CN115317542A

CN115317542A - Application of traditional Chinese medicine composition in preparation of S protein D614G resistant mutant type new coronavirus medicines

Info

Publication number: CN115317542A
Application number: CN202110507714.1A
Authority: CN
Inventors: 贾振华
Original assignee: Hebei Yiling Pharmaceutical Research Institute Co Ltd
Current assignee: Hebei Yiling Pharmaceutical Research Institute Co Ltd
Priority date: 2021-05-11
Filing date: 2021-05-11
Publication date: 2022-11-11
Also published as: WO2022237145A1

Abstract

The invention discloses application of a traditional Chinese medicine composition in preparation of an anti-S protein D614G mutant type new coronavirus medicine. The Chinese medicinal composition mainly comprises fructus forsythiae, honeysuckle, isatis root, rhubarb, patchouli, male fern rhizome, rhodiola rosea and the like, plays the integral adjustment advantage of the compound Chinese medicament, organically combines the functions of removing disease evil, relieving symptoms and adjusting immunity, realizes multi-target treatment, and proves that the Chinese medicinal composition has obvious curative effect on S protein D614G mutant type new coronavirus through cell experiments and animal experiments.

Description

Application of traditional Chinese medicine composition in preparation of S protein D614G mutant type new coronavirus resistant medicine

Technical Field

The invention relates to application of a traditional Chinese medicine composition in preparation of an anti-S protein D614G mutant type new coronavirus medicine, and belongs to the field of traditional Chinese medicines.

Background

2019 novel coronavirus (2019-nCoV), named by the World Health Organization (WHO) in 1 month of 2020. Coronaviruses are a large family of viruses known to cause the common cold and more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has not been previously discovered in humans.

SARS-CoV-2 is transmitted by aerosol, surface contact and fecal-oral routes. Infection with the virus causes a range of symptoms including fever, cough and general malaise, and more seriously, acute respiratory distress syndrome, and even death. Coronavirus is a rapidly evolving virus that has the property of infecting a wide range of hosts through different changes in the genome. SARS-CoV-2 has a number of variants, of which the variant D614G is rapidly becoming a global epidemic and has attracted considerable attention.

SARS-CoV-2 is positive strand RNA envelope virus, the spike protein on the surface of the virus exists in the form of trimer, and the S protein is responsible for binding receptor and making membrane fusion to complete the virus invasion process. The mutation of Aspartic acid (Asp) to Glycine (Gly) at position 614 outside the binding region (D614G) is rapidly becoming a global epidemic and has attracted widespread attention. By 30 days 11 months 2020, the D614G mutant accounts for 87% of the SARS-CoV-2 sequence published in the CoV-GLUE database.

Both animal experiments and human infection studies using pseudoviruses demonstrated that D614G had increased replication and infectivity compared to the wild-type strain. The serum levels of IgG, igM or IgA in the infected person were not changed. The data from the present experiments do not yet indicate that the relationship between enhanced transmission and virulence of D614G remains complex, possibly influenced by age, sex and other co-diseases, and it is not clear whether the lowest infectious dose of D614G in humans will be lower.

The D614G mutation has become the dominant strain of the SARS-CoV-2 epidemic, and more data indicate that the mutation enhances replication and infectivity. The SARS-CoV-2 lineage still accumulates nucleotide mutations at a rate of 1 to 2 mutations per month, and most of the existing strains are combined mutations based on D614G. Therefore, the mutation of D614G combined with other sites needs to be continuously monitored, and whether vaccines and antibodies have broad-spectrum protection on the variant strains is also considered to be important. The development of anti-D614 mutant drugs is imminent.

The invention is an improved invention based on the ZL03143211 patent, and the content of the patent document is cited in the whole. The above patent does not disclose the application of the Chinese medicinal composition in resisting S protein D614G mutant type new coronavirus.

Disclosure of Invention

The invention provides application of a traditional Chinese medicine composition in preparing S protein D614G mutant type new coronavirus resistant medicines, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight:

150-260 parts of forsythia, 150-260 parts of honeysuckle, 150-260 parts of isatis root, 150-260 parts of rhubarb, 30-55 parts of rhubarb

Patchouli 50-90 male fern rhizome 150-260 rhodiola root 50-90 menthol crystal 5-8

Roasted ephedra 50-90 fried bitter apricot seed 50-90 cordate houttuynia 150-260

Licorice root 50-90 and gypsum 150-260.

The traditional Chinese medicine composition disclosed by the invention preferably comprises the following raw material medicines in parts by weight:

forsythia fruit 150 honeysuckle 260 isatis root 150 rhubarb 55 patchouli 50

Rhizoma dryopteris crassirhizomae 260 rhodiola rosea 50 menthol crystal and ephedra herb 50 roasted with 8

Parched semen Armeniacae amarum 90, herba Houttuyniae 150, glycyrrhrizae radix 90 and Gypsum Fibrosum 150.

The traditional Chinese medicine composition disclosed by the invention also preferably comprises the following raw material medicines in parts by weight:

forsythia fruit 260 honeysuckle 150 isatis root 260 rhubarb 30 patchouli 90

Rhizoma Dryopteris Crassirhizomatis 150 radix Rhodiolae 90 Mentholum 5 herba Ephedrae 90

Parched semen Armeniacae amarum 50 herba Houttuyniae 260 Glycyrrhrizae radix 50 Gypsum Fibrosum 260.

forsythia fruit 170 honeysuckle 170 isatis root 170 rhubarb 34 patchouli 57

Rhizoma dryopteris crassirhizomae 170 rhodiola rosea 57 menthol crystal 5 fried ephedra 57

Parched semen Armeniacae amarum 57, herba Houttuyniae 170, glycyrrhrizae radix 57 Gypsum Fibrosum 170.

255 portions of forsythia, 255 portions of honeysuckle, 255 portions of isatis root, 255 portions of rhubarb, 51 portions of patchouli, 85

Rhizoma dryopteris crassirhizomae 255 rhodiola rosea 85 menthol crystal 7.5 mix-fried ephedra 85

Parched semen Armeniacae amarum 85 herba Houttuyniae 255 Glycyrrhrizae radix 85 Gypsum Fibrosum 255.

The traditional Chinese medicine of the invention can be replaced by traditional Chinese medicines with the same or similar effects, and the traditional Chinese medicines can be processed according to national traditional Chinese medicine processing standard or traditional Chinese medicine dictionary.

The active ingredients of the traditional Chinese medicine composition are prepared by the following steps:

(1) Weighing the traditional Chinese medicinal materials according to the weight proportion of the raw materials, and cleaning and selecting;

(2) Crushing herba Agastaches, extracting volatile oil with 5-8 times of water for 4 hr, and collecting volatile oil; filtering the extractive solution, discarding residue, and collecting filtrate;

(3) Extracting fructus forsythiae, herba Ephedrae preparata, herba Houttuyniae, and radix et rhizoma Rhei with 6-10 times of 50-90% ethanol for 2 times, each for 1-3 hr, mixing extractive solutions, filtering, and recovering ethanol to obtain filtrate;

(4) Adding 7-11 times of water into honeysuckle, gypsum, isatis root, male fern rhizome, liquorice and rhodiola rosea, decocting until boiling, adding fried bitter apricot seed, decocting for 2 times, 0.5-2.5 hours each time, combining the extracting solutions, filtering, combining the obtained filtrate with the filtrate obtained after the oil extraction of the patchouli in the step (2), concentrating into clear paste with the relative density of 1.10-1.15 measured at the temperature of 60 ℃, adding ethanol, adjusting the alcohol concentration to 70%, refrigerating, standing, filtering, and recovering the ethanol until no alcohol smell exists, thereby obtaining the clear paste for later use;

(5) Mixing the fluid extract obtained in step (4) and the ethanol extract obtained in step (3), concentrating to obtain fluid extract with relative density of 1.15-1.20 at 60 deg.C, and drying to obtain dry extract powder;

the dry paste powder obtained in the step (5), the volatile oil obtained in the step (2) and the menthol jointly form the active ingredients of the traditional Chinese medicine composition.

The medicament of the invention is in the form of capsules, tablets, powders, granules, oral liquid, soft capsules, pills, tinctures, syrups, suppositories, gels, sprays or injections.

In order to make the above dosage forms possible, pharmaceutically acceptable excipients, such as: bulking agent, disintegrating agent, lubricant, suspending agent, binder, sweetener, correctant, antiseptic, matrix, etc. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crospolyvinylpyrrolidone, low-substituted hydroxypropylcellulose, croscarmellose sodium, etc.; the lubricant comprises: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the adhesive comprises starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; the sweetener comprises: saccharin sodium, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, and the like; the flavoring agent comprises: sweeteners and various essences; the preservative comprises: nipagin, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroacetidine acetate, eucalyptus oil, etc.; the matrix comprises: PEG6000, PEG4000, insect wax, etc.

The capsule is prepared by the following steps:

(6) Adding the dry paste powder obtained in the step (5) into proper pharmaceutically acceptable auxiliary materials for granulation;

(7) And (3) dissolving the menthol and the volatile oil obtained in the step (2) in ethanol, spraying the particles obtained in the step (6), sealing, uniformly mixing, and encapsulating to obtain the capsule.

The preparation method of the granules comprises the following steps:

(1) Weighing the traditional Chinese medicinal materials according to the weight proportion of the raw materials, cleaning, and cutting off the traditional Chinese medicinal materials as appropriate;

(4) Adding 7-11 times of water into honeysuckle, gypsum, isatis root, male fern rhizome, liquorice and rhodiola rosea, decocting until boiling, adding fried bitter apricot kernel, decocting for 2 times, 0.5-2.5 hours each time, combining the extracting solutions, filtering, combining the obtained filtrate with the filtrate obtained after oil extraction of the patchouli in the step (2), concentrating into clear paste with the relative density of 1.10-1.15 measured at the temperature of 60 ℃, adding ethanol, adjusting the alcohol concentration to be 70%, refrigerating, standing, filtering, and recovering the ethanol until no alcohol smell exists, thereby obtaining the clear paste for later use;

(5) Mixing the fluid extract obtained in step (4) and the ethanol extract obtained in step (3), and concentrating to obtain soft extract with relative density of 1.25-1.35 at 60 deg.C;

(6) Adding appropriate pharmaceutically acceptable adjuvants into the soft extract obtained in step (5), and granulating;

(7) And (3) dissolving the menthol and the volatile oil obtained in the step (2) in ethanol, spraying the granules obtained in the step (6), sealing, uniformly mixing and bagging to obtain the menthol-containing capsule.

The preparation method of the preferred granules comprises the following steps:

(2) Crushing herba Agastaches, extracting volatile oil with 6 times of water for 4 hr, and collecting volatile oil; filtering the extractive solution, discarding residue, and collecting filtrate;

(3) Extracting fructus forsythiae, herba Ephedrae preparata, herba Houttuyniae, and radix et rhizoma Rhei with 8 times of 70% ethanol for 2 times, 2 hr for the first time, and 1.5 hr for the second time, mixing extractive solutions, filtering, and recovering ethanol to obtain filtrate;

(4) Adding 9 times of water into honeysuckle, gypsum, isatis root, male fern rhizome, liquorice and rhodiola rosea, decocting until boiling, adding fried bitter apricot seed, decocting for 2 times, wherein the first time is 1.5 hours, and the second time is 1 hour, combining the extracting solutions, filtering, combining the obtained filtrate with the water filtrate obtained after the oil extraction of the pogostemon cablin in the step (2), concentrating into clear paste with the relative density of 1.10-1.15 measured at the temperature of 60 ℃, adding 95% ethanol, adjusting the alcohol concentration to 70%, refrigerating, standing, filtering, and recovering the ethanol until no alcohol smell exists, so as to obtain clear paste filtrate;

(6) Adding a proper amount of pharmaceutically acceptable auxiliary materials into the thick paste obtained in the step (5), granulating, and finishing granules for later use;

(7) And (3) screening out fine powder from the granules obtained in the step (6), adding ethanol into the menthol and the volatile oil obtained in the step (2) for dissolving, spraying the fine powder, uniformly mixing with the granules obtained in the step (6), sealing for half an hour, and bagging to obtain the menthol crystal.

The preparation method of other dosage forms of the medicine comprises the following steps: the raw materials are weighed according to the proportion and prepared by a conventional preparation method, for example, a preparation process recorded in Vanbitn traditional Chinese medicine pharmacy (1 st 12 months in 1997 of Shanghai scientific Press) and prepared into a pharmaceutically acceptable conventional preparation.

Detailed Description

The following examples are intended to illustrate the preparation of the medicaments according to the invention, but they are not intended to limit the scope of the invention in any way.

Example 1

Prescription:

255 g of forsythia, 255 g of honeysuckle, 255 g of isatis root, 255 g of rhubarb and 51 g of rhubarb

Pogostemon cablin 85 g male fern rhizome 255 g rhodiola rosea 85 g menthol crystal 7.5 g

85 g of mix-fried ephedra herb, 85 g of fried bitter apricot seed, 85 g of heartleaf houttuynia herb and 255 g of heartleaf houttuynia herb

Licorice root, radix Glycyrrhizae 85 g and 255 g of gypsum.

The preparation method comprises the following steps:

(1) Weighing the traditional Chinese medicinal materials according to the prescription, and cleaning;

(4) Adding 9 times of water into honeysuckle, gypsum, isatis root, male fern rhizome, liquorice and rhodiola rosea, decocting until boiling, adding fried bitter apricot seed, decocting for 2 times, wherein the first time is 1.5 hours, and the second time is 1 hour, combining the extracting solutions, filtering, combining the obtained filtrate with the filtrate obtained after the oil extraction of the pogostemon cablin in the step (2), concentrating into clear paste with the relative density of 1.10-1.15 measured at the temperature of 60 ℃, adding ethanol, adjusting the alcohol concentration to 70%, refrigerating and standing for 24 hours, filtering, and recovering the ethanol until no alcohol smell exists, thereby obtaining the clear paste for later use;

(5) Mixing the fluid extract obtained in step (4) and the ethanol extract obtained in step (3), concentrating to obtain fluid extract with relative density of 1.15-1.20 at 60 deg.C, and spray drying to obtain dry extract powder;

(6) Adding 142 g of starch into the dry paste powder obtained in the step (5), and granulating by using 85% ethanol;

(7) And (3) dissolving the menthol and the volatile oil obtained in the step (2) in ethanol, spraying the particles obtained in the step (6), sealing, uniformly mixing, and filling into 1000 capsules to obtain the capsule.

Example 2

Prescription:

260 g of forsythia, 150 g of honeysuckle, 260 g of isatis root, 260 g of rhubarb and 30 g of rhubarb

Patchouli 90 g male fern rhizome 150 g rhodiola root 90 g menthol 5g

Mix-fried ephedra 90 g, bitter apricot kernel 50 g, cordate houttuynia 260 g

Licorice root 50 g plaster 260 g.

The preparation method comprises the following steps:

(2) Crushing herba Agastaches, extracting volatile oil with 5 times of water for 4 hr, and collecting volatile oil; filtering the extractive solution, discarding residue, and collecting filtrate;

(3) Extracting fructus forsythiae, herba Ephedrae preparata, herba Houttuyniae, and radix et rhizoma Rhei with 6 times of 50% ethanol for 2 times, 1 hr for the first time, and 2.5 hr for the second time, mixing extractive solutions, filtering, and recovering ethanol to obtain filtrate;

(4) Adding 7 times of water into honeysuckle, gypsum, isatis root, male fern rhizome, liquorice and rhodiola rosea, decocting until boiling, adding fried bitter apricot seed, decocting for 2 times, 1.5 hours for the first time and 1 hour for the second time, combining the extracting solutions, filtering, combining the obtained filtrate with the filtrate obtained after oil extraction of the pogostemon cablin in the step (2), concentrating into clear paste with the relative density of 1.10-1.15 measured at 60 ℃, adding ethanol, adjusting the alcohol concentration to 70%, refrigerating and standing for 24 hours, filtering, recovering the ethanol until no alcohol taste exists, and obtaining the clear paste for later use;

(6) Adding 151 g of starch into the dry paste powder obtained in the step (5), and granulating by using 85% ethanol;

(7) And (3) adding menthol and the volatile oil obtained in the step (2) into ethanol for dissolving, spraying the granules obtained in the step (6), sealing, uniformly mixing, and pressing into tablets to obtain 935 tablets.

Example 3

Prescription:

fructus forsythiae 185 g, honeysuckle 179 g, radix isatidis 190 g, rhubarb 36 g

Patchouli 60 g male fern rhizome 170 g rhodiola root 57 g menthol 5g

Chinese ephedra 60 g, bitter apricot kernel 60 g, cordate houttuynia 170 g

Licorice root 60 g plaster 170 g.

The preparation method comprises the following steps:

(2) Crushing herba Agastaches, adding 6 times of water to extract volatile oil for 4 hr, and collecting volatile oil with oil yield of 0.33; filtering the extract, discarding the residue, and collecting the water extract;

(4) Adding 9 times of water into honeysuckle, gypsum, isatis root, male fern rhizome, liquorice and rhodiola rosea, decocting until boiling, adding fried bitter apricot seed, decocting for 2 times, wherein the first time is 1.5 hours, and the second time is 1 hour, combining the extracting solutions, filtering, combining the obtained filtrate with the water extract obtained after the oil extraction of the pogostemon cablin in the step (2), concentrating into clear paste with the relative density of 1.10-1.15 measured at the temperature of 60 ℃, adding 95% ethanol, adjusting the alcohol concentration to 70%, refrigerating and standing for 24 hours, filtering, and recovering the ethanol until no alcohol smell exists, thus obtaining the clear paste for later use;

(5) Mixing the fluid extract obtained in step (4) and the ethanol extract obtained in step (3), concentrating to obtain soft extract with relative density of 1.25-1.30 at 60 deg.C, and spray drying to obtain dry extract powder;

(6) And (3) preparing the dry paste powder obtained in the step (5), the volatile oil obtained in the step (2) and the menthol into pills according to a conventional method to obtain pills 905.

Example 4:

the formula of the raw material medicine is as follows:

weeping forsythia 170 g honeysuckle flower 170 g mix-fried Chinese ephedra 57 g stir-fried bitter apricot kernel 57 g

170 g of gypsum, 170 g of isatis root, 170 g of male fern rhizome, 170 g of cordate houttuynia

Patchouli 57 g rhubarb 34 g rhodiola 57 g menthol 5.0 g

Licorice root, radix Glycyrrhizae 57 g

The preparation method comprises the following steps:

the extraction process comprises the following steps:

(1) Weighing the traditional Chinese medicinal materials according to the prescription, cleaning, and cutting according to the requirement;

(2) Extracting herba Agastaches with 6 times of water for 4 hr, collecting volatile oil with oil yield of 0.33%, filtering the extractive solution, removing residue, and collecting water extractive solution;

(3) Extracting fructus forsythiae, herba Ephedrae preparata, herba Houttuyniae, and radix et rhizoma Rhei with 8 times of 70% ethanol for 2 times, 2 hr for the first time, and 1.5 hr for the second time, filtering the extractive solutions, mixing the filtrates, and recovering ethanol until no ethanol smell exists;

(4) Adding 9 times of water into honeysuckle, fried bitter almond, gypsum, isatis root, male fern rhizome, liquorice and rhodiola rosea, decocting until boiling, adding the fried bitter almond, decocting for 2 times, wherein the first time is 1.5 hours, and the second time is 1 hour, filtering an extracting solution, combining filtrates, simultaneously adding an aqueous solution obtained after oil extraction of the pogostemon cablin in the step (2), concentrating into clear paste with the relative density of 1.10-1.15 determined at 60 ℃, adding 95% ethanol, stirring while adding until the alcohol concentration is 70%, refrigerating and standing for 24 hours, filtering, recovering ethanol from the filtrate until no alcohol taste exists, combining with an alcohol extracting solution, concentrating into thick paste with the relative density of 1.25-1.35 determined at 60 ℃ for later use;

(II) preparation process:

(5) The formula of the preparation is as follows: 335.5g of thick paste obtained in step (4) and 5g of menthol

0.2ml of patchouli oil obtained in the step (2), 342.5g of powdered sugar, 514.0g of dextrin

(6) Granulating: mixing sugar powder and dextrin, making soft mass with soft extract as binder, granulating with 14 mesh screen, oven drying at 60-65 deg.C, and grading with 10 mesh screen;

(7) Subpackaging: sieving to obtain fine powder, adding appropriate amount of ethanol into Mentholum and herba Agastaches volatile oil, dissolving, spraying into the fine powder, mixing with the granule, sealing for half an hour, and packaging to obtain 1000g granule.

Description of the drawings:

FIG. 1: EC (EC) ₅₀ Calculation results

FIG. 2: pulmonary index at day 3 (A) and day 6 (B) after viral infection in groups of mice

FIG. 3: pulmonary viral load of mice on days 3 (A) and 6 (B) post-virus infection

Copy number of Pneumovirus subgenomic RNA in mice on day 3 post-virus infection

B copy number of Pneumovirus subgenomic RNA on day 6 post-virus infection in mice

FIG. 4: lung HE staining score of mice on days 3 (A) and 6 (B) post-virus infection

FIG. 5:3dpi pathological change of lung (HE, 2 x) of mice with the pharmaceutical composition of the invention, ruler =100 μm

Mild pneumonia in 3 mice and moderate to severe pneumonia in 2 mice

FIG. 6: pathological changes in lungs (HE, 2 ×) in 3dpi PBS group mice (scale =100 μm)

Moderate pneumonia in 4 mice, moderate to severe pneumonia in 1 mouse

FIG. 7:6dpi pathological change of lung (HE, 2 x) of mice with the pharmaceutical composition of the invention, and scale =100 μm

Mild pneumonia in 3 mice and moderate to severe pneumonia in 2 mice

FIG. 8: pathological changes in lungs (HE, 2 ×) in 6dpi PBS group mice, scale =100 μm

Mild pneumonia in 2 mice and moderate to severe pneumonia in 3 mice

Experimental example:

to confirm the efficacy of the pharmaceutical composition of the present invention for preventing and treating pneumonia caused by S protein D614G mutant type of new coronavirus infection, the following clinical trial studies were conducted using the particles prepared according to the method of example 4 (hereinafter referred to as the present pharmaceutical composition):

1. the data and method are as follows:

test content 1: the medicinal composition particle inhibits the replication effect of wild type and S protein D614G mutant type new coronavirus in a continuous cell line

The purpose of the test is as follows: evaluation of the inhibitory Effect of the pharmaceutical composition of the present invention on wild type and S protein D614G mutant New coronavirus at cellular level

Test time: 18 days in 9 months of 2020 to 7 days in 10 months of 2020

Test materials

The invention relates to a medicine composition, a preparation method of the medicine composition with the concentration of 12 mg/mL comprises the following steps: 120 mg of the drug is dissolved in 1mL of DMSO, 9 mL of DMEM medium containing 100U/mL of double antibody (cyan, streptomycin) and 2% FBS is added, the mixture is vortexed for 5 min, and after the mixture is placed in a refrigerator at 4 ℃ for 12 h, the mixture is vortexed for 5 min again, and then the mixture is filtered by a 0.22 mu M filter.

Cells

VERO-E6 cells

4.3 Virus

4.3.1 Wild-type New coronaviruses [ 2019-nCoV, wild-type (Wt) ];

4.3.2S protein mutant New coronaviruses [ 2019-nCoV, D614G ]

4.4 reagents

4.4.1 viral dilutions: DMEM medium containing 100U/mL double antibody (penicillin, streptomycin) at final concentration and no serum;

4.4.2 drug dilutions: DMEM medium containing 100U/mL double antibody (penicillin, streptomycin) at final concentration and no serum;

4.4.3 cell culture: DMEM medium containing 100U/mL double antibody (cyan, streptomycin) and 10% FBS at final concentration;

4.4.4 Fetal Bovine Serum (FBS) (cat No. 10270-044, brand: GIBCO);

4.4.5DMEM medium (cat # C11995500BT, brand: GIBCO);

4.4.61 XPBS (pH 7.4) (cat # C10010500BT, brand: GIBCO);

4.4.70.25% pancreatin-EDTA (Trypsin Trypsin 2.5g (cat # 0458-100 g, brand: amresco), EDTA-2Na 0.34 g (cat # 0105-100 g, brand: amresco), dissolved in 1000 mL 1 XPBS, filtered through 0.22 uM filter);

4.4.8DMSO (Cat number D2650-100 mL, brand: sigma)

4.5 Consumable material

96-well flat-bottomed cell culture plates (cat # 3599, brand: costar); 15 mL centrifuge tube (Cat: CFT-011-150, brand: BIOFIL); 50 mL centrifuge tube (Cat: CFT-011-150, brand: BIOFIL); a pipette tip (Cat: T-200-Y-R-S, brand: AXYGEN); 10 mL pipette (goods number 4488, brand: costar)

4.6 Instrument

A cell incubator; -80 ℃ refrigerator; a centrifuge; inverting the microscope; a biological safety cabinet; an electric pipette; a pipette gun; 8 gun rows; a water bath kettle; oscillating instrument

5. Experimental methods

5.1 cells

VERO-E6 cells, after a monolayer of confluent particles, were subjected to an antiviral assay of the particles of the pharmaceutical composition of the invention.

5.2 viruses

New coronaviruses (Wt and D614G), 100 TCID50/well virus seeded cells.

5.3 medicaments

The experimental concentration of the particles of the pharmaceutical composition is 75, 37.5, 18.75, 9.375, 4.69, 2.34, 1.17, 0.59, 0.29 and 0.15 microgram/ml, which are 10 concentrations in total. After the virus and the drug are incubated for 1 h at 37 ℃, cells are inoculated, and a normal cell control and a positive virus control are set at the same time.

5.4 CPE Observation

After 72 h of viral infection, CPE was observed

6. Results

6.1CPE Observation: after 72 h of virus infection, the normal cell control group has no CPE; the CPE is obvious in a virus control group; different drug concentrations showed different inhibitory effects on CPE, as shown in table 1 and table 2.

6.2 median effective concentration (EC 50) and Therapeutic Index (TI) calculation: calculating the half effective concentration (EC 50) according to the results of tables 1 and 2, as shown in FIG. 1, the EC50 of the capsule particles of the pharmaceutical composition of the invention on Wt virus on Vero E6 cells is 17.77 mug/ml; the EC50 of the capsule particles of the pharmaceutical composition of the invention on the D614G virus is 35.85 mug/ml.

Based on the toxicity test results (TD 50 of 723.5 μ g/ml) of the same batch of particles of the pharmaceutical composition of the present invention on VERO-E6 cells, the therapeutic index (TI = TD50/EC 50) was calculated. The results show that the therapeutic index of the particles of the pharmaceutical composition of the present invention is 40.71 for the Wt virus and 20.18 for the D614G virus.

7. Conclusion

The drug composition particles of the invention with a certain concentration can inhibit the formation of CPE on Vero E6 cells by the new coronavirus (Wt and D614G mutant new coronavirus).

Test content 2: the effect of the pharmaceutical composition of the invention on inhibiting S protein D614G mutant neocoronavirus infection is researched by using a mouse infection model

1 purpose of the test: research on the effect of the pharmaceutical composition of the invention on inhibiting the replication of S protein D614G mutant new coronavirus in mice

2, test time: 5 days 1 month to 21 days 1 month in 2021

3 test materials

3.1 Animals: c57BL/6J, female, 6 to 8 weeks old, weight 18 to 20g

3.2 Virus: s protein mutant type new coronavirus [ 2019-nCoV, D614G ]

3.3 0.5% sodium carboxymethylcellulose (CMC-Na) solution: 0.5 g of CMC-Na is weighed and 100 ml of distilled water is added (in the preparation process, the distilled water is firstly added, then the CMC-Na is slowly added, and the mixture is dissolved overnight under magnetic stirring at 37 ℃.

3.4 pharmaceutical composition of the invention (82 mg/mL): 820 mg of the pharmaceutical composition is weighed, 1mL of 0.5% CMC-Na is used for dissolving the pharmaceutical composition in a mortar, the grinding is carried out for 1 to 3 min, then PBS (double antibody (streptomycin) containing 100U/mL) is used for 10 times dilution to reach the final concentration of 82mg/mL, and the mouse is administrated according to 0.82 g/kg.

4 test method

4.1 Animal grouping and specific experimental protocol: mice were randomly divided into 2 groups, which were a treatment group of the pharmaceutical composition of the present invention and a control group of viral infection, respectively.

4.1.1 treatment groups with the pharmaceutical composition of the invention: 13 mice

(1) Administration and counteracting toxic substance: the medicine composition of the invention is insufflated at 0.82g/kg 2 days before the infection of the virus, and the total volume of the enema is 200 mu L. After the drug composition of the invention is intragastrically administered on day 3, the drug composition is infected with virus (5 days before the infection with the virus, adenovirus is inoculated into hACE2 by nasal drip), and the drug administration is continued for 5 days after the infection with the virus, and the drug administration lasts for 8 days.

(2) Euthanasia and material selection: 5 mice were killed by dissection on day 3 and 6 post virus infection.

Blood is collected from the orbit, centrifuged at 4000 rpm, the upper serum (light yellow) is taken and subpackaged, and the mouse is marked and frozen at-80 ℃.

Weighing the lung of each mouse, and recording the weight of the lung, thereby calculating a lung index; fixing a small piece of the left lung of each mouse in 4% paraformaldehyde for pathological detection; grinding the rest lung part, wherein the grinding speed is 4 m/s, the cycle time is 10 s, the interval time is 10 s, the cycle times are 3 times, centrifuging at 7000 rpm of grinding fluid for 10 minutes, taking 140 mu L to perform nucleic acid extraction and qRT-PCR experiment, and freezing and storing the rest part in a refrigerator at-80 ℃.

(3) Clinical symptoms: and weighing the weight of the mice on the day of challenge, weighing the weight of the mice and observing clinical symptoms every day, and drawing a weight change curve of the mice until the 11 th day after virus infection.

4.1.2 Virus infection control group: 13 mice

(1) Administration and counteracting toxic substance: gavage of PBS and viral infection was performed at the same time points as the 4.1.1 treatment group with the pharmaceutical composition of the present invention;

(2) Euthanasia and material selection: 5 mice were killed by dissection on day 3 and day 6 after viral infection, and blood and organs were treated in the same manner as in 5.1.1 (2).

(3) Clinical symptoms: the weight of the mice and the observation of clinical symptoms were carried out daily, and the weight change curve of the mice was plotted until day 11 after the viral infection.

4.2 Specific experimental method

4.2.1 weight change curve: the weight of the mice was weighed daily, and a weight change curve was drawn according to the percentage of the weight of the mice in the initial weight of the experiment during the experiment. Compared with a virus infection control group, the influence of the medicaments such as the pharmaceutical composition of the invention on the weight change of mice after virus infection is analyzed.

4.2.2 Collecting serum: on day 3 and day 6 after infection with the virus, 5 mice were sacrificed from the corresponding groups, blood was collected from the orbit into 1.5mL EP tubes, centrifuged (4000 rpm,10 min), and the supernatant was aspirated into the EP tubes and stored in a refrigerator at-80 ℃.

4.2.3 Lung tissue treatment:

(1) Lung index: on days 3 and 6 after infection with the virus, the mice were sacrificed under anesthesia and 5 mice were dissected out for the respective groups. The mouse is fixed in a supine position, the ribs and pectoralis muscles at both sides are cut off, the thoracic cavity is exposed, the lung is taken out, tissues such as the trachea, the lung portal lymph node and the like are removed, the tissues are washed for 2 times by normal saline, and the surface moisture of the lung is sucked dry by filter paper, and the weight of the lung is weighed. The lung index was calculated from lung weight and mouse body weight.

Lung index = mouse lung weight (g)/mouse body weight (g) × 100%

(2) Tissue fixation and preservation: a small portion of the tissue sample from the left lung was fixed in 4% paraformaldehyde solution for subsequent use as HE.

(3) Pulmonary viral load assay: on days 3 and 6 after infection with the virus, the corresponding groups were sacrificed 5 mice, respectively. Grinding 5 mice lungs, centrifuging at 7000 rpm of grinding fluid for 10 minutes, taking 140 mu L of supernatant after centrifugation, performing nucleic acid extraction and qRT-PCR (quantitative reverse transcription-polymerase chain reaction) experiments, and detecting the copy numbers of N genes, ORF1ab genes and S genes in the grinding fluid by using (1) a biological original kit; (2) and detecting the copy number of the viral subgenomic RNA in the grinding fluid.

5. Results

5.1 Body weight changes in mice

After viral infection, mice in the non-dosed PBS group lost weight at 1dpi, and lost to the nadir (by 7.5%) at 2dpi, after which time weight began to rise. The body weight of the mice treated by the pharmaceutical composition starts to rise at 1dpi, and the body weight of the mice treated by the pharmaceutical composition is lower than that of the mice not administered with PBS after 7 dpi.

5.2 Change in pulmonary index

On days 3 and 6 after infection of the mice with the virus, the lung index of the treatment group of the pharmaceutical composition of the present invention was statistically insignificant (t-test, P > 0.05) compared to the non-administration PBS control group (fig. 2).

5.3 Lung viral load assay

On day 3 after infection of the mice with the virus, the pulmonary viral load of the mice in the treatment group with the pharmaceutical composition of the invention was statistically insignificant compared to the non-administered PBS group (fig. 3a, t-test, P > 0.05). On day 6 after infection with virus, the lung viral load of the treatment group with the pharmaceutical composition of the present invention was lower than that of the PBS group (FIG. 3B, rank sum test, P-cloth 0.05).

5.4 Pathological examination results of the treatment group and PBS control group (FIGS. 4, 5, 6, 7, 8)

6. Conclusion

6.1 analysis of the body weight change curve of mice showed: after viral infection, the body weight of the mice recovered and increased faster than that of the PBS-naive group.

6.2 Analysis of the lung index showed: the lung index of the treatment group was not statistically different from that of the non-administered PBS group.

6.3 Analysis of the lung viral load in mice showed: on day 6 post-infection, the pharmaceutical composition of the present invention has an inhibitory effect on the replication of the S protein mutant D614G neocoronavirus.

6.4 Histopathological analysis of mouse lung: on days 3 and 6 after infection, the lung pathological damage of the mice treated by the pharmaceutical composition of the invention is slightly lighter than that of the mice not administered with PBS.

The comprehensive analysis of the clinical symptom changes such as the mouse body weight after the comprehensive virus infection, the lung index, the lung virus load result and the HE staining result shows that: the pharmaceutical composition can inhibit the replication of S protein D614G mutant new coronavirus in the lung of a mouse.

Claims

1. The application of a traditional Chinese medicine composition in preparing S protein D614G mutant type new coronavirus resistant medicines is characterized by being prepared from the following raw material medicines in parts by weight:

150-260 parts of fructus forsythiae, 150-260 parts of honeysuckle, 150-260 parts of isatis root, 150-260 parts of rhubarb, 30-55 parts of rhubarb

Herba Agastaches 50-90 rhizoma Dryopteris Crassirhizomatis 150-260 radix Rhodiolae 50-90 Mentholum 5-8

Licorice root 50-90 and gypsum 150-260.

2. The application of the traditional Chinese medicine composition according to claim 1 is characterized by being prepared from the following raw material medicines in parts by weight:

forsythia fruit 150 honeysuckle 260 isatis root 150 rhubarb 55 patchouli 50

3. The application of the traditional Chinese medicine composition is characterized by being prepared from the following raw medicines in parts by weight:

forsythia fruit 260 honeysuckle 150 isatis root 260 rhubarb 30 patchouli 90

Rhizoma Dryopteris Crassirhizomatis 150 radix Rhodiolae 90 Mentholum 5 processed herba Ephedrae 90

4. The application of the traditional Chinese medicine composition according to claim 1 is characterized by being prepared from the following raw material medicines in parts by weight:

forsythia fruit 170 honeysuckle 170 isatis root 170 rhubarb 34 patchouli 57

5. The application of the traditional Chinese medicine composition according to claim 1 is characterized by being prepared from the following raw material medicines in parts by weight:

6. The use according to any one of claims 1 to 5, characterized in that the active ingredients of the Chinese medicinal composition are prepared by the following steps:

(1) Weighing the traditional Chinese medicinal materials according to the weight proportion of the raw materials, and cleaning;

(3) Extracting fructus forsythiae, herba Ephedrae preparata, herba Houttuyniae, and radix et rhizoma Rhei with 6-10 times of 50-90% ethanol for 2 times, each for 1-3 hr, mixing extractive solutions, filtering, and recovering ethanol to obtain extractive solution;

7. Use according to any one of claims 1 to 5, characterized in that the pharmaceutical dosage form is a capsule, tablet, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection.

8. Use according to claim 7, characterized in that the capsules are prepared by the following steps:

(3) Extracting fructus forsythiae, herba Ephedrae preparata, herba Houttuyniae, and radix et rhizoma Rhei with 6-10 times of 50-90% ethanol for 2 times, each for 1-3 hr, mixing extractive solutions, filtering, recovering ethanol, and collecting filtrate;

9. Use according to claim 7, characterized in that the granules are made by the following steps:

(1) Weighing the traditional Chinese medicinal materials according to the weight proportion of the raw materials, cleaning, and cutting according to the condition;

(5) Mixing the fluid extract obtained in step (4) with the ethanol extractive solution obtained in step (3), concentrating to obtain fluid extract with relative density of 1.15-1.20 at 60 deg.C, and drying to obtain dry extract powder;

(6) Adding a proper amount of pharmaceutically acceptable auxiliary materials into the dry paste powder obtained in the step (5) for granulation;

10. The use according to claim 9, characterized in that the preparation method of the granules is made by the following steps:

(2) Crushing herba Agastaches, extracting volatile oil with 6 times of water for 4 hr, and collecting volatile oil with oil yield of 0.33 + -0.05%; filtering the extract, discarding the residue, and collecting the water extract;

(4) Adding 9 times of water into honeysuckle, gypsum, isatis root, male fern rhizome, liquorice and rhodiola rosea, decocting until boiling, adding fried bitter apricot seed, decocting for 2 times, wherein the first time is 1.5 hours, and the second time is 1 hour, combining the extracting solutions, filtering, combining the obtained filtrate with the water extract obtained after the oil extraction of the pogostemon cablin in the step (2), concentrating into clear paste with the relative density of 1.10-1.15 measured at the temperature of 60 ℃, adding 95% ethanol, adjusting the alcohol concentration to 70%, refrigerating, standing, filtering, and recovering the ethanol until no alcohol smell exists, thus obtaining the clear paste;

(7) And (3) screening out fine powder from the granules obtained in the step (6), adding menthol and the volatile oil obtained in the step (2) into ethanol for dissolving, spraying the fine powder, uniformly mixing with the granules obtained in the step (6), sealing for half an hour, and bagging to obtain the menthol-sealed capsule.