WO2022237145A1

WO2022237145A1 - Use of traditional chinese medicine composition in preparation of drug for resisting novel coronavirus with d614g mutation in s protein

Info

Publication number: WO2022237145A1
Application number: PCT/CN2021/136478
Authority: WO
Inventors: 贾振华
Original assignee: 石家庄以岭药业股份有限公司
Priority date: 2021-05-11
Filing date: 2021-12-08
Publication date: 2022-11-17
Also published as: CN115317542A

Abstract

The use of a traditional Chinese medicine composition in the preparation of a drug for resisting novel coronavirus with a D614G mutation in the S protein. The traditional Chinese medicine composition is prepared from Forsythiae fructus, Lonicerae japonicae flos, Isatidis radix, Rhei radix et rhizoma, Pogostemonis herba, Dryopteridis crassirhizomatis rhizoma, Rhodiolae crenulatae radix et rhizoma, L-menthol, Ephedrae herba, Armeniacae semen amarum, Houttuyniae herba, Glycyrrhizae radix et rhizoma and Gypsum fibrosum; and has the effects of removing pathogens, relieving symptoms and regulating immunity.

Description

Application of a traditional Chinese medicine composition in the preparation of anti-S protein D614G mutant new coronavirus drug

technical field

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

Background technique

The 2019 novel coronavirus (2019-nCoV) was named by the World Health Organization (WHO) in January 2020. Coronaviruses are a large family of viruses known to cause colds as well as more serious illnesses such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has never been found in humans before.

SARS-CoV-2 is transmitted by aerosol, surface contact, and fecal-oral routes. Infection with the virus can cause a series of symptoms such as fever, cough and general malaise, and more severe cases can lead to acute respiratory distress syndrome and even death. Coronaviruses are rapidly evolving viruses that have the property of infecting a wide range of hosts through different changes in the genome. Many mutant strains of SARS-CoV-2 have emerged, among which the mutant strain D614G has rapidly become a global epidemic strain and has attracted widespread attention.

SARS-CoV-2 is a positive-strand RNA enveloped virus. The spike protein on the surface of the virus exists in the form of a trimer, and the S protein is responsible for binding the receptor and performing membrane fusion to complete the virus invasion process. The mutation (D614G) in which the 614th aspartic acid (Asp) becomes glycine (Glycine, Gly) outside the binding region quickly became a global popular strain and attracted widespread attention. As of November 30, 2020, the D614G mutant accounted for 87% of the SARS-CoV-2 sequences published in the CoV-GLUE database.

Whether using pseudovirus to infect animal experiments or human infection studies, it is shown that the replication and infectivity of D614G are higher than those of wild strains. Serum levels of IgG, IgM, or IgA did not change in infected individuals. Existing experimental data cannot explain the relationship between D614G's enhanced transmissibility and virulence is still complex and may be affected by age, gender and other comorbidities. It is unclear whether the lowest infectious dose of D614G in humans will be more effective. Low.

The D614G mutation has become the prevailing strain of SARS-CoV-2, and more data show that this mutation enhances replication and infectivity. The SARS-CoV-2 lineage still accumulates nucleotide mutations at a rate of 1-2 mutations per month, and most of the existing strains are combined mutations based on D614G. Therefore, continuous monitoring is required for the mutation of D614G combined with other sites, and attention should also be paid to whether vaccines and antibodies have broad-spectrum protection against mutant strains. It is imminent to carry out the research and development of anti-D614 mutant strain drugs.

The present invention is an improved invention based on the ZL03143211 patent, and the content recorded in this patent document is quoted in full here. The above-mentioned patent does not disclose the application of the traditional Chinese medicine composition in anti-S protein D614G mutant new coronavirus.

Contents of the invention

The invention provides an application of a traditional Chinese medicine composition in the preparation of anti-S protein D614G mutant new coronavirus medicine, the traditional Chinese medicine composition is made of the following raw materials by weight:

The weight ratio of the crude drug of the Chinese medicine composition of the present invention is preferably:

The weight ratio of the crude drug of the Chinese medicine composition of the present invention is also preferably:

The traditional Chinese medicine of the present invention can be replaced by traditional Chinese medicines with the same or similar effects, and these medicinal materials can be processed according to the "National Traditional Chinese Medicine Processing Standard" or "Chinese Medicine Dictionary".

The active ingredient of Chinese medicine composition of the present invention is made by following steps:

(1) Take the Chinese medicinal materials according to the weight ratio of the crude drug, and select them;

(2) Patchouli is broken, add 5-8 times the amount of water to extract the volatile oil, the oil extraction time is 4 hours, collect the volatile oil, and set aside; after the extract is filtered, the residue is discarded, and the filtrate is set aside;

(3) forsythia, roasted ephedra, houttuynia cordata, rhubarb, extracted twice with 50-90% ethanol of 6-10 times, each time for 1-3 hours, the extracts were combined and filtered, the ethanol was recovered, and the filtrate was used for subsequent use;

(4) Honeysuckle, gypsum, isatidis, Mianma Guanzhong, licorice, rhodiola, add 7-11 times the amount of water and boil until boiling, add fried bitter almonds, decoct twice, each time for 0.5-2.5 hours, extract Liquid is merged and filtered, and the filtrate after gained filtrate and step (2) Patchouli extract oil is merged, is concentrated into the clear ointment that measures relative density at 60 ℃ and is 1.10-1.15, adds ethanol, is adjusted to alcohol concentration and is 70%, refrigerated Place, filter, recover ethanol until there is no alcohol smell, and get clear paste for later use;

(5) Combine the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrate to a clear paste with a relative density of 1.15-1.20 at 60° C., dry to obtain dry paste powder, and set aside;

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

The dosage form of the medicine of the present invention is capsule, tablet, powder, granule, oral liquid, soft capsule, pill, tincture, syrup, suppository, gel, spray or injection.

In order to realize the above dosage forms, it is necessary to add pharmaceutically acceptable adjuvants when preparing these dosage forms, such as: fillers, disintegrating agents, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives , matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, Cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, croscarmellose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agent Including: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropylmethylcellulose, etc.; binders include starch slurry, polyvinylpyrrolidone, hydroxypropylmethylcellulose, etc.; sweeteners include: Sodium saccharin, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various essences; preservatives include: paraben, benzoic acid, sodium benzoate, sorbic acid and other Salts, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil, etc.; substrates include: PEG6000, PEG4000, insect wax, etc.

Wherein capsule is made by following steps:

(5) The clear cream obtained in step (4) is combined with the alcohol extract obtained in step (3), concentrated to a clear cream with a relative density of 1.15-1.20 measured at 60° C., dried to obtain a dry cream powder, and set aside;

(6) adding appropriate pharmaceutically acceptable auxiliary materials to the dry cream powder obtained in step (5) to granulate;

(7) Add ethanol to dissolve menthol and the volatile oil obtained in step (2), spray into the granules obtained in step (6), airtightly mix, pack into capsules, and obtain.

Wherein the preparation method of granule is made by following steps:

(1) Weigh the Chinese herbal medicines according to the weight ratio of the raw materials, select them cleanly, and break them as appropriate;

(4) Honeysuckle, gypsum, isatidis, Mianma Guanzhong, licorice, rhodiola, add 7-11 times the amount of water and boil until boiling, add fried bitter almonds, decoct twice, each time for 0.5-2.5 hours, extract The liquids are combined and filtered, and the filtrate obtained from the step (2) patchouli extract oil is combined with the filtrate, and concentrated into a clear paste with a relative density of 1.10-1.15 when measured at 60° C., adding ethanol, adjusting to an alcohol concentration of 70%, and refrigerated Place, filter, recover ethanol until there is no alcohol smell, and get clear paste for later use;

(5) Combine the clear ointment obtained in step (4) with the ethanol extract obtained in step (3), concentrate to a thick ointment with a relative density of 1.25-1.35 at 60° C., and set aside;

(6) adding appropriate pharmaceutically acceptable auxiliary materials to the thick paste obtained in step (5) to granulate;

(7) Add menthol and the volatile oil obtained in step (2) into ethanol to dissolve, spray into the particles obtained in step (6), airtightly mix, pack into bags, and obtain.

The preparation method of preferred granule is:

(2) Patchouli is broken, add 6 times the amount of water to extract the volatile oil, the oil extraction time is 4 hours, collect the volatile oil, and set aside; after the extract is filtered, the residue is discarded, and the filtrate is set aside;

(3) forsythia, sun-burned ephedra, houttuynia cordata, rhubarb, extracted twice with 8 times the amount of 70% ethanol, the first time for 2 hours, the second time for 1.5 hours, the extracts were combined and filtered, the ethanol was recovered, and the filtrate was used for subsequent use;

(4) Honeysuckle, gypsum, isatidis, Mianmaguanzhong, licorice, rhodiola rosea, add 9 times the amount of water and boil until boiling, add fried bitter almonds, and boil for 2 times, the first time is 1.5 hours, the second time is 1 hour hour, the extracts are combined and filtered, and the water filtrate after the gained filtrate is combined with the step (2) Patchouli oil extraction, is concentrated into a clear paste that measures the relative density at 60°C and is 1.10-1.15, adds 95% ethanol, and adjusts to alcohol The concentration is 70%, put it in cold storage, filter, recover the ethanol until it has no alcohol smell, and obtain the clear cream filtrate;

(6) Add the thick paste obtained in step (5) into appropriate pharmaceutically acceptable adjuvants to granulate, granulate, and set aside;

(7) Sieve the granules obtained in step (6) out of fine powder, add menthol and the volatile oil obtained in step (2) into ethanol to dissolve, spray into the fine powder, after mixing evenly, mix evenly with the granules obtained in step (6), and seal the semi- Hours, bagged, that is.

The preparation method of other dosage forms of the medicine of the present invention is: take the crude drug in proportion, and prepare by a conventional preparation method, for example, the preparation process recorded in Fan Biting's "Chinese Medicine Pharmacy" (Shanghai Science Press, December 1997, 1st edition), Made into pharmaceutically acceptable conventional dosage forms.

Description of drawings

Figure 1: _EC50 calculation results.

Figure 2: Lung index of mice in each group on day 3 (A) and day 6 (B) after virus infection.

Figure 3: Viral load in the lungs of mice on day 3 (A) and day 6 (B) after virus infection;

The copy number of lung virus subgenomic RNA of A mouse on the 3rd day after virus infection;

B The copy number of viral subgenomic RNA in the lungs of mice on the 6th day after virus infection.

Figure 4: HE staining scores of lungs of mice on day 3 (A) and day 6 (B) after virus infection.

Figure 5: 3dpi pathological changes (HE, 2×) of the mouse lungs of the pharmaceutical composition group of the present invention, scale bar=100 μm;

Three mice had mild pneumonia, and two mice had moderate to severe pneumonia.

Figure 6: Pathological changes in the lungs of mice in the 3dpi PBS group (HE, 2×), scale bar = 100 μm;

Four mice had moderate pneumonia, and one mouse had moderate to severe pneumonia.

Fig. 7: 6dpi pathological changes (HE, 2×) of mouse lungs in the pharmaceutical composition group of the present invention, scale bar=100 μm;

Three mice had mild pneumonia, and two mice had moderate to severe pneumonia.

Figure 8: Pathological changes in the lungs of mice in the 6dpi PBS group (HE, 2×), scale bar = 100 μm;

2 mice had mild pneumonia and 3 mice had moderate to severe pneumonia.

Detailed ways

The following examples are used to illustrate the preparation of the medicament of the present invention, but they cannot limit the scope of the present invention in any way.

Example 1

prescription:

Preparation:

(1) Take Chinese medicinal materials according to the above prescription, and select them;

(4) Honeysuckle, gypsum, isatidis, Mianmaguanzhong, licorice, rhodiola rosea, add 9 times the amount of water and boil until boiling, add fried bitter almonds, and boil for 2 times, the first time is 1.5 hours, the second time is 1 hour hours, the extracts are combined and filtered, and the resulting filtrate is combined with the filtrate after step (2) Patchouli oil extraction, and concentrated into a clear paste with a relative density of 1.10-1.15 when measured at 60° C. Add ethanol to adjust to an alcohol concentration of 70 %, refrigerated for 24 hours, filtered, recovered ethanol until it has no alcohol smell, and cleared ointment for later use;

(5) The clear ointment obtained in step (4) is combined with the alcohol extract obtained in step (3), concentrated to a clear ointment with a relative density of 1.15-1.20 measured at 60° C., spray-dried to obtain dry ointment powder, and set aside;

(6) Add 142 grams of starch to the dry paste powder obtained in step (5), and granulate with 85% ethanol;

(7) Add menthol and volatile oil obtained in step (2) into ethanol to dissolve, spray into the granules obtained in step (6), airtightly mix, pack into 1000 capsules, and obtain final product.

Example 2

prescription:

Preparation:

(2) Patchouli is broken, add 5 times the amount of water to extract the volatile oil, the oil extraction time is 4 hours, collect the volatile oil, and set aside; after the extract is filtered, the residue is discarded, and the filtrate is set aside;

(3) forsythia, sun-burned ephedra, houttuynia cordata, rhubarb, extracted twice with 6 times the amount of 50% ethanol, the first time for 1 hour, and the second time for 2.5 hours, the extracts were combined and filtered, the ethanol was recovered, and the filtrate was used for subsequent use;

(4) Honeysuckle, gypsum, isatidis, Mianmaguanzhong, licorice, rhodiola, add 7 times the amount of water and boil until boiling, add fried bitter almonds, and boil twice, the first time 1.5 hours, the second time 1 hours, the extracts are combined and filtered, and the resulting filtrate is combined with the filtrate after step (2) Patchouli oil extraction, and concentrated into a clear paste with a relative density of 1.10-1.15 when measured at 60° C. Add ethanol to adjust to an alcohol concentration of 70 %, refrigerated for 24 hours, filtered, recovered ethanol until it has no alcohol smell, and cleared ointment for later use;

(5) Combine the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrate to a clear paste with a relative density of 1.15-1.20 measured at 60° C., spray dry to obtain dry paste powder, and set aside;

(6) Add 151 grams of starch to the dry paste powder obtained in step (5), and granulate with 85% ethanol;

(7) Add ethanol to dissolve the menthol and the volatile oil obtained in step (2), spray into the granules obtained in step (6), seal, mix, and press into tablets to obtain 935 tablets.

Example 3

prescription:

Preparation:

(2) Patchouli is broken, add 6 times the amount of water to extract the volatile oil, the oil extraction time is 4 hours, collect the volatile oil, the oil yield is 0.33, and set aside; after the extract is filtered, the residue is discarded, and the water extract is set aside;

(4) Honeysuckle, gypsum, isatidis, Mianma Guanzhong, licorice, rhodiola, add 9 times the amount of water and boil until boiling, add fried bitter almonds, and boil twice, the first time 1.5 hours, the second time 1 hour, the extracts are combined and filtered, and the water extract after the obtained filtrate is combined with the step (2) Patchouli oil extraction oil, is concentrated into a clear paste that measures a relative density of 1.10-1.15 at 60° C., adds 95% ethanol, and adjusts to The alcohol concentration is 70%, put it in the refrigerator for 24 hours, filter, recover the ethanol until there is no alcohol smell, and get the clear paste for later use;

(5) The clear paste obtained in step (4) is combined with the alcohol extract obtained in step (3), concentrated to a thick paste with a relative density of 1.25-1.30 measured at 60° C., spray-dried to obtain a dry paste powder, and set aside;

(6) The dry paste powder obtained in step (5), the volatile oil obtained in step (2) and menthol are made into pills in a conventional manner to obtain 905 pills.

Example 4:

The raw material drug formula is:

Preparation:

(1) Extraction process:

(1) Take the Chinese herbal medicines according to the above-mentioned prescription amount, cleanly select them, and break them as appropriate;

(2) Patchouli was added with 6 times the amount of water to extract the volatile oil, the oil extraction time was 4 hours, the volatile oil was collected, and the oil yield was 0.33%, the extract was filtered and set aside, the residue was discarded, and the water extract was set aside;

(3) Forsythia, Sun-dried Ephedra, Houttuynia cordata, and rhubarb, extract twice with 8 times the amount of 70% ethanol, the first time for 2 hours, and the second time for 1.5 hours, filter the extract, combine the filtrates, and recycle the ethanol to no mellow, spare;

(4) Honeysuckle, fried bitter almonds, gypsum, isatidis, Mianma Guanzhong, licorice, rhodiola, add 9 times the amount of water and boil until boiling, add fried bitter almonds, and cook twice, the first time 1.5 hours, For the second 1 hour, the extract was filtered, and the filtrate was combined and added to the aqueous solution after the step (2) patchouli oil was extracted at the same time, concentrated to 60 ° C to measure the relative density of 1.10-1.15 clear paste, add 95% ethanol, and stir while adding , to an alcohol concentration of 70%, refrigerated for 24 hours, filtered, and the filtrate recovered ethanol until it had no alcohol smell, combined with the alcohol extract, concentrated until concentrated to 60°C to measure the relative density of 1.25-1.35 thick paste, and set aside;

(2) Preparation process:

(5) The preparation formula is: 335.5g thick cream obtained in step (4), 5g menthol

Step (2) obtained patchouli oil 0.2ml powdered sugar 342.5g dextrin 514.0g

(6) Granulation: mix powdered sugar and dextrin evenly, use thick paste as a binder to make soft materials, granulate with a 14-mesh screen, dry at 60-65°C, and granulate with a 10-mesh screen;

(7) Packing: Sieve out an appropriate amount of fine powder, add menthol and patchouli volatile oil to appropriate amount of ethanol, dissolve, spray into the fine powder, mix evenly, and mix evenly with the granules, seal it for half an hour, pack it into a bag, and obtain , the above prescription can be made into granules 1000g.

Experimental example:

In order to confirm the curative effect of the Chinese medicine composition of the present invention in preventing and treating the pneumonia drug of S protein D614G mutant new coronavirus infection, the following clinical trials were carried out with the granules (hereinafter referred to as the drug of the present invention) prepared by the method of Example 4:

1 Materials and methods:

Test content 1: The pharmaceutical composition particles of the present invention inhibit the replication effect of wild-type and S protein D614G mutant new coronaviruses in passaged cell lines

The purpose of the test: to evaluate the inhibitory effect of the pharmaceutical composition of the present invention on the wild type and the S protein D614G mutant new coronavirus at the cellular level

Test time: September 18, 2020 to October 7, 2020

experiment material

Medicine: pharmaceutical composition of the present invention, 12mg/mL pharmaceutical composition of the present invention configuration method: 1mL DMSO dissolves 120mg medicine, then adds 9mL DMEM culture medium-containing 100U/mL double antibody (penicillin, streptomycin) and 2%FBS, Vortex for 5 min, place in a refrigerator at 4°C for 12 h, then vortex for 5 min, and filter with a 0.22 μM filter.

cell

VERO-E6 cells

4.3 Viruses

4.3.1 Wild-type novel coronavirus [2019-nCoV, Wild-type (Wt)];

4.3.2 S protein mutant new coronavirus [2019-nCoV, D614G]

4.4 Reagents

4.4.1 Virus dilution: DMEM medium, containing double antibodies (penicillin and streptomycin) at a final concentration of 100U/mL, without serum;

4.4.2 Drug diluent: DMEM medium, containing double antibodies (penicillin and streptomycin) at a final concentration of 100U/mL, without serum;

4.4.3 Cell culture medium: DMEM medium, containing double antibodies (penicillin and streptomycin) at a final concentration of 100U/mL and 10% FBS;

4.4.4 Fetal bovine serum (FBS) (article number: 10270-044, brand: GIBCO);

4.4.5 DMEM medium (article number: C11995500BT, brand: GIBCO);

4.4.6 1×PBS (pH 7.4) (article number: C10010500BT, brand: GIBCO);

4.4.7 0.25% trypsin-EDTA (Trypsin trypsin 2.5g (article number: 0458-100g, brand: Amresco), EDTA-2Na 0.34g (article number: 0105-100g, brand: Amresco), dissolved in 1000mL 1×PBS , 0.22uM filter);

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

4.5 Consumables

96-well flat bottom cell culture plate (Cat. No.: 3599, brand: costar); 15mL centrifuge tube (Cat. No.: CFT-011-150, brand: BIOFIL); 50mL centrifuge tube (Cat. No.: CFT-011-150, brand: BIOFIL); Pipette Tips (Cat. No.: T-200-Y-R-S, Brand: AXYGEN); 10mL Pipettes (Cat. No.: 4488, Brand: costar)

4.6 Instruments

Cell incubator; -80°C refrigerator; centrifuge; inverted microscope; biological safety cabinet; electric pipette gun; pipette gun; 8-channel row gun; water bath; oscillator

5. Experimental method

5.1 cells

VERO-E6 cells were covered with a monolayer to carry out the antiviral experiment of the pharmaceutical composition particles of the present invention.

5.2 Viruses

SARS-CoV-2 (Wt and D614G), 100TCID50/well virus-inoculated cells.

5.3 Drugs

The experimental concentration of the pharmaceutical composition granules of the present invention is 75, 37.5, 18.75, 9.375, 4.69, 2.34, 1.17, 0.59, 0.29, 0.15 μg/ml, a total of 10 concentrations. The virus was incubated with the drug at 37°C for 1 hour, and then the cells were inoculated, and a normal cell control and a positive virus control were set at the same time.

5.4 CPE Observation

After 72 hours of virus infection, observe the CPE

6. Results

6.1 CPE observation: 72 hours after virus infection, the normal cell control group had no CPE; the virus control group had obvious CPE; different drug concentrations showed different inhibitory effects on CPE, see Table 1 and Table 2 for details.

6.2 half effective concentration (EC50) and therapeutic index (TI) calculation: calculate half effective concentration (EC50) according to table 1 and table 2 result, as shown in Figure 1, on Vero E6 cell, pharmaceutical composition capsule particle of the present invention is opposite to The EC50 of the Wt virus is 17.77 μg/ml; the EC50 of the capsule particles of the pharmaceutical composition of the present invention to the D614G virus is 35.85 μg/ml.

Based on the toxicity test results (TD50 of 723.5 μg/ml) of the same batch of pharmaceutical composition particles of the present invention to VERO-E6 cells, the therapeutic index (TI=TD50/EC50) was calculated. The results show that the therapeutic index of the pharmaceutical composition particles of the present invention to the Wt virus is 40.71, and the therapeutic index to the D614G virus is 20.18.

7. Conclusion

A certain concentration of the pharmaceutical composition particles of the present invention can inhibit the formation of CPE on Vero E6 cells by the new coronavirus (Wt and D614G mutant new coronavirus).

Table 1. The pharmaceutical composition of the present invention inhibits the results of Wt virus forming CPE on Vero E6 cells

("+" with CPE means negative for antiviral infection; "-" without CPE means positive for antiviral infection)

Table 2. The pharmaceutical composition of the present invention inhibits the D614G virus to form the result of CPE on Vero E6 cells

("+" with CPE means negative for antiviral infection, "-" without CPE means positive for antiviral infection)

Test content 2: Using a mouse infection model to study the effect of the pharmaceutical composition of the present invention on inhibiting the infection of the S protein D614G mutant new coronavirus

1 Test purpose: To study the effect of the pharmaceutical composition of the present invention on inhibiting the replication of S protein D614G mutant new coronavirus in mice

2 Test time: January 5, 2021 ~ January 21, 2021

3 test material

3.1 Animal: C57BL/6J, female, 6-8 weeks old, weighing 18-20g

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

3.3 0.5% carboxymethylcellulose sodium (CMC-Na) solution: Weigh 0.5g CMC-Na, add 100ml distilled water (during the configuration process, you need to add distilled water first, then slowly add CMC-Na, stir and dissolve overnight at 37°C) .

3.4 The pharmaceutical composition of the present invention (82mg/mL): Weigh 820mg of the pharmaceutical composition of the present invention, first dissolve the medicine in a mortar with 1mL 0.5% CMC-Na, grind for 1-3min, and then use PBS (containing 100U/mL The double antibody (penicillin streptomycin) was diluted 10 times to a final concentration of 82mg/mL, and mice were administered at 0.82g/kg.

4 test method

4.1 Animal grouping and specific experimental scheme: the mice were randomly divided into two groups, which were respectively the pharmaceutical composition treatment group of the present invention and the virus infection control group.

4.1.1 The pharmaceutical composition treatment group of the present invention: 13 mice

(1) Administration and challenge: 2 days before virus infection, the pharmaceutical composition of the present invention was gavaged at 0.82 g/kg, with a total volume of 200 μL. On the 3rd day, the drug composition of the present invention was inoculated with the virus (inoculated with adenovirus to transduce hACE2 5 days before the virus infection), and the drug was continued for 5 days after the virus infection, and the drug was administered for 8 days in total.

(2) Euthanasia and sampling: 5 mice were sacrificed on the 3rd and 6th day after virus infection.

Blood was taken from the orbit, centrifuged at 4000rpm, and the upper serum (light yellow) was taken for subpackage, and the number of mice was labeled and frozen in a -80°C refrigerator.

Take the lung of each mouse, weigh it first, record the lung weight, and calculate the lung index; take a small piece of the left lung of each mouse and fix it in 4% paraformaldehyde for pathological detection; the remaining lung part is ground and ground The speed is 4m/s, the cycle time is 10s, the interval time is 10s, and the number of cycles is 3 times. The grinding liquid is centrifuged at 7000rpm for 10 minutes, and 140 μL is taken for nucleic acid extraction and qRT-PCR experiments. The rest is stored in a -80°C refrigerator.

(3) Clinical symptoms: On the day of challenge, the mice were weighed and the clinical symptoms were observed every day until the 11th day after virus infection, and the weight change curve of the mice was drawn.

4.1.2 Virus infection control group: 13 mice

(1) Administration and challenge: at the same time point as the 4.1.1 pharmaceutical composition treatment group of the present invention, intragastric administration of PBS and virus infection;

(2) Euthanasia and material collection: 5 mice were killed on the 3rd and 6th day after virus infection, and the blood and organs were treated the same as 5.1.1(2).

(3) Clinical symptoms: The mice were weighed and clinical symptoms were observed every day until the 11th day after virus infection, and the body weight change curve of the mice was drawn.

4.2 Specific experimental methods

4.2.1 Body weight change curve: The mice were weighed every day, and the body weight change curve was drawn according to the percentage of the mouse weight during the experiment to the initial body weight of the experiment. Compared with the virus-infected control group, the effects of drugs such as the pharmaceutical composition of the present invention on the body weight changes of mice after virus infection were analyzed.

4.2.2 Serum collection: On the 3rd and 6th day after virus infection, 5 mice in the corresponding groups were sacrificed respectively, blood was collected from the orbits in 1.5mL EP tubes, the blood was centrifuged (4000rpm, 10min), and the supernatant was drawn to Store in EP tubes at -80°C.

4.2.3 Lung tissue processing:

(1) Lung index: On the 3rd and 6th day after virus infection, mice were sacrificed under anesthesia, and 5 mice in corresponding groups were dissected respectively. The mice were fixed in the supine position, the ribs and pectoralis muscles on both sides were cut off, the chest cavity was exposed, and the lungs were taken out. The trachea, hilar lymph nodes and other tissues were removed, washed twice with normal saline, and the water on the surface of the lungs was blotted dry with filter paper, and the weight of the lungs was weighed. Calculate lung index based on lung weight and mouse body weight.

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

(2) Tissue fixation and preservation: A small part of the left lung tissue samples were fixed in 4% paraformaldehyde solution, which will be used in subsequent HE.

(3) Determination of lung virus load: 5 mice in the corresponding groups were sacrificed on the 3rd day and 6th day after virus infection. The lungs of 5 mice were taken for grinding, and the grinding solution was centrifuged at 7000rpm for 10 minutes. After centrifugation, 140 μL of the supernatant was taken for nucleic acid extraction and qRT-PCR experiments. The N gene, ORF1ab gene, S Gene copy number; ②Detect the copy number of viral subgenomic RNA in the grinding solution.

5. Results

5.1 Changes in body weight of mice

After virus infection, the body weight of the mice in the non-administered PBS group decreased at 1dpi, dropped to the lowest point (7.5%) at 2dpi, and then began to increase in body weight. The weight of the mice in the pharmaceutical composition treatment group of the present invention began to increase at 1dpi, and the body weight of the mice in the pharmaceutical composition treatment group of the present invention was lower than that of the non-administered PBS group after 7dpi.

5.2 Changes in lung index

On the 3rd day and the 6th day after the mice were infected with the virus, there was no statistical significance in lung index between the pharmaceutical composition treatment group of the present invention and the control group without administration of PBS (t test, P>0.05) ( FIG. 2 ).

5.3 Determination of lung viral load

On the 3rd day after the mice were infected with the virus, there was no statistical significance in the viral load of the lungs of the mice treated with the pharmaceutical composition of the present invention compared with the PBS-free group (A, t test of FIG. 3 , P>0.05). On the 6th day after virus infection, the lung viral load of the pharmaceutical composition treatment group of the present invention was lower than that of the PBS group (B of FIG. 3 , rank sum test, P<0.05).

5.4 Pathological test results of treatment group and PBS control group (Figure 4, Figure 5, Figure 6, Figure 7, Figure 8)

6 Conclusion

6.1 Analysis of the body weight change curve of the mice showed that after being infected with the virus, the body weight of the mice recovered and increased faster than those of the PBS group without administration of the pharmaceutical composition of the present invention.

6.2 Lung index analysis showed that there was no statistical difference between the lung index of the treatment group and the non-administered PBS group.

6.3 Analysis of the viral load in the lungs of mice showed that the pharmaceutical composition of the present invention had an inhibitory effect on the replication of the S protein mutant D614G novel coronavirus on the 6th day after infection.

6.4 Histopathological analysis of mouse lungs: on the 3rd and 6th days after infection, the pathological damage of the lungs of the mice in the pharmaceutical composition-treated group of the present invention was slightly milder than that in the non-administered PBS group.

Comprehensive analysis of changes in clinical symptoms such as body weight of mice after virus infection, lung index, lung viral load results, and HE staining results showed that the pharmaceutical composition of the present invention can inhibit the replication of the S protein D614G mutant new coronavirus in the lungs of mice.

Claims

An application of a traditional Chinese medicine composition in the preparation of an anti-S protein D614G mutant new coronavirus drug, which is characterized in that it is made of the following raw materials in parts by weight:
The application according to claim 1, characterized in that it is made from the bulk drug of the following parts by weight:
The application according to claim 1, characterized in that it is made from the bulk drug of the following parts by weight:
The application according to claim 1, characterized in that it is made from the bulk drug of the following parts by weight:
The application according to claim 1, characterized in that it is made from the bulk drug of the following parts by weight:
According to the application described in any one of claims 1-5, it is characterized in that the active ingredient of the Chinese medicine composition is made by the following steps:

(1) Take the Chinese medicinal materials according to the weight ratio of the crude drug, and select them;

(2) Patchouli is broken, add 5-8 times the amount of water to extract the volatile oil, the oil extraction time is 4 hours, collect the volatile oil, and set aside; after the extract is filtered, the residue is discarded, and the filtrate is set aside;

(3) Forsythia, Sunburned Ephedra, Houttuynia cordata, Rhubarb, extract twice with 6-10 times of 50-90% ethanol, each time for 1-3 hours, combine and filter the extracts, recover ethanol, and use the ethanol extract for later use ;

(4) Honeysuckle, gypsum, isatidis, Mianma Guanzhong, licorice, rhodiola, add 7-11 times the amount of water and boil until boiling, add fried bitter almonds, decoct twice, each time for 0.5-2.5 hours, extract Liquid is merged and filtered, and the filtrate after gained filtrate and step (2) Patchouli extract oil is merged, is concentrated into the clear ointment that measures relative density at 60 ℃ and is 1.10-1.15, adds ethanol, is adjusted to alcohol concentration and is 70%, refrigerated Place, filter, recover ethanol until there is no alcohol smell, and get clear paste for later use;

(5) Combine the clear paste obtained in step (4) with the alcohol extract obtained in step (3), concentrate to a clear paste with a relative density of 1.15-1.20 at 60° C., dry to obtain dry paste powder, and set aside;

The dry paste powder obtained in step (5), the volatile oil obtained in step (2) and menthol jointly constitute the active ingredients of the traditional Chinese medicine composition.
According to the application described in any one of claims 1-5, it is characterized in that the pharmaceutical dosage form is capsule, tablet, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray doses or injections.
application according to claim 7, is characterized in that capsule is made by following steps:

(1) Take the Chinese medicinal materials according to the weight ratio of the crude drug, and select them;

(2) Patchouli is broken, add 5-8 times the amount of water to extract the volatile oil, the oil extraction time is 4 hours, collect the volatile oil, and set aside; after the extract is filtered, the residue is discarded, and the filtrate is set aside;

(3) forsythia, roasted ephedra, houttuynia cordata, rhubarb, extracted twice with 50-90% ethanol of 6-10 times, each time for 1-3 hours, the extracts were combined and filtered, the ethanol was recovered, and the filtrate was used for subsequent use;

(4) Honeysuckle, gypsum, isatidis, Mianma Guanzhong, licorice, rhodiola, add 7-11 times the amount of water and boil until boiling, add fried bitter almonds, decoct twice, each time for 0.5-2.5 hours, extract Liquid is merged and filtered, and the filtrate after gained filtrate and step (2) Patchouli extract oil is merged, is concentrated into the clear ointment that measures relative density at 60 ℃ and is 1.10-1.15, adds ethanol, is adjusted to alcohol concentration and is 70%, refrigerated Place, filter, recover ethanol until there is no alcohol smell, and get clear paste for later use;

(5) The clear cream obtained in step (4) is combined with the alcohol extract obtained in step (3), concentrated to a clear cream with a relative density of 1.15-1.20 measured at 60° C., dried to obtain a dry cream powder, and set aside;

(6) adding appropriate pharmaceutically acceptable auxiliary materials to the dry cream powder obtained in step (5) to granulate;

(7) Add ethanol to dissolve menthol and the volatile oil obtained in step (2), spray into the granules obtained in step (6), airtightly mix, pack into capsules, and obtain.
The application according to claim 7, characterized in that the granules are made from the following steps:

(1) Weigh the Chinese herbal medicines according to the weight ratio of the raw materials, select them cleanly, and break them as appropriate;

(2) Patchouli is broken, add 5-8 times the amount of water to extract the volatile oil, the oil extraction time is 4 hours, collect the volatile oil, and set aside; after the extract is filtered, the residue is discarded, and the filtrate is set aside;

(3) forsythia, roasted ephedra, houttuynia cordata, rhubarb, extracted twice with 50-90% ethanol of 6-10 times, each time for 1-3 hours, the extracts were combined and filtered, the ethanol was recovered, and the filtrate was used for subsequent use;

(4) Honeysuckle, gypsum, isatidis, Mianma Guanzhong, licorice, rhodiola, add 7-11 times the amount of water and boil until boiling, add fried bitter almonds, decoct twice, each time for 0.5-2.5 hours, extract Liquid is merged and filtered, and the filtrate of the gained filtrate and the step (2) patchouli after extracting oil is merged, and is concentrated into the clear cream that measures relative density at 60 ℃ and is 1.10-1.15, adds ethanol, adjusts to alcohol concentration and is 70%, refrigerated Place, filter, recover ethanol until there is no alcohol smell, and get clear paste for later use;

(5) Combine the clear cream obtained in step (4) with the alcohol extract obtained in step (3), concentrate to a clear cream with a relative density of 1.15-1.20 measured at 60° C., dry to obtain dry cream powder, and set aside;

(6) adding appropriate pharmaceutically acceptable auxiliary materials to the dry cream powder obtained in step (5) to granulate;

(7) Add menthol and the volatile oil obtained in step (2) into ethanol to dissolve, spray into the particles obtained in step (6), airtightly mix, pack into bags, and obtain.
According to the described application of claim 9, it is characterized in that the preparation method of granule is made by the following steps:

(1) Weigh the Chinese herbal medicines according to the weight ratio of the raw materials, select them cleanly, and break them as appropriate;

(2) Patchouli is broken, add 6 times the amount of water to extract the volatile oil, the oil extraction time is 4 hours, collect the volatile oil, the oil yield is 0.33±0.05%, and set aside; after the extract is filtered, the residue is discarded, and the water extract is set aside ;

(3) forsythia, sun-burned ephedra, houttuynia cordata, rhubarb, extracted twice with 8 times the amount of 70% ethanol, the first time for 2 hours, the second time for 1.5 hours, the extracts were combined and filtered, the ethanol was recovered, and the filtrate was used for subsequent use;

(4) Honeysuckle, gypsum, isatidis, Mianmaguanzhong, licorice, rhodiola rosea, add 9 times the amount of water and boil until boiling, add fried bitter almonds, and boil for 2 times, the first time is 1.5 hours, the second time is 1 hour hour, the extracts are combined and filtered, and the water extract after the obtained filtrate is combined with the step (2) Patchouli oil extraction oil, is concentrated into a clear paste that measures a relative density of 1.10-1.15 at 60° C., adds 95% ethanol, and adjusts to The alcohol concentration is 70%, put it in cold storage, filter, recover the ethanol until it has no alcohol smell, and get a clear paste;

(5) Combine the clear ointment obtained in step (4) with the ethanol extract obtained in step (3), concentrate to a thick ointment with a relative density of 1.25-1.35 at 60° C., and set aside;

(6) Add the thick paste obtained in step (5) into appropriate pharmaceutically acceptable adjuvants to granulate, granulate, and set aside;

(7) Sieve the granules obtained in step (6) out of fine powder, add menthol and the volatile oil obtained in step (2) into ethanol to dissolve, spray into the fine powder, after mixing evenly, mix evenly with the granules obtained in step (6), and seal the semi- Hours, bagged, that is.