WO2021179950A1 - Use of pharmaceutical composition in preparing anti-viral drug - Google Patents

Abstract

A use of a pharmaceutical composition in preparing an anti-viral drug, the pharmaceutical composition being made from Ephedra sinica, gypsum, Forsythia, Scutellaria baicalensis, white mulberry root bark, bitter apricot kernel, radix peucedani, pinelliae rhizoma, chenpi, fritillary bulb, greater burdock fruit, Flos Lonicerae japonicae, Rheum officinale, Chinese bellflower, and Glycyrrhiza uralensis.

Description

Application of pharmaceutical composition in preparing antiviral drugs

This application claims the priority of the Chinese patent application 202010153827.1 with the title of "Application of a Chinese Medicine Composition in the Preparation of Antiviral Drugs" filed to the Chinese Patent Office on March 7, 2020, the content of which is incorporated by reference in its entirety. Into this article.

Technical field

The invention relates to the application of a pharmaceutical composition in the preparation of antiviral drugs, and belongs to the field of medicine.

Background technique

Viruses are the smallest kind of pathogenic microorganisms. They multiply in cells. The core is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and the outer shell is protein, which does not have a cell structure. Most viruses lack an enzyme system and cannot live independently. They must rely on the host's enzyme system to reproduce (replicate) themselves. Viral nucleic acids are sometimes integrated in cells and are difficult to eliminate. Therefore, the development of antiviral drugs has been slow.

Viral infection refers to the process by which viruses invade the body through multiple channels and multiply in susceptible host cells. Human viruses refer to viruses that can infect humans or have disease-causing effects on humans. The essence of virus infection is the process of interaction between virus and body, virus and susceptible cells. Viral infections often cause varying degrees of damage or viral diseases due to different types of viruses and body conditions. Virus pathogenicity begins by invading the host and infecting cells, and the pathogenic effect is manifested in both the human body and the cells.

Viral diseases are a class of highly contagious diseases. The antiviral chemical drugs currently on the market are divided into the following categories:

1. Penetration and shelling inhibitors: amantadine, rimantadine, enfuvirtide, maraviro

2. DNA polymerase inhibitors: acyclovir, ganciclovir, valacyclovir, famciclovir, foscarnet sodium

3. Reverse transcriptase inhibitor:

Nucleosides: lamivudine, zidovudine, emtricitabine, tenofovir, adefovir dipivoxil

Non-nucleoside: efavirenz, nevirapine

4. Protein inhibitor: Saquinavir

5. Neuraminidase inhibitors: Oseltamivir, Zanamivir

6. Broad-spectrum antiviral drugs: ribavirin, interferon

However, the existing antiviral drugs are not yet satisfactory in terms of effectiveness and/or safety. For example, there is still a lack of effective and reliable drugs for killing or inhibiting a variety of viruses, or some drugs have effects but have obvious side effects. For example, ribavirin, a commonly used antiviral chemical drug in clinical practice, can cause heart damage when used in large doses, and can also cause a decrease in white blood cells and lead to reversible anemia. . The toxicity and side effects of chemical drugs limit their application to a certain extent.

Therefore, there is still an urgent need for safe and effective antiviral drugs.

The present invention is an improved invention based on the pharmaceutical composition disclosed in the patent of CN101549060A, and the content recorded in the patent document is quoted here in full. The aforementioned patent does not disclose that the pharmaceutical composition has an antiviral effect.

Summary of the invention

The inventor unexpectedly discovered in research that the pharmaceutical composition of the present invention has an excellent antiviral effect, and the pharmaceutical composition is made of the following raw materials in parts by weight:

Ephedra 52-86; Gypsum 194-324; Forsythia 194-324; Scutellaria 78-130; Mulberry Bark 194-324; Bitter Almond 78-130; Qianhu 78-130; Pinellia 78-130; Tangerine Peel 78-130 Fritillaria 78-130; Arctium lappa 78-130; Honeysuckle 78-130; Rhubarb 39-65; Platycodon 46-76; Licorice 39-65.

The weight ratio of the bulk drug of the pharmaceutical composition of the present invention is preferably:

Ephedra 52; Gypsum 324; Forsythia 194; Scutellaria 78; Mulberry 194; Bitter Almond 130; Qianhu 78; Pinellia 130; Tangerine Peel 78; Fritillaria 78; Burdock Seed 130; Honeysuckle 130; Rhubarb 39; Platycodon 76; Licorice 65.

The weight ratio of the bulk drug of the pharmaceutical composition of the present invention is also preferably:

Ephedra 86; Gypsum 194; Forsythia 324; Scutellaria 130; Mulberry Bark 324; Bitter Almond 78; Qianhu 130; Pinellia 78; Tangerine Peel 130; Fritillaria 130; Burdock 78; Honeysuckle 78; Rhubarb 65; Platycodon 46; Licorice 39.

The weight ratio of the bulk drug of the pharmaceutical composition of the present invention is also preferably:

Ephedra 69; Gypsum 259; Forsythia 259; Scutellaria baicalensis 104; Mulberry bark 259; Bitter almond 104; Qianhu 104; Pinellia 104; Licorice 52.

The weight ratio of the bulk drug of the pharmaceutical composition of the present invention is also preferably:

Ephedra 55; Gypsum 254; Forsythia 318; Scutellaria baicalensis 107; Mulberry bark 203; Bitter almond 107; Qianhu 82; Pinellia 105; Tangerine peel 84; Fritillaria 125; Burdock 122; Honeysuckle 113; Rhubarb 42; Licorice 50.

Preferably, in the pharmaceutical composition of the present invention, the bitter almonds are fried bitter almonds, the fritillary is Fritillaria fritillary, the honeysuckle is the Lonicera japonica, and the Pinellia ternata is the Qing Pinellia.

The pharmaceutical composition of the present invention has been confirmed by in vitro studies and clinical experiments to effectively kill a variety of viruses, and the effect is significant.

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

A. Weigh Zhe Fritillaria according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, sauteed bitter almonds, Qing Pinellia, burdock seeds, rhubarb, and 40-70% ethanol for 2 times reflux extraction, 1-4 hours each time, and 8-10 for the first time. Double the amount, add 6-9 times the amount for the second time, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C and heat the clear cream with a relative density of 1.14-1.16 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, tangerine peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportions of the prescriptions. Add water and decoct twice for 1-4 hours each time, and add 9-11 times the amount for the first time. Add 7-9 times the amount for the second time, combine the decoction, filter, concentrate to 60°C and measure the clear ointment with a relative density of 1.14-1.16, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture for use;

D. Mix the fine powder obtained in step A and the clear ointment mixture obtained in step C to obtain the active ingredient of the pharmaceutical composition.

The dosage form of the medicine of the present invention can be capsules, tablets, powders, granules, oral liquids, soft capsules, pills, tinctures, syrups, suppositories, gels, sprays or injections.

In order to realize the above dosage forms, it is preferable to add pharmaceutically acceptable excipients when preparing these dosage forms, such as fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, correctives, and preservatives. , Substrate, 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, hydroxypropyl methylcellulose, etc.; binders include starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: Saccharin sodium, aspartame, sucrose, cyclamate, glycyrrhetinic acid, etc.; correctives include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and other Salts, benzalkonium bromide, chloroethyl acetate, eucalyptus oil, etc.; bases include: PEG6000, PEG4000, insect wax, etc.

The tablet can be made by a method including the following steps:

A. Weigh Zhe Fritillaria according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, sauteed bitter almonds, Qing Pinellia, burdock seeds, rhubarb, and 40-70% ethanol for 2 times reflux extraction, 1-4 hours each time, and 8-10 for the first time. Double the amount, add 6-9 times the amount for the second time, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C and heat the clear cream with a relative density of 1.14-1.16 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, tangerine peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportions of the prescriptions. Add water and decoct twice for 1-4 hours each time, and add 9-11 times the amount for the first time. Add 7-9 times the amount for the second time, combine the decoction, filter, concentrate to 60°C and measure the clear ointment with a relative density of 1.14-1.16, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture for use;

D. Spray drying the cleansing paste mixture obtained in step C, and collect the spray powder for later use;

E. The spray powder obtained in step D and the fine powder obtained in step A are prepared by using ethanol as a binder to prepare a soft material, and then sieved to granulate; it is obtained by pressing according to the conventional method of pharmacy.

The preferred preparation method of the tablet may be a method including the following steps:

A. Weigh Zhe Fritillaria according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, sauteed bitter almonds, Qing Pinellia, burdock seed, rhubarb and reflux with 50% ethanol for 2 times, 3 hours each time, 10 times the amount for the first time, and the second time Add 6 times the amount, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C and heat the clear cream with a relative density of 1.15 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescription. Add water and decoct twice for 2 hours each time. Add 10 times the amount for the first time and add it for the second time 7 times the amount, the decoction is combined, filtered, concentrated to 60°C to measure the clear ointment with a relative density of 1.15, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture for use;

D. Spray drying the cleansing paste mixture obtained in step C, and collect the spray powder for later use;

E. The spray powder obtained in step D and the fine powder obtained in step A are used to prepare a soft material with ethanol as a binder, sieved, granulated, dried, and then sieved, and then mixed with sodium starch glycolate, microcrystalline cellulose, and magnesium stearate. Evenly, press the tablet and get it.

To prepare other dosage forms of the drug of the present invention, the raw materials can be weighed in proportion and prepared by conventional preparation methods, for example, according to the preparation described in Fan Biting's "Chinese Medicine Pharmacy" (Shanghai Science Press, December 1997, 1st edition) Process, made into a pharmacologically acceptable conventional dosage form.

Experiments have confirmed that the pharmaceutical composition of the present invention can inhibit influenza virus, especially influenza A virus; the pharmaceutical composition of the present invention can inhibit the new coronavirus.

Description of the drawings

Figure 1 shows the protective effect of the pharmaceutical composition of the present invention on mice infected with influenza virus.

Detailed ways

The following examples are used to illustrate the preparation of the pharmaceutical composition of the present invention, but it should be understood that they do not limit the scope of the present invention.

Example 1

prescription:

Ephedra 52g; gypsum 324g; forsythia 194g; skullcap 78g; mulberry 194g; bitter almond 130g; Qianhu 78g; pinellia 130g; tangerine peel 78g; zhejiang fritillary 78g; burdock seed 130 grams; 130 grams of licheniflora; 39 grams of rhubarb; 76 grams of platycodon; 65 grams of licorice. Preparation:

A. Weigh Zhe Fritillaria according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, bitter almonds, pinellia, burdock seeds, rhubarb, and 50% ethanol to extract 2 times, each 3 hours, 10 times the amount for the first time, and 6 times for the second time. Double the volume, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to measure a clear paste with a relative density of 1.15 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescription. Add water and decoct twice for 2 hours each time. Add 10 times the amount for the first time and add it for the second time 7 times the amount, the decoction is combined, filtered, concentrated to 60°C to measure the clear ointment with a relative density of 1.15, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture for use;

D. Spray drying the cleansing paste mixture obtained in step C, and collect the spray powder for later use;

E. Prepare a soft material with the spray powder obtained in step D and the fine powder obtained in step A using 80% ethanol as a binder, sieving and granulating, drying at 60 degrees, and sizing. Add sodium carboxymethyl starch, microcrystalline cellulose, and magnesium stearate, mix well, and prepare tablets according to the conventional preparation method to obtain.

Example 2

prescription:

Ephedra 86g; Gypsum 194g; Forsythia 324g; Scutellaria baicalensis Georgi 130g; Mulberry bark 324g; Fried bitter almond 78g; Qianhu 130g; Pinellia ternata 78g; Tangerine peel 130g; Fritillaria 130g; Burdock seed 78 grams; 78 grams of Lonicera japonica; 65 grams of rhubarb; 46 grams of platycodon; 39 grams of licorice. Preparation:

A. Weigh the mother-of-pearl according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, sauteed bitter almonds, pinellia, burdock seeds, rhubarb in proportion to the formula, add 40% ethanol and reflux for extraction 2 times, each 4 hours, the first time is 8 times the amount, the second time Add 9 times the amount, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C and heat the clear cream with a relative density of 1.14 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescriptions. Add water and decoct twice for 4 hours each time. Add 9 times the amount for the first time and add it for the second time 7 times the amount, the decoction is combined, filtered, concentrated to 60°C to measure the clear ointment with a relative density of 1.16, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture for use;

D. Spray drying the cleansing paste mixture obtained in step C, and collect the spray powder for later use;

E. Prepare a soft material with the spray powder obtained in step D and the fine powder obtained in step A using 80% ethanol as a binder, sieving and granulating, drying at 60 degrees, and sizing. Add sodium carboxymethyl starch, microcrystalline cellulose, and magnesium stearate, mix well, and prepare tablets according to the conventional preparation method to obtain.

Example 3

prescription:

Ephedra 69g; Gypsum 259g; Forsythia 259g; Scutellaria baicalensis Georgi 104g; Mulberry bark 259g; Fried bitter almonds 104g; Qianhu 104g; Qing Pinellia 104g; Tangerine peel 104g; Zhejiang Fritillaria 104g; 104 grams of burdock seeds; 104 grams of lichen; 52 grams of rhubarb; 61 grams of platycodon; 52 grams of licorice.

Preparation:

A. Weigh Zhe Fritillaria according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, sauteed bitter almonds, clear pinellia, burdock seeds, rhubarb in proportion to the formula, add 70% ethanol and reflux for extraction 2 times, 1 hour each time, 10 times the amount for the first time, and the second time Add 6 times the amount each time, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to measure a clear paste with a relative density of 1.16 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, Chenpi, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescription, add water and decoct twice for 1 hour each time, add 11 times the amount for the first time, and add it for the second time. 7 times the amount, the decoction is combined, filtered, concentrated to 60°C to measure the clear ointment with a relative density of 1.14, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture for use;

D. Spray drying the cleansing paste mixture obtained in step C, and collect the spray powder for later use;

E. The spray powder obtained in step D and the fine powder obtained in step A are prepared using 80% ethanol as a binder to prepare a soft material, sieved and granulated, dried at 60 degrees, and then granulated. Add sodium carboxymethyl starch, microcrystalline cellulose, and magnesium stearate, mix well, and prepare tablets according to the conventional preparation method to obtain.

Example 4:

The API formula is:

Ephedra 55g; Plaster 254g; Forsythia 318g; Scutellaria baicalensis 107g; Mulberry 203g; Fried bitter almonds 107g; Qianhu 82g; Pinellia ternata 105g; Tangerine peel 84g; 122 grams of seeds; 113 grams of lichen; 42 grams of rhubarb; 60 grams of platycodon; 50 grams of licorice.

Preparation:

A. Weigh Zhe Fritillaria according to the proportion of the group, crush it into fine powder, and set aside;

B. Weigh ephedra, forsythia, sauteed bitter almonds, pinellia, burdock seeds, rhubarb in proportion to the formula, add 60% ethanol and reflux for extraction 2 times, each 2 hours, the first time is 9 times the amount, the second time Add 7 times the amount, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to measure a clear paste with a relative density of 1.15 for use;

C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, Lonicera japonica, Platycodon grandiflorum, and licorice according to the proportion of the prescription, add water and decoct twice for 2.5 hours each time, add 10 times the amount for the first time, and add it for the second time. 7 times the amount, the decoction is combined, filtered, concentrated to 60°C to measure the clear ointment with a relative density of 1.14, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture for use;

D. Spray drying the cleansing paste mixture obtained in step C, and collect the spray powder for later use;

E. Prepare a soft material with the spray powder obtained in step D and the fine powder obtained in step A using 80% ethanol as a binder, sieving and granulating, drying at 60 degrees, and sizing. Add sodium carboxymethyl starch, microcrystalline cellulose, and magnesium stearate, mix well, and prepare tablets according to the conventional preparation method to obtain.

Example 5:

Ephedra 62g Plaster 220g Forsythia 256g Radix Scutellaria 90g Mulberry Bark 300g Bitter Almond 90g Qian Hu 90g Pinellia 90g Tangerine Peel 100g Fritillaria 100g Burdock Seed 100g Honeysuckle 100g Rhubarb 50g Platycodon 66g Licorice 50g

The above medicinal materials can be obtained as capsules according to conventional methods.

Example 6:

Ephedra 68g Plaster 215g Forsythia 215g Radix Scutellariae 100g Mulberry Bark 220g Bitter Almond 90g Qian Hu 90g Pinellia 90g Tangerine Peel 90g Fritillaria 90g Burdock Seed 90g Honeysuckle 90g Rhubarb 50g Platycodon 50g Licorice 50g

The above medicinal materials are prepared into granules according to conventional methods.

Example 7:

Ephedra 50g Plaster 200g Forsythia 300g Radix Scutellaria 100g Mulberry Bark 250g Bitter Almond 100g Qianhu 100g Pinellia 100g Tangerine Peel 100g Fritillaria 100g Burdock Seed 100g Honeysuckle 100g Rhubarb 50g Platycodon 50g Licorice 50g

The above medicinal materials can be obtained as injections according to conventional methods.

Example 8:

Ephedra 60 g Plaster 200 g Forsythia suspense 200 g Scutellaria baicalensis Georgi 95 g Mulberry bark 230 g Bitter almond 95 g Qianhu 95 g Pinellia 95 g Tangerine peel 95 g Fritillaria 95 g Burdock seed 95 g Honeysuckle 95 g Rhubarb 50 g Platycodon 50 g Licorice 50g

The above medicinal materials can be obtained as pills according to conventional methods.

Experimental example of biological activity:

In order to confirm that the pharmaceutical composition of the present invention has the antiviral effect, the pharmaceutical composition of the present invention prepared in Example 3), that is, the granules (hereinafter referred to as the drug of the present invention) after the granulation in step E of Example 3 and before the tableting are performed The following pharmacological test studies were carried out:

Experimental study on the anti-mouse influenza A virus of the pharmaceutical composition of the present invention

I. Experimental study on anti-mouse influenza A virus in vitro of the drug combination of the present invention

Experimental Materials

1. Drugs and reagents:

(1) The test drug is the pharmaceutical composition of the present invention (prepared in Example 3): the pharmaceutical composition is brown-yellow particles, and each gram of particles is equivalent to 4.095 grams of crude drug. The daily dosage is 22g crude drug/person, orally, the human weight is 60kg, which is equivalent to 0.367g pharmaceutical composition/kg body weight.

(2) The positive control drug is Shuanghuanglian Oral Liquid, produced by Harbin Pharmaceutical Group Sanjing Pharmaceutical Co., Ltd.

2. Virus strains and cell strains:

(1) Virus strain: Influenza virus subtype murine lung-adapted strain (FM1), provided by the Institute of Chinese Medicine, China Academy of Chinese Medical Sciences. After the 9-day-old chicken embryo allantoic cavity was successively passaged twice, the virus liquid was taken to measure the hemagglutination titer of 1:512, and the aliquots were frozen and stored at -76°C for later use.

(2) Cell line: Canine kidney cell line (MDCK) was purchased from the cell room of the Academy of Military Medical Sciences, and used in this room after routine freezing, resuscitation, and passage.

3. Medium: DMEM medium, product of GBICO company. The DMEM growth medium is a DMEM culture medium containing 10% newborn calf serum; the DMEM maintenance medium is a serum-free DMEM medium containing 0.002 mg/ml trypsin.

4. Apparatus: The inverted microscope is a product of Olympus, Japan, and the CalaxyS CO ₂ incubator is a product of RSBiotech.

Methods and results

1. Cell preparation: subculture the MDCK cells for 3-5 days, make them into slices, when the boundary is clear, the three-dimensional sense and the refractive index are strong, digest with trypsin, wait until the needle-like holes appear on the cell surface, suck up the digestion solution, and take a count Disperse the cells with ml of culture medium, count them, and dilute with culture medium to about 3×10 ⁸ /L, then inoculate them in a 96-well culture plate, and wait for the cells to grow into a single layer.

2. Preparation of the pharmaceutical composition of the present invention: Weigh 4.88 g of the extract powder of the pharmaceutical composition of the present invention and dissolve it in 40 ml of physiological saline, which is equivalent to 500 mg crude drug/ml. After autoclaving and storing in a refrigerator at 4°C for 5 days, centrifuged at 5000 rpm for 20 minutes, and the supernatant was taken for in vitro experiments.

3. Determination of TCID50 of influenza virus: Use serum-free DMEM medium to dilute influenza virus FM1 mouse lung-adapted strain continuously by 10 times, and inoculate the virus of each dilution into 3×10 ⁸ /L MDCK cells, each Make 6 replicate wells in parallel with each dilution, and set a normal cell control at the same time. After adsorbing at 37°C for 2 hours, aspirate the virus solution and replace it with a maintenance solution to continue the culture. Observe the cytopathic effect (CPE) under an inverted microscope every day, and record the degree of disease and the number of holes. The cell control has no obvious degeneration, and the virus infected cytopathic disease. Cell pores with a rate of 50% and above are diseased holes, and those with a cytopathic rate of less than 50% are non-pathological holes. The TCID50 (median tissue culture infective dosage) of the virus is calculated according to the Reed-Muench method. The results showed that the TCID50 of the influenza virus strain used in the experiment was 5×10 ^-4 .

4. Cytotoxicity determination of the pharmaceutical composition of the present invention:

When the MDCK cells grow in the best state, digest the cells with 0.02% EDTA and 0.5% trypsin, adjust the cells to the required concentration with the growth medium, and add the cell liquid to a 96-well cell culture plate, 100 μL/well, at 37°C , Cultivate to a dense cell monolayer in a 5% CO _{2 incubator.} Dilute the pharmaceutical composition of the present invention to different concentrations with the maintenance solution, respectively 500mg/ml, 250mg/ml, 125mg/ml, 62.5mg/ml, 31.3mg/ml, 15.6mg/ml, 7.8mg/ml, 3.9mg /ml; add to the cell culture plate, 100μL/well, the final concentration of the drug in the culture well is 250mg/ml, 125mg/ml, 62.5mg/ml, 31.3mg/ml, 15.6mg/ml, 7.8mg/ml , 3.9mg/ml, 1.9mg/ml; Shuanghuanglian takes the original solution as the original concentration, dilutes them in sequence and adds them to the culture wells. Make 3 replicate wells in parallel for each drug dilution concentration, and set up a normal cell control at the same time. Cultivate in a 37°C 5% CO ₂ incubator, observe the cytopathic effect (CPE) under the microscope every day, calculate the cytotoxicity caused by the drug solution, and calculate the maximum non-toxic concentration. The results show that the maximum non-toxic concentration of Shuanghuanglian oral liquid is a 1:1 dilution of the original solution, and the maximum non-toxic concentration of the pharmaceutical composition of the present invention is 150 mg/ml.

4. The effect of the pharmaceutical composition of the present invention on the cytopathic effect of mouse influenza virus:

Inoculate 2×10 ⁶ /ml MDCK on a 96-well culture plate, 100 μL/well, and place it at 37°C with 5% CO _{2 for} 24 hours to form a cell monolayer. Wash the cells twice with serum-free DMEM solution, add 50×TCID50 (50 times the 50% cytopathic concentration) virus solution, 100μL/well, 37℃5% CO ₂ incubator for 2h, and then aspirate the virus solution , Adding 5 doses of the pharmaceutical composition of the present invention that are double-diluted in the non-toxic concentration range, respectively, 125, 63, 31, 16 and 8 mg/ml. Incubate for 7 days at 37°C with 5% CO ₂ and observe the cytopathic condition (CPE) under the microscope every day. Each dose of the drug was added to 4 holes. The experiment also set up a cell control group, a Shuanghuanglian control group and a virus control group. CPE was observed at the same time as the experimental group. The degree of CPE is expressed by the proportion of CPE cells, which is judged according to the following 6-level criteria: (-) Cells grow normally without lesions; (±) Cytopathies are less than 10% of the entire cell monolayer; (+) Cytopathies are less than 25% of the entire cell monolayer; (++) 25%-50% of cells produce lesions; (+++) 50%-75% of cells produce lesions; (++++) 75%-100% of cells produce lesions. According to the degree of CPE, the Reed-Muench method is used to calculate the half effective concentration (IC ₅₀ ) of the drug. The results show that, in the medicament of the present invention 31-125mg / ml concentration of influenza virus induced cytopathic has some protective effect IC ₅₀ = 65.4mg / ml. _{The IC 50} of Shuanghuanglian Oral Liquid is about 1:8 dilution. See Table 1.

Table 1: Protective effect of the pharmaceutical composition of the present invention on virus-induced cytopathic changes

in conclusion

(1) The TCID50 of the mouse influenza virus is 5×10 ^-4 .

(2) The concentration of the positive control drug Shuanghuanglian oral liquid is in the range of 1:8 dilution to 1:1 dilution, all have the effect of inhibiting the pathological changes caused by influenza virus on cells, and its IC ₅₀ is about 1:8 dilution.

(3) The 31-125 mg/ml concentration of the drug of the present invention has a protective effect on cytopathic changes caused by influenza virus, and its IC ₅₀ is 65.4 mg/ml.

II. Experimental study on anti-mouse influenza A virus in vivo of the drug combination of the present invention

Experimental Materials

1. Animals: ICR mice, half male and half male, weighing 13-15g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number SCXK (Beijing 2005-2006).

2. Drugs and reagents:

(1) The test drug is a pharmaceutical composition of the present invention, which is the same as in vitro anti-mouse influenza A virus test study I. Use 0.5% CMC-Na to make the required concentration before using this product.

(2) The positive control drug is Shuanghuanglian oral liquid, which is the same as in vitro anti-mouse influenza A virus test study I.

3. Virus strain: The same in vitro anti-mouse influenza A virus test study I.

4. Apparatus: The 400R high-speed refrigerated centrifuge is a product of Heraeus, Germany, and the AE100 analytical balance is a product of METTLER, Switzerland.

Methods and results

1, virulence assay: Mice were lightly anesthetized with ether, nasal infection, each nostril was added 50μl of different dilutions of virus, Percent mortality was recorded, and to calculate the minimum lethal dose LD _50. The results showed that the LD ₅₀ of the virus used in this experiment was 2.5×10 ^-5 .

2. The protective effect of the pharmaceutical composition of the present invention on death caused by infection of mice with influenza virus murine lung-adapted strain FM1:

(1) Animal grouping and treatment: 78 ICR mice were randomly divided into 5 groups, namely the influenza model control group (gavage equal volume 0.5% CMC-Na); Shuanghuanglian group (gavage 10ml/kg, equivalent to 10 times the daily dosage per person); the pharmaceutical composition group of the present invention is administered intragastrically, and the dosages are respectively 1.9g/kg (equivalent to 5 times the daily dosage of a person) and 3.7g/kg (equivalent to 10 times the daily dosage of a person). Times), 7.4g/kg (equivalent to 20 times the human consumption). The mice in each group were administered continuously for 5 days, and the administration volume was 0.4ml/20g body weight.

(2) The protective effect of the pharmaceutical composition of the present invention on the death of mice after influenza virus infection: mice in each group were given intragastric administration the day before infection for 5 consecutive days, and the viral pneumonia model group was given an equal volume of solvent. 2 days after administration, the mice were anesthetized with ether shallow intranasally infected with influenza virus strain rat lung adaptation FM1, virus inoculation amount of 10 times LD _50, mice were observed daily after the infection, death of the mice were recorded The test was terminated 2 weeks after infection, and the survival time of mice that did not die was calculated as 14 days. For statistical processing, SPSS software was used for chi-square test and t-test. The results show that both large and medium doses of the pharmaceutical composition of the present invention can significantly reduce the mortality of mice infected with influenza virus, and can significantly prolong the survival time of mice infected with the virus. The results are shown in Tables 2, 3 and Figure 1.

Table 2: Protective effect of the pharmaceutical composition of the present invention on death of mice infected with influenza virus

Table 3: Protective effect of the pharmaceutical composition of the present invention on death of mice infected with influenza virus

Note: Compared with the control group, *p<0.05, **p<0.01.

3. The effect of the pharmaceutical composition of the present invention on influenza virus pneumonia in mice:

(1) Animal grouping and treatment: 70 ICR mice were randomly divided into 6 groups, namely the normal control group (gavage equal volume 0.5% CMC-Na); Viral pneumonia model group (gavage equal volume 0.5% CMC) -Na), Shuanghuanglian group (gave 10ml/kg, equivalent to 10 times the daily dosage of human); the pharmaceutical composition group of the present invention, administered by gavage, the dosage is 1.9g/kg (equivalent to daily dosage per person) 5 times the amount), 3.7g/kg (equivalent to 10 times the daily dosage of a person), 7.4g/kg (equivalent to 20 times the daily dosage of a person). Except for the normal control group, the other groups were all nasally inhaled influenza virus to prepare a virus-infectious pneumonia model.

(2) The effect of the pharmaceutical composition of the present invention on the lung index of mice with viral pneumonia: mice in each group were given intragastric administration on the day before infection for 5 consecutive days. The normal control group and the viral pneumonia model group were given an equal volume of solvent . Day 2 after administration, in addition to the normal control group, the mice were anesthetized with ether shallow intranasally infected with influenza virus strain rat lung adaptation FM1, virus inoculation amount of 10 times LD _50, dissected 5 days after infection, lung Weigh, and calculate the lung index according to the following formula. The results showed that the lung index of the virus model group was significantly increased compared with the normal control group, and the large and medium doses of the pharmaceutical composition of the present invention could significantly reduce the lung index of the model mice. Statistical analysis was performed with SPSS software for t test. The results are shown in Table 4.

Table 4: Effect of the pharmaceutical composition of the present invention on the lung index of mice with viral pneumonia

Note: The virus model group is compared with the normal control group, ▲▲p<0.01, and each administration group is compared with the model group, **p<0.01.

(3) The effect of the pharmaceutical composition of the present invention on the virus titer in the lungs of mice with viral pneumonia: measured by hemagglutination method. Mice administration and preparation of viral pneumonia model are the same as above. Five days after virus inoculation, the mice were sacrificed by neck removal. The lungs were dissected and the lungs were grinded by a tissue homogenizer. The 10% lung tissue suspension was made with normal saline. After the plate, add 0.2ml 1% chicken red blood cell suspension to each well, mix well, leave it at room temperature for 30 minutes, observe and record the hemagglutination titer. The end point is when red blood cells agglutinate (++), and the reciprocal of the suspension dilution is used to indicate the virus titer in the mouse lung. The results show that the virus content in the mouse lungs in the large and medium dose groups of the pharmaceutical composition of the present invention is significantly lower than that in the model group. Statistical analysis was performed with SPSS software for t test. The results are shown in Table 5.

Table 5: The effect of the pharmaceutical composition of the present invention on the virus titer in the lungs of mice with viral pneumonia

Note: The virus model group is compared with the normal control group, ▲▲p<0.01, and each administration group is compared with the model group, *p<0.05, **p<0.01.

in conclusion

(1) times the LD 10 in mice infected with influenza virus strain FM1 ₅₀ rat lung accommodate up to 100% mortality.

(2) The 10ml/kg dose of Shuanghuanglian can significantly reduce the mortality of mice infected with the virus to 53% and prolong the survival time of mice.

(3) The 7.4g/kg and 3.7g/kg doses of the pharmaceutical composition of the present invention can reduce the mortality of mice infected with influenza virus by 50% and 63%, respectively, p<0.01 compared with the model group. The three doses of 7.4g/kg, 3.7g/kg and 1.9g/kg of the pharmaceutical composition of the present invention can significantly prolong the survival time of virus-infected mice, and compared with the model group, p<0.01.

(4) The lung index of influenza virus infection model mice was significantly higher than that of normal control mice, p<0.01. In addition, the virus titer in the lungs of model mice reached 34.7±9.2, and no influenza virus was detected in the lungs of normal control mice.

(5) The 10ml/kg dose of Shuanghuanglian can significantly reduce the lung index and the virus titer in the lungs of virus-infected mice, p<0.01.

(6) The 7.4g/kg and 3.7g/kg doses of the pharmaceutical composition of the present invention can significantly inhibit the lung index and the virus titer in the lungs of virus-infected mice. Compared with the model group, p<0.01.

The above-mentioned in vitro anti-influenza virus experiment results show that the 31-125mg/ml concentration of the pharmaceutical composition of the present invention has a protective effect on the cytopathic effect caused by influenza virus, and its IC ₅₀ is 65.4 mg/ml; moreover, the in vivo anti-influenza effect of the pharmaceutical composition of the present invention The effect of influenza virus is better than that of in vitro.

The in vivo anti-influenza virus of the drug combination of the present invention shows that the dosage of 7.4g/kg and 3.7g/kg can reduce the mortality of mice infected with influenza virus by 50% and 63%, respectively, which is p<0.05 compared with the model group. The 7.4g/kg, 3.7g/kg and 1.9g/kg doses of the pharmaceutical composition of the present invention can significantly prolong the survival time of virus-infected mice, and compared with the model group, p<0.01. The 7.4g/kg and 3.7g/kg doses of the pharmaceutical composition of the present invention can significantly inhibit the lung index and the virus titer in the lungs of virus-infected mice. Compared with the model group, p<0.01 and p<0.05.

It can be seen from the above experimental results that the pharmaceutical composition of the present invention has an in vitro antiviral effect; the pharmaceutical composition of the present invention has significant resistance to mice when administered in large doses (7.4g/kg) and medium doses (3.7g/kg) in vivo. The role of golden influenza virus infection is reflected in reducing the mortality of mice, prolonging survival time, alleviating lung lesions and reducing the virus content in the lungs.

Experiment III. The effect of the drug combination of the present invention on the new coronavirus (SARS-COV-2) in vitro

1 Material

1.1 Medicine The pharmaceutical composition of the present invention (prepared in Example 3, as described above).

1.2 Cells: VeroE6 cells, preserved by the Virus Room of the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health.

1.3 Virus: SARS-CoV-2, with a titer of TCID50 = 10 ^-6 /100μL, and stored at -80°C in the BSL-3 Laboratory of Guangzhou Customs Technology Center (Respiratory Disease State Key Laboratory of Highly Curable Pathogenic Microorganisms Laboratory). The virus titer used is 100TCID50.

2 Method

2.1 Preparation of the test drug: The drug composition of the present invention is dissolved in dimethyl sulfoxide (DMSO), and ultrasonic is used to aid the dissolution. After centrifugation, the supernatant is taken and filtered for later use, and diluted with the culture medium to the required concentration. 2.2 Determination of the in vitro toxicity of the drug combination of the present invention by the MTT method

Inoculate VeroE6 cells in a 96-well plate, add 100μL of VeroE6 cells at a concentration of 2×10 ⁵ cells/mL to each well, set up 4 multiple wells, and _{culture at 37℃ with 5% CO 2. When the} cells grow into a monolayer, the cells are divided into blanks. Group (without cells, only medium), solvent control group (DMSO group), and pharmaceutical composition group of the present invention. The cells were incubated with 5% CO ₂ and 37°C for 48 hours. 4 hours before the end of the experiment, 100uL of 1mg/mL MTT solution was added, and the culture was continued for 4 hours. Then, the culture was terminated. Measure the absorbance value of each well at 490nm wavelength on a full-wavelength scanner, and calculate the TC ₅₀ value of the drug with GraphPad Prism 6.0.

2.3 In vitro anti-influenza virus activity screening

The antiviral activity of the drug was tested by the cytopathic inhibition method. Inoculate VeroE6 cells in a 96-well plate, add 100μL of VeroE6 cells at a concentration of 2×10 ⁵ cells/mL to each well, set up 4 multiple wells, _{culture with 5% CO 2} at 37°C, and discard the culture solution when the cells grow into a monolayer. Wash the cell surface twice with PBS. The cells are divided into a blank group, a solvent control group, a virus control (negative control) group and a pharmaceutical composition group of the present invention. Add 100uL/well of SARS-CoV-2 virus dilution solution containing 100TCID50 to VeroE6 cells, incubate in an incubator, discard the virus solution after 2 hours, add medium containing the drug to be tested, and continue to incubate in the incubator for 48 hours and observe daily And record the cell pathology. The degree of cell lesions is recorded according to the following 6-level standards: "-" no lesions; "±" means cytopathic less than 10%; "+" means cytopathic greater than or equal to 10% but less than 25%; "++" means Cell lesions are greater than or equal to 25% but less than 50%; "+++" means that cells have lesions greater than or equal to 50% but less than 75%; "++++" means that more than 75% of cells have lesions. GraphPad Prism 6.0 was used to calculate the half inhibitory concentration (IC ₅₀ ).

2.4 Data analysis: Calculate the selection index SI, SI=TC ₅₀ /IC ₅₀ .

3 results

3.1 The toxicity test result of the pharmaceutical composition of the present invention on VeroE6 cells The TC ₅₀ of the pharmaceutical composition of the present invention on VeroE6 cells is 3383 μg/ml. See Table 6.

Table 6 Toxicity of the pharmaceutical composition of the present invention on VeroE6 cells

3.2 Experimental results of the pharmaceutical composition of the present invention against SARS-CoV-2 virus The IC ₅₀ of the pharmaceutical composition of the present invention against SARS-CoV-2 virus is 583 μg/ml. See Table 7. After calculation, SI = TC ₅₀ /IC ₅₀ =5.8.

Table 7 The effect of the pharmaceutical composition of the present invention on SARS-CoV-2 virus

4 Conclusion

The pharmaceutical composition of the present invention has the effect of anti-SARS-CoV-2 virus, and its selection index SI is 5.8.

Claims (13)

1. An application of a pharmaceutical composition in the preparation of antiviral drugs, wherein the pharmaceutical composition is made of the following raw materials in parts by weight:

   Ephedra 52-86; Gypsum 194-324; Forsythia 194-324; Scutellaria 78-130; Mulberry Bark 194-324; Bitter Almond 78-130; Qianhu 78-130; Pinellia 78-130; Tangerine Peel 78-130 Fritillaria 78-130; Arctium lappa 78-130; Honeysuckle 78-130; Rhubarb 39-65; Platycodon 46-76; Licorice 39-65.
2. The application according to claim 1, wherein the pharmaceutical composition is made of the following raw materials in parts by weight:

   Ephedra 52; Gypsum 324; Forsythia 194; Scutellaria 78; Mulberry 194; Bitter Almond 130; Qianhu 78; Pinellia 130; Tangerine Peel 78; Fritillaria 78; Burdock Seed 130; Honeysuckle 130; Rhubarb 39; Platycodon 76; Licorice 65.
3. The application according to claim 1, wherein the pharmaceutical composition is made of the following raw materials in parts by weight:

   Ephedra 86; Gypsum 194; Forsythia 324; Scutellaria 130; Mulberry Bark 324; Bitter Almond 78; Qianhu 130; Pinellia 78; Tangerine Peel 130; Fritillaria 130; Burdock 78; Honeysuckle 78; Rhubarb 65; Platycodon 46; Licorice 39.
4. The application according to claim 1, wherein the pharmaceutical composition is made of the following raw materials in parts by weight:

   Ephedra 69; Gypsum 259; Forsythia 259; Scutellaria baicalensis 104; Mulberry bark 259; Bitter almond 104; Qianhu 104; Pinellia 104; Licorice 52.
5. The application according to claim 1, wherein the pharmaceutical composition is made of the following raw materials in parts by weight:

   Ephedra 55; Gypsum 254; Forsythia 318; Scutellaria baicalensis 107; Mulberry bark 203; Bitter almond 107; Qianhu 82; Pinellia 105; Tangerine peel 84; Fritillaria 125; Burdock 122; Honeysuckle 113; Rhubarb 42; Licorice 50.
6. The use according to any one of claims 1 to 5, wherein the bitter almond is fried bitter almond, the fritillary is Fritillaria fritillary, the honeysuckle is the Lonicera japonica, and the Pinellia ternata is the Qing Pinellia.
7. The use according to any one of claims 1-6, wherein the active ingredient of the pharmaceutical composition is made by the following steps:

   A. Weigh the mother-of-pearl according to the proportion of the group, crush it into fine powder, and set aside;

   B. Weigh ephedra, forsythia, bitter almonds, pinellia, burdock seeds, rhubarb and 40-70% ethanol for 2 times reflux extraction, 1-4 hours each time, and 8-10 times the amount for the first time , Add 6-9 times the amount for the second time, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to heat the clear cream with a relative density of 1.14-1.16 for use;

   C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, honeysuckle, platycodon, and licorice according to the ratio of the prescription, add water and decoct twice for 1-4 hours each time, add 9-11 times the amount for the first time, and then add 9-11 times the amount for the second Add 7-9 times the amount each time, combine the decoction, filter, concentrate to 60°C and measure the clear ointment with a relative density of 1.14-1.16, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture;

   D. Mix the fine powder obtained in step A and the clear cream mixture obtained in step C to obtain the active ingredient of the pharmaceutical composition.
8. The use according to any one of claims 1 to 6, wherein the medicine is a tablet, capsule, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray or injection form.
9. The use according to claim 8, wherein the tablet is made by a method including the following steps:

   A. Weigh the mother-of-pearl according to the proportion of the group, crush it into fine powder, and set aside;

   B. Weigh ephedra, forsythia, bitter almonds, pinellia, burdock seeds, rhubarb and 40-70% ethanol for 2 times reflux extraction, 1-4 hours each time, and 8-10 times the amount for the first time , Add 6-9 times the amount for the second time, combine the extracts, filter, and recover the ethanol from the filtrate under reduced pressure, and concentrate it to 60°C to heat the clear cream with a relative density of 1.14-1.16 for use;

   C. Weigh gypsum, Morus alba peel, Peucedanum, orange peel, honeysuckle, platycodon, and licorice according to the ratio of the prescription, add water and decoct twice for 1-4 hours each time, add 9-11 times the amount for the first time, and then add 9-11 times the amount for the second Add 7-9 times the amount each time, combine the decoction, filter, concentrate to 60°C and measure the clear ointment with a relative density of 1.14-1.16, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture;

   D. Spray drying the cleansing paste mixture obtained in step C, and collect the spray powder for later use;

   E. The spray powder obtained in step D and the fine powder obtained in step A are used to prepare a soft material using ethanol as a binder, and then sieved and granulated; and tableting.
10. The use according to claim 9, wherein the tablet is made by a method including the following steps:

    A. Weigh the mother-of-pearl according to the proportion of the group, crush it into fine powder, and set aside;

    B. Weigh ephedra, forsythia, bitter almond, pinellia, burdock, and rhubarb in proportion to the formula, add 50% ethanol and reflux for extraction 2 times, each 3 hours, 10 times the amount for the first time, and the second time 6 times the amount, the extracts are combined, filtered, the filtrate is decompressed to recover ethanol, and concentrated to a clear paste with a relative density of 1.15 at 60°C for use;

    C. Weigh gypsum, Morus alba peel, Peucedanum, Chenpi, Honeysuckle, Platycodon grandiflorum, and licorice according to the ratio of the prescriptions. Add water and decoct twice for 2 hours each time, 10 times the amount for the first time and 7 times for the second time. Measure, combine the decoction, filter, concentrate to 60°C and measure the clear ointment with a relative density of 1.15, and combine with the clear ointment obtained in step B to obtain a clear ointment mixture;

    D. Spray drying the cleansing paste mixture obtained in step C, and collect the spray powder for later use;

    E. The spray powder obtained in step D and the fine powder obtained in step A are used to prepare a soft material with ethanol as a binder, sieved, granulated, dried, and then sieved, and then mixed with sodium starch glycolate, microcrystalline cellulose, and magnesium stearate. Evenly, press tablets.
11. The use according to any one of claims 1-6, wherein the medicine is used for anti-influenza virus.
12. The use according to claim 11, wherein the influenza virus is influenza A virus.
13. The use according to any one of claims 1-6, wherein the pharmaceutical composition is used for anti-new coronavirus.

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