was performed with a Bio-Plex Manager.

5. Observation of pathological changes

After fixing brain and lung tissues, dehydration, paraffin embedding, slicing, HE staining and sealing were performed, and histopathological changes were observed under an optical microscope.

6. Statistical analysis

Data were analyzed using GraphPad Prism 8.0 software. Brain index, lung index, virus titer and inflammatory factor expression are expressed as mean ± SD, and One-Way ANOVA was used for group-to-group differential analysis; survival curve analysis was performed using Log-rank (Mantel-Cox) Test. p <0.05 is a significant difference.

3. Test results

1. Influence of traditional Chinese medicine composition on weight loss and death of OC43 infected suckling mice

Weight loss after the mice were infected with OC43 compared to normal group (fig. 5A); there was no significant improvement in weight loss in the RDV (25 mg/kg/d) and XF (2.9 mL/kg, 5.8mL/kg, and 11.6 mL/kg) mice compared to the model group (FIG. 5A). Fig. 5B shows that the model group of rats all died on day 6 after challenge; compared to the model group, RDV group and (p < 0.01) XF (2.9 mL/kg/d) group (p < 0.05) significantly improved the survival curve of the rats (FIG. 5B).

2. Inhibition of OC43 proliferation in brain tissue of suckling mice by traditional Chinese medicine composition

RDV (25 mg/kg/d, p < 0.001) and XF (2.9 mL/kg/d, p <0.05;5.8mL/kg/d, p < 0.01) significantly reduced the brain tissue virus titer of the rats compared to the model group (FIG. 6).

3. Inhibition of up-regulated expression of OC43 infected mice brain and lung inflammatory factors by traditional Chinese medicine composition

The expression levels of IFN-gamma, IL-6, TNF-alpha, IP-10, CXCL-1, MCP-1 and MIP-1 alpha were significantly up-regulated in the brain tissue of the mice infected with OC43 compared to the normal group (FIGS. 7A-G); RDV (25 mg/kg/d) had a significant inhibitory effect on the expression of IL-6 (p < 0.05), IP-10 (p < 0.01) and CXCL-1 (p < 0.05) (FIG. 7B, D, E). XF intervention had significant inhibitory effects on IFN-gamma (2.9 mL/kg/d, p <0.01;11.6mL/kg/d, p < 0.001), TNF-alpha (2.9 and 11.6mL/kg/d, p < 0.001) and IP-10 (2.9 mL/kg/d, p <0.01;11.6mL/kg/d, p < 0.05) (FIG. 7A, C, D), with no significant inhibitory effects on other inflammatory factors.

FX (2.9 mL/kg/d) significantly reduced TNF- α expression in lung tissue (FIG. 8) (p < 0.05) compared to the infected group.

4. Improving effect of traditional Chinese medicine composition on pathological changes of brain and lung tissues caused by OC43 infected suckling mice

OC43 infected mice were seen with brain cell shrinkage and necrosis, with inflammatory infiltrates predominantly neutrophils, while lymphocyte infiltrates were seen (fig. 9a, b). The high dose of XF (11.6 mL/kg/D) had a significant improvement on the brain lesions described above (FIG. 9D), the medium dose of XF (5.8 mL/kg/D) had a second improvement to the high dose (FIG. 9E), while the low doses of XF (2.9 mL/kg/D) and RDV (25 mg/kg/D) had pathological changes similar to those of the infected group (FIGS. 9C, F). In addition, no obvious pathological changes to the lung tissue of mice were observed as normal (fig. 9G), slight red blood cell exudation and cellulose exudation were seen in OC 43-infected mice, and slight hyperemia was seen in the lumen of blood vessels (fig. 9H). Three doses of XF improved the pathology described above (fig. 9j, k, l), whereas the improvement in RDV was more pronounced than XF (fig. 9I).

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. Application of infantile wind-heat clearing agent in preparing anti-coronavirus medicine is provided.

2. The use of the pediatric wind-heat clearing agent according to claim 1 for the preparation of an anti-coronavirus medicament, wherein the coronavirus comprises one or more of OC43, wild strain SARS-CoV-2 and Omicron variant.

3. The use of the pediatric wind-heat clearing agent according to claim 2 for preparing an anti-coronavirus medicament, wherein the anti-coronavirus medicament comprises: a medicament for preventing or treating coronavirus infection.

4. The use of the pediatric wind-heat clearing agent according to claim 3 for preparing an anti-coronavirus medicament, wherein the medicament for preventing or treating coronavirus infection comprises: inhibiting coronavirus replication, inhibiting inflammatory factor overexpression, and improving survival quality of individuals after coronavirus infection.

5. The use of the pediatric wind-heat clearing agent according to claim 3 for preparing an anti-coronavirus medicament, wherein the anti-coronavirus medicament comprises the pediatric wind-heat clearing agent as an active ingredient.

6. The use of the pediatric wind-heat clearing agent according to claim 3 for preparing an anti-coronavirus medicament, wherein the anti-coronavirus medicament comprises one or more of powder, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, external use agents, suppositories and sterile injection solutions.

7. The use of the pediatric wind-heat clearing agent according to claim 4 for preparing an anti-coronavirus drug, wherein Vero E6 cells are used as cells of an experimental model, and the anti-coronavirus drug CC ₅₀ 40.14mg/mL; antiviral action IC of the anti-coronavirus drug on coronavirus wild strain SARS-CoV-2 ₅₀ Antiviral action against coronavirus Omicron variant IC at 3.491mg/mL ₅₀ 2.415mg/mL.

8. The use of the pediatric wind-heat clearing agent according to claim 4 for preparing an anti-coronavirus medicament, wherein the anti-coronavirus medicament has a coronavirus wild strain therapeutic index SI of 11.50; the therapeutic index SI of the pediatric wind-heat clearing agent on the coronavirus Omicron variant strain is 16.62.

9. The use of the pediatric wind-heat clearing agent according to claim 4 for preparing an anti-coronavirus drug, wherein the anti-coronavirus drug can significantly inhibit over-expression of cytokines TNF-alpha, IL-6, MCP-1 and IP-10 induced by coronavirus infection at a concentration of 12.5-3.13 mg/mL.

10. The use of the pediatric wind-heat clearing agent according to claim 4 for preparing an anti-coronavirus drug, wherein the anti-coronavirus drug has a significant inhibitory effect on the expression of IFN- γ, TNF- α and IP-10 in brain tissue;

the anti-coronavirus drug can obviously reduce the expression of TNF-alpha in lung tissues.