CN107982356B

CN107982356B - Oral medicine for treating respiratory tract infection

Info

Publication number: CN107982356B
Application number: CN201610947000.1A
Authority: CN
Inventors: 马耀茹; 杨成; 赵卉
Original assignee: Chongqing Hilan Pharmaceutical Co ltd
Current assignee: CHONGQING HILAN PHARMACEUTICAL Co.,Ltd.
Priority date: 2016-10-26
Filing date: 2016-10-26
Publication date: 2021-06-25
Anticipated expiration: 2036-10-26
Also published as: CN107982356A

Abstract

An oral medicine for treating respiratory tract infection is a medicament prepared by traditional Chinese medicine raw materials of 10-30 parts of fructus forsythiae, 5-25 parts of lonicera confusa, 5-20 parts of schizonepeta, 5-20 parts of mulberry leaf, 5-25 parts of radix bupleuri, 5-20 parts of scutellaria baicalensis, 10-25 parts of folium isatidis, 5-20 parts of fructus viticis, 5-20 parts of burdock and 1-10 parts of liquorice according to a conventional preparation method. The drug effect test shows that the drug can inhibit and kill common pathogenic bacteria and viruses of respiratory tract infection in vitro, block the infection of the viruses to cells and inhibit the proliferation of the viruses in the cells; can inhibit the infection of bacteria and viruses in mice and reduce the lung inflammation caused by the infection of bacteria and viruses; the composition has obvious antipyretic effect on rabbit fever models caused by typhoid fever and paratyphoid fever; the anti-inflammatory effect on a mouse peritoneal capillary permeability model and the anti-inflammatory effect on a mouse auricle swelling model caused by xylene are obvious; and has significant analgesic effect.

Description

Oral medicine for treating respiratory tract infection

Technical Field

The invention belongs to the technical field of medical preparations containing raw materials or reaction products of the raw materials and unknown structures, and particularly relates to a plant-derived material.

Background

Respiratory tract infection is the most common clinical respiratory tract disease, the onset is especially common in the crossing time of autumn, winter, spring, summer and autumn with seasonal changes, and the respiratory tract infection is particularly harmful to children, old people, weak people and other people, and pulmonary heart disease, respiratory failure or even death is often induced. Clinically, cough, expectoration, headache, pharyngalgia, hoarseness, fever, chills and tremor, general malaise and so on are the main manifestations.

At present, the medicines for treating respiratory tract infection in the market are mainly antibiotics or selective sex hormone. With the emergence of bacterial drug resistance and the side effect of clinical use of a large amount of antibacterial drugs, the development of natural traditional Chinese medicine preparations is particularly important and urgent. Some Chinese patent medicines aiming at respiratory tract infection, such as acute bronchitis syrup, loquat cough syrup and the like, are available on the market at present, but the medicines are mainly designed aiming at a certain stage of the respiratory tract infection, such as acute tracheitis, bronchitis and the like, or aiming at certain symptoms in the respiratory tract infection, such as cough, expectoration and the like, and the medicines are used for treating a certain symptom or a certain pathological link and are expensive.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the medicines and provide an oral medicine for treating respiratory tract infection, which aims at the whole pathological link of respiratory tract infection and the symptoms, signs or symptoms and signs to be appeared, quickly relieves the pain of a patient, eliminates or prevents the symptoms and signs of the patient, and has the advantages of convenient clinical use, low price and small side effect.

The technical scheme adopted for solving the technical problems is that the oral medicament is prepared from the following traditional Chinese medicine raw materials in parts by mass according to a conventional preparation method:

10-30 parts of fructus forsythiae and 5-25 parts of lonicera confusa

5-20 parts of schizonepeta, 5-20 parts of mulberry leaves

5-25 parts of radix bupleuri and 5-20 parts of radix scutellariae

10-25 parts of folium isatidis and 5-20 parts of fructus viticis

5-20 parts of burdock, 1-10 parts of liquorice;

the preferred traditional Chinese medicine raw materials for preparing the medicine of the invention are as follows in parts by mass:

10-15 parts of fructus forsythiae and 8-15 parts of lonicera confusa

5-10 parts of schizonepeta, 5-10 parts of mulberry leaves

8-15 parts of radix bupleuri and 5-10 parts of scutellaria baicalensis

10-15 parts of folium isatidis and 5-10 parts of fructus viticis

5-10 parts of burdock and 1-5 parts of liquorice;

the optimal mass ratio of the traditional Chinese medicine raw materials for preparing the medicine is as follows:

13 parts of fructus forsythiae and 11 parts of lonicera confusa

9 parts of schizonepeta and 9 parts of mulberry leaves

11 parts of radix bupleuri and 9 parts of radix scutellariae

13 parts of dyers woad leaf, 9 parts of fructus viticis

9 parts of burdock fruit and 3 parts of liquorice.

The oral medicament prepared from the components by a conventional method is a tablet, a granule, a capsule or an oral liquid in pharmaceutics.

The medicine in the proportion is precise and appropriate in compatibility, great in medicine specificity, and has the key of keeping the pathogenesis in mind, and the forsythia and the lonicera confusa are pungent in flavor and cool in property, and clear away heat and toxic materials; folium mori, scutellaria, radix bupleuri and schizonepeta are used for dispelling wind and heat and clearing internal heat; folium isatidis is used for cooling blood and detoxifying, fructus viticis is used for clearing head and eyes, and burdock is used for detoxifying and relieving sore throat; licorice root, radix Glycyrrhizae coordinates the properties of the drugs, protects the stomach and regulates the middle, relieves sore throat and cough. In the formula, wind dispelling and exterior syndrome relieving are compatible with heat clearing and detoxifying, and the formula has the functions of dispelling wind heat externally and clearing heat and toxic materials internally, although pungent and cool, the method emphasizes on clearing upper-jiao wind heat, has no defect of bitter cold injury, combines the effects of dispelling wind and clearing heat, and is a good formula for treating respiratory tract infection.

The preparation method of the oral liquid of the invention comprises the following steps:

the traditional Chinese medicines are proportioned, 12 times of water is added for soaking for 2 hours for the first time, the mixture is decocted for 1.0 hour, 10 times of water is added for decocting for 1.0 hour for the second time, the filtrate is filtered, the filtrate is combined and concentrated to the relative density of 1.10-1.20 (measured at 60 ℃), ethanol is added to ensure that the ethanol content reaches 70 percent, the mixture is kept stand for 24 hours, the filtration is carried out, the ethanol is recovered from the filtrate under reduced pressure until no alcohol smell exists, the concentration is continued, the refrigeration is carried out, the filtration is carried out, the pH value is adjusted to 7.0, distilled water is added to 1000 mL, the. Each bottle is filled with 10 milliliters (mL), and each milliliter contains 0.96 g of traditional Chinese medicine raw materials.

The preparation process of the medicine granule of the invention is as follows:

the traditional Chinese medicine raw materials and the mass ratio of the traditional Chinese medicine raw materials used by the medicine granule of the invention are completely the same as those used by the medicine oral liquid of the invention, the extraction process steps of the traditional Chinese medicine raw materials are the same as those of the traditional Chinese medicine raw materials of the preparation process of the oral liquid of the invention, and the auxiliary materials and other process steps are carried out according to the conventional preparation process of the granule. Each bag weighs 10 g, and each gram contains 0.96 g of traditional Chinese medicine raw materials.

The preparation method of the medicine tablet of the invention is as follows:

the traditional Chinese medicine raw materials and the mass ratio of the traditional Chinese medicine raw materials used by the medicine oral liquid are completely the same as those used by the medicine oral liquid, the extraction process steps of the traditional Chinese medicine raw materials are the same as those of the traditional Chinese medicine raw materials of the preparation process of the oral liquid, and the auxiliary materials and other process steps are carried out according to the conventional preparation process of the tablet. Each tablet is 0.5 g, and each gram contains 4.8 g of crude drug.

The preparation method of the medicine capsule comprises the following steps:

the traditional Chinese medicine raw materials and the mass ratio of the traditional Chinese medicine raw materials used by the medicine capsule of the invention are completely the same as those used by the medicine oral liquid of the invention, the extraction process steps of the traditional Chinese medicine raw materials are the same as those of the traditional Chinese medicine raw materials of the preparation process of the oral liquid of the invention, and the auxiliary materials and other process steps are carried out according to the conventional preparation process of the capsule. Each granule is 0.4 g, and each gram contains 6 g of crude drug.

The drug effect test shows that the drug can inhibit and kill common pathogenic bacteria and viruses of respiratory tract infection in vitro, block the infection of the viruses to cells and inhibit the proliferation of the viruses in the cells; can inhibit the infection of bacteria and viruses in mice and reduce the lung inflammation caused by the infection of bacteria and viruses; the composition has obvious antipyretic effect on rabbit fever models caused by typhoid fever and paratyphoid fever; the anti-inflammatory effect on a mouse peritoneal capillary permeability model and the anti-inflammatory effect on a mouse auricle swelling model caused by xylene are obvious; and has significant analgesic effect.

Detailed Description

The present invention will be described in further detail with reference to examples, but the present invention is not limited to these examples.

Example 1

The traditional Chinese medicine raw materials and auxiliary materials used for producing 1000 mL of the medicine oral liquid product of the invention and the mixture ratio thereof are as follows:

forsythia fruit 130 g

Lonicera confusa 110 g

Schizonepeta tenuifolia, herba Schizonepetae 90 g

Mulberry leaf 90 g

Bupleurum root, radix bupleuri 110 g

Baikal skullcap root, radix Scutellariae 90 g

Folium Isatidis 130 g

Vitex rotundifolia fruit 90 g

Great burdock achene 90 g

Licorice root, radix Glycyrrhizae 30 g

Distilled water was added to 1000 mL.

The preparation method comprises the following steps:

The traditional Chinese medicine raw materials and auxiliary materials used for producing 1000 g of the granular product of the invention and the mass ratio thereof are as follows:

forsythia fruit 130 g

Lonicera confusa 110 g

Schizonepeta tenuifolia, herba Schizonepetae 90 g

Mulberry leaf 90 g

Bupleurum root, radix bupleuri 110 g

Baikal skullcap root, radix Scutellariae 90 g

Folium Isatidis 130 g

Vitex rotundifolia fruit 90 g

Great burdock achene 90 g

Licorice root, radix Glycyrrhizae 30 g

Dextrin was added to 1000 g.

The preparation process is carried out according to the preparation process of the granules. Each bag weighs 10 g, and each gram contains 0.96 g of traditional Chinese medicine raw materials.

The traditional Chinese medicine raw materials and auxiliary materials used for producing 1000 tablets of the medicine tablet product of the invention and the mixture ratio thereof are as follows:

67.7 g weeping forsythia capsule

Honeysuckle flower 57.3 g

Schizonepeta tenuifolia, herba Schizonepetae 46.9 g

Mulberry leaf 46.9 g

57.3 g of Chinese thorowax root

46.9 g of Baikal skullcap root

Folium Isatidis 67.7 g

Vitex rotundifolia 46.9 g

46.9 g of burdock

Licorice root, radix Glycyrrhizae 15.6 g

Starch was added to 500 g.

The preparation process is carried out according to the preparation process of the tablet. Each tablet weighs 0.5 g, and each gram contains 4.8 g of traditional Chinese medicine raw materials.

The traditional Chinese medicine raw materials and auxiliary materials used for producing 1000 capsules of the medicine capsule product of the invention and the mixture ratio thereof are as follows:

54.2 g weeping forsythia capsule

Flos Lonicerae 45.8 g

37.5 g of herba Schizonepetae

Mulberry leaf 37.5 g

Bupleurum root, radix bupleuri 45.8 g

Baikal skullcap root 37.5 g

Folium Isatidis 54.2 g

Vitex rotundifolia 37.5 g

Burdock fruit 37.5 g

Licorice root, radix Glycyrrhizae 12.5 g

Starch was added to 400 g.

The preparation process is carried out according to the preparation process of the capsule. Each granule is 0.4 g, and each gram contains 6 g of traditional Chinese medicine raw materials.

In the mixture ratio of the embodiment, the traditional Chinese medicine raw materials comprise the following components in parts by mass:

13 parts of fructus forsythiae and 11 parts of lonicera confusa

9 parts of schizonepeta and 9 parts of mulberry leaves

11 parts of radix bupleuri and 9 parts of radix scutellariae

13 parts of dyers woad leaf, 9 parts of fructus viticis

9 parts of burdock fruit and 3 parts of liquorice.

Example 2

171.4 g of weeping forsythia

Lonicera confusa 85.7 g

Schizonepeta tenuifolia, herba Schizonepetae 85.7 g

Mulberry leaf 85.7 g

Bupleurum root, radix bupleuri 85.7 g

Baikal skullcap root 85.7 g

Folium Isatidis 171.4 g

Vitex rotundifolia 85.7 g

Arctium lappa fruit 85.7 g

Licorice root, radix Glycyrrhizae 17.1 g

Distilled water was added to 1000 mL.

The preparation process is the same as that of the oral liquid in example 1. Each bottle is filled with 10 mL of the traditional Chinese medicine raw materials, and each milliliter of the traditional Chinese medicine raw materials contains 0.96 g.

171.4 g of weeping forsythia

Lonicera confusa 85.7 g

Schizonepeta tenuifolia, herba Schizonepetae 85.7 g

Mulberry leaf 85.7 g

Bupleurum root, radix bupleuri 85.7 g

Baikal skullcap root 85.7 g

Folium Isatidis 171.4 g

Vitex rotundifolia 85.7 g

Arctium lappa fruit 85.7 g

Licorice root, radix Glycyrrhizae 17.1 g

Dextrin was added to 1000 g.

89.3 g of weeping forsythia

Flos Lonicerae 44.6 g

Schizonepeta tenuifolia, herba Schizonepetae 44.6 g

Mulberry leaf 44.6 g

Bupleurum root, radix bupleuri 44.6 g

Baikal skullcap root 44.6 g

Folium Isatidis 89.3 g

Vitex rotundifolia 44.6 g

Great burdock achene 44.6 g

8.9 g of licorice root

Starch was added to 500 g.

forsythia fruit 71.4 g

Honeysuckle flower 35.7 g

Schizonepeta tenuifolia, herba Schizonepetae 35.7 g

Mulberry leaf 35.7 g

35.7 g of radix bupleuri

Baikal skullcap root 35.7 g

Folium Isatidis 71.4 g

Vitex trifolia 35.7 g

Arctium lappa fruit 35.7 g

Licorice root, radix Glycyrrhizae 7.1 g

Starch was added to 400 g.

10 parts of forsythia suspense and 5 parts of lonicera confusa

5 parts of schizonepeta and 5 parts of mulberry leaves

Bupleurum root 5 parts, baikal skullcap root 5 parts

10 parts of dyers woad leaf and 5 parts of fructus viticis

5 parts of burdock and 1 part of liquorice.

Example 3

134 g of weeping forsythia fruit

Flos Lonicerae 112 g

Schizonepeta tenuifolia (Nepeta cataria L.) 89 g

Mulberry leaf 89 g

Bupleurum root, radix bupleuri 112 g

Baikal skullcap root 89 g

Folium Isatidis 112 g

Vitex rotundifolia (L.) DC 89 g

Burdock fruit 89 g

Licorice root, radix Glycyrrhizae 45 g

Distilled water was added to 1000 mL.

134 g of weeping forsythia fruit

Flos Lonicerae 112 g

Schizonepeta tenuifolia (Nepeta cataria L.) 89 g

Mulberry leaf 89 g

Bupleurum root, radix bupleuri 112 g

Baikal skullcap root 89 g

Folium Isatidis 112 g

Vitex rotundifolia (L.) DC 89 g

Burdock fruit 89 g

Licorice root, radix Glycyrrhizae 45 g

Dextrin was added to 1000 g.

69.8 g of weeping forsythia

Honeysuckle flower 58.1 g

Schizonepeta tenuifolia, herba Schizonepetae 46.5 g

Mulberry leaf 46.5 g

58.1 g of bupleurum root

46.5 g Baikal skullcap root

58.1 g of dyers woad leaf

Vitex rotundifolia 46.5 g

46.5 g of burdock

Licorice root, radix Glycyrrhizae 23.3 g

Starch was added to 500 g.

forsythia fruit 55.8 g

Flos Lonicerae 46.5 g

37.2 g of herba Schizonepetae

Mulberry leaf 37.2 g

46.5 g of bupleurum root

Baikal skullcap root 37.2 g

Folium Isatidis 46.5 g

Vitex rotundifolia 37.2 g

Burdock fruit 37.2 g

Licorice root, radix Glycyrrhizae 18.6 g

Starch was added to 400 g.

30 parts of fructus forsythiae and 25 parts of lonicera confusa

20 parts of schizonepeta, 20 parts of mulberry leaves

Bupleurum root 25 parts, baikal skullcap root 20 parts

Folium isatidis 25 parts, fructus viticis 20 parts

20 parts of burdock and 10 parts of liquorice.

In order to verify the treatment effect of the drug of the invention on respiratory tract infection, the applicant entrusts the pharmaceutical college of the university of transport of west ampere with the oral liquid of the drug of the invention (named as the oral liquid of the drug of the invention at the time of testing) prepared according to the formulation of the example 1 of the invention, and carries out pharmacodynamic tests, and the test conditions are as follows:

purpose of the experiment

The pharmacological effect of the medicament is observed through pharmacological experiments, and certain experimental basis is provided for further clinical trial research.

2 materials of experiment

2.1 test strains

Staphylococcus aureus ATCC25923 Staphylococcus epidermidis CMCC26069

Beta hemolytic streptococcus CMCC32210 pneumococcal CMCC31003

Klebsiella pneumoniae ATCC10031 Haemophilus influenzae CMCC58539

Escherichia coli ATCC25922 Pseudomonas aeruginosa ATCC27853

Candida albicans CMCC85021

Quality control reference strain for drug sensitive experiment

The freeze-dried products of the 9 standard strains are purchased from China medical bacteria collection center of Beijing institute of technology for biological product drug assay, China academy of medical sciences. All the bacteria are recovered and passaged for the second generation, and are prepared into concentrated bacteria liquid with serum glycerin strain preserving liquid, and the concentrated bacteria liquid is frozen and preserved at minus 80 ℃, and is used in the experiment after subculturing for 2 generations before the experiment.

2.2 Experimental strains and cell lines

Influenza A virus H1N1 mouse lung adapted strain FM1 (FM 1) adenovirus type 3 (AD 3)

MDCK (Canine Kidney cell) strain Hep-2 (lip cancer cell) strain

The virus strains and cell strains are purchased from Beijing institute of Virus of Chinese academy of science and medicine. The cell is frozen and stored by passage of liquid nitrogen. Resuscitating before experiment.

2.3 drugs and reagents

2.3.1 the invention medicine, batch number: 20130301. 1ml of oral liquid is equivalent to 0.96 g of crude drug. Each bag is 10 mL. Human clinical planned usage dosage: it is administered orally at a dose of 10 mL, 2 times daily.

2.3.2 Aspirin effervescent tablet, Aslicon pharmaceuticals, Inc., national drug Standard H32026201, batch number: 1210115.

2.3.3 morphine hydrochloride injection, Shenyang first pharmaceutical Co., Ltd, batch No.: 130104-1, specification: 10 mg/mL.

2.3.4 glacial acetic acid, Yangjin Yanghua Chemicals, Inc., batch No. 20120614.

2.3.50.9% physiological saline, national drug group chemical reagents ltd, lot number: F20121220.

2.4 animals and instruments

2.4.1 animals: ICR mice, SPF grade, male and female. KM mice, SPF grade, female. Healthy rabbits, SPF grade, male and female.

All provided by the experimental animal center of the medical college of the western-safety transportation university, the experimental animals produce license numbers: SCXK (shan): 2012-: SYXK (shan) 2012 and 005.

2.4.2 Instrument:

JM type electronic balance, yao jiming weighing and checking equipment limited.

AUY120 electronic balance, manufactured by Shimadzu corporation, Japan.

UV1600 proportion double-beam ultraviolet visible spectrophotometer, manufactured by Fuli Analyzer of Zhejiang.

RB-200 Intelligent hotplate Instrument, Gendtis Hispanics science Inc.

YXQ-LS-50 SII type vertical steam sterilizer, Shanghai Bingshi medical equipment factory.

BIO 1500-II-A2 type biological safety cabinet, Shanghai Zhengcata Chua air purification equipment Co.

HERA Cell 150i carbon dioxide incubator, Thermo corporation, USA.

DHP-9162 electric heating constant temperature incubator, Shanghai-Heng scientific instruments Co.

HW-100 model constant temperature water bath, Nanjing Tester laboratory equipments Ltd.

1-200. mu.l of a tuneable micropipette, Mettler Toledo, Switzerland.

McFarland (0.5-5 McFarland), Wenzhou Congtai Biotechnology, Inc., 201309-JC 16.

A disposable 9cm sterile culture dish, Jiangsu health medical supplies Co. Batch number: 20130515.

disposable 96-well cell culture plates, Costar, usa. Batch number: 201309.

Miller-Haiton broth (MHB), Shanghai science insect Biotechnology development, Inc., 130707.

Miller-Hayton agar (MHA), Shanghai Ke insect Biotechnology development, Inc., 130913.

Chocolate-colored blood plates, Shanghai Zhongke insect Biotechnology development, Inc., 130913.

The culture medium is prepared according to the instruction and sterilized for standby.

Sheep's yang whole blood, 25 mL/bag, Kantai Biotech technologies, Inc., 20131120.

Blood plating: adding 5% of sheep positive whole blood into MHA heated to melt and insulated at 45 ℃, quickly mixing, and pouring into a culture dish. Condensing and storing at 4 ℃ for later use. The blood plate was used within 4 days after preparation.

Chocolate-colored blood plates: adding 5% of sheep positive whole blood into heated and melted 60 ℃ insulated MHA, shaking in a water bath at 85 ℃ for 10 minutes until the blood cells are completely discolored, and pouring into a culture dish. Condensing and storing at 4 ℃ for later use. The chocolate-colored blood plate is used within 4 days after being prepared.

Model BS223S electronic balance, Sartorius, germany.

CKX31-C11BF inverted microscope, Olympus, Japan.

12 well cell culture plates, Costar, usa.

96-well hemagglutination reaction plate, Jiangsu health medical supplies Co.

PRMI1640 medium, Gibco, Lot 20121021.

Hank's Balanced liquid, Shanghai Danry Biotech Co., Ltd., batch No. 20130615.

Mycoplasma-free super newborn bovine serum, hangzhou biotechnology limited, zhejiang, lot No. 20120520.

Cell culture solution: 10% newborn bovine serum, 10 IU/mL penicillin, 1. mu.g/mL streptomycin and 1 mg/mL glutamine are added into the PRMI1640 liquid.

Cell maintenance solution: 2% newborn bovine serum, 10 IU/mL penicillin, 1. mu.g/mL streptomycin and 1 mg/mL glutamine are added into the PRMI1640 liquid.

Chicken red blood cells: collecting healthy cock blood, anticoagulating heparin, adding erythrocyte Ahi preservation solution at a ratio of 1:20, and preserving at 4 deg.C (preservation period of 7 days). The chicken erythrocyte suspension with the concentration of 0.5 percent is prepared after being washed three times by physiological saline at 1500 rpm/10 min/time before the experiment and is used within 4 hours.

2.5 test conditions

The room temperature is 22-24 ℃, the relative air humidity is 40-70%, the temperature of a central air conditioner is adjusted, the air is ventilated, the male and female animals are fed in separate cages, the cleanness is kept, the rat house is cleaned once a day, and the light/dark time is 12/12 hours.

3 test dose selection basis and statistical method of test results

The acute toxicity test result of the medicine of the invention shows that: after the medicine clear paste is administrated to mice by gastric lavage, LD can not be detected₅₀The maximum dose was found to be 229.68 g crude drug/kg.

The formula is derived from clinical proved formulas of old traditional Chinese medicine, and the clinical planned dosage is as follows: the medicine of the invention is 10 mL once, 2 times a day, and 1mL of liquid medicine is equivalent to 0.96 g of crude medicine. The dose for pharmacodynamic tests was pre-tested and was designed as: mice: 2.5 mL/kg, 5 mL/kg, 10 mL/kg, as the crude drugs respectively: 2.4 g crude drug/kg, 4.8 g crude drug/kg, 9.6 g crude drug/kg. Rabbit: 1.25 mL/kg, 2.5 mL/kg, 5 mL/kg, as the crude drugs respectively: 1.2 g crude drug/kg, 2.4 g crude drug/kg, 4.8 g crude drug/kg. Experimental data are expressed as mean ± standard deviation (

S) indicates that t-test was performed between groups to check the significance of drug action. The medicine of the present invention is diluted with pure water into required dosage in different concentration for pharmacodynamic test.

4 methods and results

4.1 in vitro antibacterial test of the inventive drugs

The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the drug of the invention against the major pathogens causing acute upper respiratory infections in vitro were determined.

4.1.1 test strains

Staphylococcus aureus ATCC25923 Staphylococcus epidermidis CMCC26069

Beta hemolytic streptococcus CMCC32210 pneumococcal CMCC31003

Klebsiella pneumoniae ATCC10031 Haemophilus influenzae CMCC58539

Escherichia coli ATCC25922 Pseudomonas aeruginosa ATCC27853

Candida albicans CMCC85021

Quality control reference strain for drug sensitive experiment

4.1.2 Experimental methods

According to the principle of antibacterial in vitro antibacterial experiment of antibacterial drugs and the technology of microbiological examination in the guideline of the medical and political administration of the ministry of health of the people's republic of China for the pre-clinical research of New drugs (Western medicines), MIC is determined by adopting a continuous two-fold gradient agar plate dilution method, and MBC is determined by adopting a liquid transfer plate method.

4.1.2.1MIC determination

Preparation of bacterial liquid

Dipping a small amount of the strain liquid which is frozen and preserved, carrying out passage inoculation on MHA plates (beta hemolytic streptococcus, pneumococcus inoculation blood plates, haemophilus influenzae inoculation chocolate-stained blood plates) by using a separation and marking method, and culturing in a common incubator at 37 ℃ for 48 h. Individual colonies were selected and passaged on MHB (S.beta.haemolyticus, pneumococcus, Haemophilus influenzae inoculated into MHB containing 5% calf serum) and cultured at 37 ℃ for 24 hours. Bacteria were counted using a McLeod turbidimetry and made up with MHB to 106 CFU/mL of bacteria solution for use.

② preparation of medicated flat plate

The medicine is sucked into a sterile triangular flask in sterile operation, and MHA heated and melted at 50 ℃ for heat preservation is added for continuous two-time gradient dilution for 8 gradients. The gradient MHA is poured into a 9cm culture dish and 20 mL/dish, and is kept stand and condensed for later use.

MIC determination of Streptococcus hemolyticus, pneumococcus, and Haemophilus influenzae drug-containing plates were prepared using MHA containing 5% calf serum, and the procedure was as described above.

The concentrations of the medicines of the invention are 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, 0.0078 and 0.0039 (mL/mL) in sequence, and the flat plate containing the medicines is used within 4 hours after preparation.

Culture determination of MIC value

And (4) making partitions and marks on the back of the prepared drug-containing flat plate. And dropwise adding 20 mul of experimental bacteria liquid to the surface of the drug-containing flat plate according to the mark. A bacteria control (bacteria were added dropwise to the drug-free plate) and a media blank were set up. The mixture was allowed to stand at room temperature for 20 minutes. The plate was placed upside down in an incubator and incubated at 37 ℃ for 48 hours to observe the results of the experiment. And judging that the bacteria are positive in bacteriostasis if no bacteria grow in the inoculated bacteria area, and judging that the bacteria are negative in bacteriostasis if bacterial colonies or bacterial mosses appear. The minimum drug concentration for no bacterial growth in the inoculated area is the MIC.

4.1.2.2MBC determination

Firstly, preparing liquid medicine

Sterile procedure the drug of the invention was pipetted into a sterile tube and serially diluted in 8 gradients in two-fold gradients with MHB from 1: 2. Each gradient sample was added dropwise to a 96 well cell culture plate, 0.2 mL/well. Bacterial controls and media controls were set up.

② the action of the drugs with bacteria

Adding 20 mul/hole of experimental bacteria liquid, fully mixing uniformly, and then placing at room temperature for 24 hours.

③ culture and determination of MBC value

The working solution 20. mu.L in the wells was pipetted and diluted into 0.2mL of MHB to remove the effect of the drug. 20 μ L of the diluted working solution was spotted as a marker on HBA plates (Streptococcus hemolyticus, pneumococcus inoculated blood plates, Haemophilus influenzae inoculated chocolate-stained blood plates) containing no drug, and allowed to stand at room temperature for 20 minutes. The plate was placed upside down in an incubator and incubated at 37 ℃ for 48 hours to observe the results of the experiment. The minimum drug concentration for the bacteria-free growth zone is MBC.

4.1.3 results of the experiment

The MIC and MBC of the drug of the invention to 9 experimental bacteria standard strains are shown in Table 1.

TABLE 1 in vitro antibacterial test results of the inventive drugs

Name of bacterium	MIC (g crude drug/mL)	MBC (g crude drug/mL)
			Staphylococcus aureus	0.0299	0.2393
Staphylococcus epidermidis	0.1196	0.2393
			Beta hemolytic streptococcus	0.0299	0.0598
Pneumococcus	0.0598	0.1196
			Haemophilus influenzae	0.0598	0.2393
Klebsiella pneumoniae	0.1196	0.4785
			Escherichia coli	0.1196	0.2393
Pseudomonas aeruginosa	0.4785	0.957
			Candida albicans	0.4785	0.957

As shown in Table 1, the medicament has bacteriostatic and sterilizing effects on main pathogenic bacteria causing respiratory tract infection, and the MBC is 2-8 times of the MIC.

Among the experimental bacteria, Escherichia coli, Pseudomonas aeruginosa and Candida albicans are quality control reference strains for drug sensitivity test, non-respiratory tract infection bacteria, and MIC and MBC values thereof are used as references.

4.2 in vitro antiviral experiments of the inventive drug

The minimum antiviral concentrations of the inventive drugs against influenza virus (RNA virus) and adenovirus (DNA virus) causing acute upper respiratory infection in vitro cell culture were determined.

4.2.1 Experimental strains and cell lines

MDCK (Canine Kidney cell) strain Hep-2 (lip cancer cell) strain

4.2.2 Virus Strain titer and minimum infectivity assay

4.2.2.1 Experimental methods

According to the antiviral experimental requirements in the 'Chinese medicine new drug research guideline', the minimum antiviral effective concentration of the drug is detected by adopting a tissue cell culture method.

Virus recovery and amplification

The frozen virus was thawed, diluted 10-fold with Hank's solution, and inoculated into a well-grown cell monolayer T-type culture flask at 1 mL/flask. FM1 was inoculated with MDCK and AD3 was inoculated with Hep-2. Adsorbing at 37 ℃ for 30 min, adding 10 mL of cell maintenance solution/bottle, incubating at 37 ℃ for 96 hours, and harvesting the virus. The virus is continuously passaged and revived for two generations on the cell, and the virus is harvested and split charged for standby at minus 86 ℃.

Virus fluid titer determination

FM1 titer determination using hemagglutination

FM1 virus was diluted in 1/20 as a two-fold serial gradient and the diluted virus was added to a 96-well hemagglutination plate at 0.1 mL/well. Then 0.1mL of 0.5% chicken red blood cell suspension is added per hole, the mixture is shaken and evenly mixed, and then is kept stand for 40 min at room temperature, and the result is observed.

And (4) judging a result: the maximum dilution of virus fluid capable of forming a circular agglutination (++) is one hemagglutination unit. The titer of the virus solution is the reciprocal of the dilution. The hemagglutination test results are shown in Table 2.

Titer determination by plaque formation assay

Ten times of continuous gradient dilution is carried out on AD3 from 1/10, the virus liquid of each dilution is inoculated into a Hep-2 cell monolayer 12-hole plate, 2 holes are added in parallel, each hole is filled with 0.5mL, and the adsorption is carried out for 30 min at 37 ℃. The well was discarded, and 1.0 mL/well of a cell maintenance medium containing 0.5% agarose was added, as a control for normal cells. 5% CO₂And cultured at 37 ℃ for 96 h. 0.5% Tulip staining and the number of viral plaques formed was counted under the lens. 1-10 plaques in the well served as the final well. The average number of plaques of the final well x the inverse of the dilution fold = titer of the virus fluid. The results of the plaque formation experiments are shown in table 3.

(iii) minimal viral Infection (IC)₁₀₀) Measurement of

FM1 minimum infection determination

Mixing FM1 virus liquid (5.12X 10)³10 times of continuous gradient dilution is started, the solution is added into a MDCK cell monolayer 96-well plate, 4 wells are parallelly added into each concentration of virus solution, each concentration of virus solution is 0.1 mL/well, and the solution is adsorbed at 37 ℃ for 30 min. The well was discarded and 0.2mL of cell maintenance solution was added per well. Normal cell controls were set. 5% CO₂And cultured at 37 ℃ for 96 h. The minimum FM1 concentration that gives cells the appearance of ++++ CPE was the minimum amount of viral infection when viewed under an inverted microscope. The results are shown in Table 4.

Determination of minimum infection

Mixing AD3 virus solution 3.5 × 10⁵PFU/mL was initially diluted in 10-fold serial gradients and added to a Hep-2 cell monolayer 96-well plate with 4 wells per concentration of virus solution in parallel, 0.1 mL/well, adsorbed at 37 ℃ for 30 min. The well was discarded and 0.2mL of cell maintenance solution was added per well. Normal cell controls were set. 5% CO₂And cultured at 37 ℃ for 96 h. The minimum AD3 concentration at which the cells exhibit ++++ CPE was observed under an inverted microscope as the minimum amount of viral infection. The results are shown in Table 5.

4.2.2.1 results of the experiment

See tables 2, 3, 4, 5.

TABLE 2 influenza virus FM1 Titers assay-hemagglutination test results

Degree of dilution

1/20

1/40

1/80

1/160

1/320

1/640

1/1280

Degree of aggregation

++++

+++

Degree of dilution

1/5120

1/10240

1/20480

1/41960

Control

Degree of aggregation

++

+

-

The +++ blood cells are all agglutinated into a disk shape and evenly spread on the bottom of the hole

The + 80% hemagglutination is in the shape of a disk and spread at the bottom of the hole, and the non-agglutinated hemagglutination is in the shape of a solid circle at the center

+ + 50-60% hemagglutination, typically in the form of a hollow ring

+ small amount of hemagglutination, and a coarse-edged solid circle spread in the center of the bottom of the well

Negative for agglutination, with the cells laying as a solid circle with well-defined edges in the center of the well bottom

TABLE 3 adenovirus AD3 Titer assay-plaque formation test results

Not counting: viral infection of cells is excessive, plaques fuse into sheets, no single plaque can be counted

TABLE 4 minimum infection assay for MDCK by FM1

-

Good growth of the partial cells, typical morphology ± most cells, few cells with lesions

+ 20% of cells developing CPE + + 50% of cells developing CPE

+ + + 80% of cells were CPE + + + + all cells were CPE

CPE cytopathic effect. The cell morphology is significantly altered: cells become round, swell or contract, the light transmittance is changed, the cell membrane boundary is fuzzy, a large amount of particle content exists in cytoplasm, the cells are fused, and the cells die and fall off.

TABLE 5 determination of the minimum infection of Hep-2 by AD3

As can be seen from tables 2, 3, 4 and 5, the titer of influenza virus strain FM 1: 5.12X 10³Hemagglutination unit/mL (HCU/mL); adenovirus type 3 titers: 3.5X 10⁷Plaque forming units/mL (PFU/mL); minimal infection of MDCK by FM1The amount is 0.512 HCU/mL; the minimum infection amount of AD3 to Hep-2 is 3.5X 10²PFU/mL。

4.2.3 in vitro determination of the Effect of the anti-influenza Virus strain FM1

4.2.3.1 Experimental methods

Firstly, the toxic action of the medicine on MDCK is measured (maximum nontoxic concentration TC)₀Measurement)

The drug of the present invention was diluted with cell culture fluid to drug solutions of concentrations 1/10, 1/15, 1/20, 1/30, 1/40, 1/60, 1/80, 1/120, each of which was inoculated to a monolayer of 4-well MDCK cells, 0.2mL per well. A normal cell control was set up. Put 5% CO_2、Cultured at 37 ℃ for 96h, and observed under an inverted microscope. Cell morphology typically, the maximum concentration of drug that occurs without cytopathic effect (CPE) is the maximum concentration at which the drug is non-toxic to the cells. The results of the MDCK cytotoxicity test on the drug of the invention are shown in Table 6.

② measuring the minimum direct virus inactivation concentration of the liquid medicine

The medicine is diluted by 8 gradients in a two-fold continuous gradient manner from the maximum ten-fold nontoxic concentration (1/2), and is subpackaged in a 1.5 mL sterile centrifuge tube and a 0.5/tube. 51.2 HCU/mL (100 IC) was added₁₀₀Concentration) 0.5mL of FM1 virus solution is mixed with the liquid medicine and acted for 1 h at room temperature. The working solution is diluted by 10 times by Hank's solution (the maximum drug concentration is 1/20) and inoculated into a MUCK cell monolayer 96-well plate, and the working solution with each concentration is inoculated into 4 wells in parallel at 0.1 mL/well and adsorbed at 37 ℃ for 30 min. The well was discarded and 0.2mL of cell maintenance solution was added per well. Put 5% CO_2、The medium is cultured for 96 hours at 37 ℃, observed under an inverted microscope, and the minimum concentration of the medicine without CPE is the minimum direct inactivation concentration of the medicine to FM 1. The results are shown in Table 7.

② measuring the minimum inhibitory concentration of the liquid medicine on the proliferation of the virus in the cells

FM1 virus solution was diluted to 5.12 HCU/mL (10 × IC) with Hank's solution₁₀₀Concentration), inoculating to a MDCK cell monolayer 96-well plate, adsorbing at 37 ℃ for 30 min, and removing liquid in the well, wherein each well is 0.1 mL. Diluting the medicinal liquid with cell maintenance solution from maximum nontoxic concentration (1/20) by twice continuous gradient, adding into MDCK cell monolayer adsorbed with FM1 virus, and diluting with the same diluentThe release-degree liquid medicine is added into 4 holes in parallel, and each hole is 0.2 mL. Virus control and normal cell control were set. 5% CO₂And cultured at 37 ℃ for 96 h. The cell morphology was observed under an inverted microscope and the minimum concentration of drug without CPE was the minimum inhibitory concentration of the drug on FM1 virus proliferation. The results are shown in Table 8.

Measuring the minimum preventing concentration of the medicine liquid to the virus infected cell

The liquid medicine is diluted by cell maintenance liquid in twice continuous gradient from the maximum nontoxic concentration (1/20), each gradient diluted liquid medicine is added to MDCK cell monolayer 96 pore plate, 4 pores are added in parallel in each dilution, and each dilution is 0.2 mL/pore. 5% CO₂Incubate at 37 ℃ for 12 h. Discarding the solution, adding 5.12 HCU/mL FM1 virus solution 0.1 mL/well, and adsorbing at 37 deg.C for 30 min. The well was discarded and 0.2mL of cell maintenance solution was added per well. Virus control and normal cell control were set. 5% CO₂And cultured at 37 ℃ for 96 h. Cell morphology was observed under an inverted microscope and the minimum concentration of drug without CPE was the minimum prophylactic concentration for FM1 infection with MDCK. The results are shown in Table 9.

4.2.3.2 results of the experiment

See tables 6, 7, 8, 9.

TABLE 6 toxicity test results of the inventive drugs on MDCK cell lines

Degree of dilution

1/10

1/15

1/20

1/30

1/40

1/60

1/80

1/120

Control

①

++

±

-

CPE ②

++

+

-

③

++

+

-

④

++

+

-

TABLE 7 direct inactivation of FM1 by the drug of the present invention

Degree of dilution	1/2	1/4	1/8	1/16	1/32
						①	-	-	-	-	-
CPE ②	-	-	-	-	-
						③	-	-	-	-	+
④	-	-	-	-	-
						Degree of dilution	1/64	1/128	1/256	FM1 control	MDCK control
①	+++	++++	++++	++++	-
						CPE ②	+++	++++	++++	++++	-
③	+++	++++	++++	++++	-
						④	++	++++	++++	++++	-

TABLE 8 inhibition of proliferation of FM1 in MDCK cells by the drugs of the present invention

TABLE 9 preventive Effect of the drugs of the present invention on FM 1-infected MDCK cells

As is clear from the results in tables 6, 7, 8 and 9, the maximum nontoxic concentration of the drug of the present invention to MUCK cell line is 1/20 dilution, i.e., 0.0479 g crude drug/mL; the minimum direct inactivation concentration of the medicine to FM1 is 1/32 dilution (0.0299 g crude drug/mL); the minimum inhibitory concentration of the drug of the invention on the proliferation of FM1 in MDCK is 1/160 dilution (0.0060 g crude drug/mL); the minimum prevention concentration of the medicine of the invention on FM1 infected MDCK cells is 1/40 dilution (0.0239 g crude drug/mL).

4.2.4 determination of in vitro anti-adenovirus type 3 action

4.2.4.1 Experimental methods

The maximum nontoxic concentration TC of the liquid medicine to Hep-2 cells₀Measurement of

The drug of the invention is diluted into drug solutions with concentrations of 1/10, 1/15, 1/20, 1/30, 1/40, 1/60, 1/80 and 1/120 by using cell culture solution, and each drug solution with concentration is respectively inoculated into a 96-well single-layer Hep-2 cell monolayer plate with 4 wells, wherein each well is 0.2 mL. A normal cell control was set up. Put 5% CO_2、Cultured at 37 ℃ for 96 h. The morphology of the cells is typical when observed under an inverted microscope, and the maximum concentration of the drug without cytopathic effect (CPE) is the maximum nontoxic concentration of the drug to the cells. The toxicity test results of the drug of the invention on Hep-2 cells are shown in Table 10.

The medicine is diluted by 8 gradients in a twofold continuous gradient way from the maximum ten times of nontoxic concentration (1/1.5 dilution), and is subpackaged in a 1.5 mL sterile centrifuge tube and a 0.5/tube. Adding 3.510⁴PFU/mL（100×IC₁₀₀Concentration) AD3 virus solution 0.5 was mixed with each of the gradient diluted solutions and allowed to act at room temperature for 1 hour. The working solution is diluted by 10 times (the maximum drug concentration is 1/15) by Hank's solution and inoculated on a Hep-2 cell monolayer 96-well plate, the working solution with each concentration is inoculated on 4 wells in parallel at 0.1 mL/well and adsorbed for 30 min at 37 ℃. The well was discarded and 0.2mL of cell maintenance solution was added per well. Virus control and normal cell control were set. 5% CO₂And cultured at 37 ℃ for 96 h. Cell morphology was observed under an inverted microscope and the minimum concentration of drug without CPE was the minimum effective direct inactivation concentration of the drug for AD 3. The results are shown in Table 11.

Measuring the minimum inhibition concentration of the liquid medicine to the virus proliferation in the cell

AD3 was diluted to 3.5X 10 with Hank's solution³PFU/mL（10×IC₁₀₀Concentration), inoculating into a Hep-2 cell monolayer 96-well plate, adsorbing at 37 ℃ for 30 min, and removing liquid in the well. The liquid medicine is diluted by cell maintenance liquid in a two-fold continuous gradient manner from the maximum nontoxic concentration (1/15 dilution), and is added into a Hep-2 cell monolayer adsorbed with AD3 virus, and the liquid medicine with the same dilution degree is added into 4 holes in parallel, wherein each hole is 0.2 mL. Virus control and normal cell control were set. 5% CO₂And cultured at 37 ℃ for 96 h. The cell morphology was observed under an inverted microscope, and the minimum concentration of drug without CPE was the minimum inhibitory concentration of the drug on the proliferation of AD3 virus. The results are shown in Table 12.

Fourthly, determining the minimum prevention concentration of the liquid medicine to the virus infected cells

The liquid medicine is diluted by cell maintenance liquid in twice continuous gradient from the maximum nontoxic concentration (1/15), each gradient diluted liquid medicine is added to Hep-2 cell monolayer, 4 holes are added in parallel for each dilution, and each hole is 0.2 mL. 5% CO₂Incubate at 37 ℃ for 12 h. Discarding the liquid medicine in the hole, adding 3.5X 10³PFU/mL AD3 virus solution 0.1 mL/well, adsorbed at 37 ℃ for 30 min. The well was discarded and 0.2mL of cell maintenance solution was added per well. Virus control and normal cell control were set. 5% CO₂And cultured at 37 ℃ for 96 h. Cell morphology was observed under an inverted microscope and the minimum concentration of drug without CPE was the minimum effective prophylactic concentration for proliferation of AD3 virus. The results are shown in Table 13.

4.2.4.2 results of the experiment

See tables 10, 11, 12, 13.

TABLE 10 toxicity test results of the inventive drugs against Hep-2 cell line

Degree of dilution

1/10

1/15

1/20

1/30

1/40

1/60

1/80

1/120

Control

①

+

-

CPE ②

+

-

③

++

-

④

+

-

TABLE 11 direct inactivation of AD3 by the drugs of the present invention

TABLE 12 inhibition of AD3 proliferation in Hep-2 cells by the drugs of the present invention

TABLE 13 Experimental results of the preventive effect of the inventive drugs on AD 3-infected Hep-2 cells

As is clear from the results in tables 10, 11, 12 and 13, the maximum nontoxic concentration of the drug of the present invention against the Hep-2 cell line was 1/15 dilution (0.0638 g crude drug/mL); the minimum direct inactivation concentration of the medicament to AD3 is 1/24 dilution (0.0399 g crude drug/mL); the minimum inhibitory concentration of the drug of the invention to the proliferation of AD3 in Hep-2 is 1/60 dilution (0.016 g crude drug/mL); the minimum prevention concentration of the drug of the invention on AD3 infected Hep-2 cells is 1/30 dilution (0.0319 g crude drug/mL).

4.2.5 nodules

The results of the in vitro antiviral pharmacodynamics experiments of the medicament of the invention are summarized in table 14.

TABLE 14 summary of the results of the in vitro antiviral pharmacodynamic experiments for the inventive drugs

4.3 in vivo antibacterial and antiviral experiments

4.3.1 Experimental strains

(1) Staphylococcus aureus ATCC25923 strain

Staphylococcus aureus was inoculated to blood plates and cultured at 37 ℃ for 18 h. Eluting bacterial colony with sterile normal saline, performing turbidimetry, and preparing into 10 with sterile normal saline⁸CFU/mL bacterial solution is reserved.

(2) A type H₁N₁Influenza virus mouse lung adapted strain FM₁Plant strain

The virus was detoxified and the virus titer was determined to be 5X 10³HCU/mL. Subpackaging at-80 deg.C for use.

4.3.2 Experimental methods

Test strain half number Lethal Dose (LD) of ICR mice₅₀) Measurement of

①FM₁For ICR mouse LD₅₀Measurement of

Mice were randomly grouped into groups of 10 mice each, half male and female.

5×10³HCU/mL FM₁The virus solution was diluted 10-fold in serial gradients with sterile physiological saline, and a group of mice were infected with each gradient concentration of virus solution. The mice were anesthetized with ether, virus solution was slowly dropped into the nasal cavity of the mice at 25 μ L/time, and normal control was inhaled by dropping normal saline into the nasal cavity. The infection was repeated at 3 hour intervals. The mice in each group were raised and observed for morbidity and mortality within 7 days. The results are shown in Table 15.

TABLE 15 measurement of lethal dose of FM 1-infected ICR mice

Group of	Animal number (only)	Dose of infection(HCU/kg)	Number of dead animals	Mortality (P)
					C	10	-	0	0
A1	10	1250	9	0.9
					A2	10	125	4	0.4
A3	10	12.5	1	0.1
					A4	10	1.25	0	0

Calculating LD by improved Kouyan method₅₀。FM₁LD of viral strains on ICR mice₅₀It was 157.3655 HCU/kg.

② staphylococcus aureus ATCC25923 strain to ICR mouse LD₅₀Measurement of

Mice were randomly grouped into groups of 10 mice each, half male and female.

1×10⁸The CFU/mL bacterial solution was diluted with sterile physiological saline in 10-fold serial gradients, and a group of mice was infected with each gradient concentration of bacterial solution. The mice were anesthetized with ether, the bacteria solution was slowly dropped into the nasal cavity of the mice at 25. mu.L/time, and normal control was inhaled with normal saline. The infection was repeated at 3 hour intervals. The mice in each group were raised and observed for morbidity and mortality within 7 days. The results are shown in Table 16.

TABLE 16 determination of the amount of mortality caused by Staphylococcus aureus ATCC25923 strain infecting ICR mice

Group of	Animal number (only)	Infectious dose (CFU/kg)	Number of dead animals	Mortality (P)
					C	10	-	0	0
B1	10	2.5×10⁸	10	1.0
					B2	10	2.5×10⁷	10	1.0
B3	10	2.5×10⁶	8	0.8
					B4	10	2.5×10⁵	3	0.3

Calculating LD by improved Kouyan method₅₀. LD of Staphylococcus aureus ATCC25923 Strain on ICR mice₅₀Is 6.27971X 10⁵CFU/kg。

The invention also discloses a therapeutic effect of the medicine on an experimental bacteria infection model

Grouping animals

Mice were randomly grouped into groups of 10 mice each, half male and female. FM₁Virus infection 5 experimental groups were set up: FM₁Infection model group, ribavirin control group, and large, medium and small dose groups of the medicine of the invention. Staphylococcus aureus infections 5 experimental groups were set up: staphylococcus aureus infection model group, amberErythromycin B control group, and large, medium and small dosage groups of the medicine of the invention. A normal control group.

② infection and administration

FM1 infected 5 experimental mice were lightly anesthetized and infected with LD by nasal drip inhalation₅₀Quantity FM₁Virus (5X 10)³HCU/mL FM₁The frozen stock solution of the virus is diluted with 1/80 sterile normal saline and is dripped into the nose to inhale 50 mu L of the frozen stock solution per 20 g of the body weight). Gavage administration was started 24 h after infection. The ribavirin control group is administered with 50 mg/mL ribavirin tablet solution 0.5 mL/20 g (1.25 g/kg), the large, medium and small doses of the drug are administered with 1/2.5 dilution, 1/5 dilution and 1/10 dilution of the drug 0.5 mL/20 g, and the administration amounts are respectively 9.6 g crude drug/kg, 4.8 g crude drug/kg and 2.4 g crude drug/kg. The infection model group was given 0.5 mL/20 g saline.

Mice of 5 experimental groups infected with staphylococcus aureus were lightly anesthetized and infected with LD by nasal drip inhalation₅₀Staphylococcus aureus (2.5X 10)⁵CFU/mL Staphylococcus aureus solution nasal drop 50 u L/20 g body weight). Gavage administration was started 24 h after infection. The erythromycin ethylsuccinate control group is administered with 0.5 mL/20 g (0.5 g/kg) of 20 mg/mL erythromycin ethylsuccinate tablet solution, and the large, medium and small doses of the drug of the invention are respectively administered with 0.5 mL/20 g of the drug of the invention diluted by 1/2.5, 1/5 and 1/10, and the administration amounts are respectively 9.6 g crude drug/kg, 4.8 g crude drug/kg and 2.4 g crude drug/kg. The infection model group was given 0.5 mL/20 g saline.

The normal control group was administered with 50. mu.L/20 g of normal saline by nasal drip, and after 24 hours, the normal control group was administered with 0.5 mL/20 g of normal saline by gavage.

The administration is once daily for 7 days.

③ measurement of pulmonary index

7 days after dosing, mice were sacrificed by cervical dislocation. Weighing the weight, picking the lung and weighing. The lung index was calculated.

Lung index = lung weight (mg)/body weight (g)

The mean and standard deviation of the pulmonary index of each group were determined (

s), t-test between groups.

And calculating the lung index inhibition rate of the treatment group.

Lung index inhibition rate = (model group lung index mean-experimental group lung index mean)/(model group lung index mean-normal group lung index mean) × 100%

(iii) detection of pathogens in Lung tissue

100 mg of lung is weighed, 5mL of sterile physiological saline is added, and a tissue homogenizer is used for homogenizing at 20000 rpm/min by using 6 g of a dispersing head. Standing for 30 min, and collecting supernatant for detecting pathogenic bacteria in lung homogenate.

Staphylococcus aureus detection

The tissue homogenate supernatant was diluted in 10-fold serial gradient. 0.2mL of each gradient dilution homogenate was added to a 12-well plate. Heating to melt mannitol high salt agar culture medium, cooling to 50 deg.C, adding into 12-well culture plate 1.0 mL/well, and rapidly mixing with homogenate. And (5) standing, condensing, and culturing at 37 ℃ for 48h to observe the result. The number of colonies was counted under a colony counter, and the number of colonies of 10 to 100 colonies/well was used as the colony count result. The number of colonies/4 Xdilution is the number of viable Staphylococcus aureus bacteria per mg of lung tissue (CFU/mg).

1 detection

Diluting the supernatant of the tissue homogenate by using normal saline for twice continuous gradient, adding the diluted supernatant into a 96-hole hemagglutination reaction plate with 0.1 mL/hole, adding 0.5% chicken erythrocyte suspension with 0.1 mL/hole, shaking and uniformly mixing, standing for 40 min, and observing the hemagglutination result. The end point was the maximum dilution at which + + circular agglutination was formed. Dilution fold number of end well is FM per mg lung tissue₁Viral titer (HCU/mg).

4.3.3 results of the experiment

The drug of the invention is p-A type H₁N₁Influenza virus mouse lung adapted strain FM₁The results of the antiviral experiments on ICR-infected mice are shown in Table 17, and the results of the antibacterial experiments on ICR-infected mice infected with Staphylococcus aureus ATCC25923 are shown in Table 18.

TABLE 17 anti-influenza virus FM drugs of the invention₁Infection with viral infectionICR mouse experimental results (n =10,

s）

p <0.01 in comparison to infection model group

Table 18 results of ICR mouse infection with inventive drug against staphylococcus aureus ATCC25923 (n =10,

s）

p <0.01 in comparison to infection model group

The results in tables 17 and 18 show that the titer of FM1 in lung homogenate of a large-dose and medium-dose mouse of the drug is remarkably lower than that of a model group (P < 0.01), the lung index is remarkably lower than that of the model group (P < 0.01), and the inhibition rate of the lung index reaches 50-70%; the quantity of staphylococcus aureus in the lung homogenate of the mouse with each dose of the medicament is obviously lower than that of a model group (P < 0.01), the lung index is obviously lower than that of the model group (P < 0.01), and the inhibition rate of the lung index reaches 30-50%.

4.4 study on the influence of the drug of the invention on the fever and body temperature of rabbits caused by typhoid and paratyphoid

4.4.1 Experimental methods

Screening qualified rabbits: healthy white rabbits are taken, the SPF grade is achieved, the weight is 1.8-2.0 kg, and the male rabbit and the female rabbit are half in weight. Anal temperature was measured twice, and the mean was taken as normal body temperature. The rabbit with normal body temperature of 38.5-39.5 deg.c is taken for experiment.

36 rabbits with qualified body temperature are taken and randomly divided into 6 groups according to the body weight, namely a blank control group, a model control group and a positive drug control group, wherein the drug of the invention is small, medium and large in dosage, and each group comprises 6 rabbits. 1mL/kg of physiological saline is injected into ear margin vein of a blank control group, 1mL/kg of typhoid bacteria liquid is injected into ear margin vein of a model group, a positive group and small, medium and large dose groups of the medicine respectively, and the medicine is respectively administrated by gastric perfusion after the body temperature of the model group, the positive group and the small, medium and large dose groups of the medicine is raised to be higher than 1 ℃ (about 1-1.5 hours). The blank group and the model control group are given with 5 mL/kg of normal saline, the positive drug control group is given with 0.1 g/kg of aspirin, the small, medium and large dosage groups of the drug are respectively given with 1.2 g of crude drug/kg, 2.4 g of crude drug/kg and 4.8 g of crude drug/kg on the basis of pre-test, and the administration volume is 5 mL/kg. Anal temperature was measured 30, 60, 90, 120 min after administration.

4.8.2 results of the experiment

See table 19.

Table 19 effect of the inventive drug on the hyperthermic body temperature of paratyphoid typhoid in rabbits (n =6,

s,℃)

note: t-test, P <0.05 compared to model control; p <0.01

From the results in table 19, the body temperature of the positive drug control group at 30, 60, 90, 120 min after administration was significantly decreased (P <0.05 or 0.01) compared to the model control group; compared with a model control group, the body temperature of the medicine of the invention 30 min, 60 min, 90 min and 120 min after the administration of the small, medium and large dose groups is obviously reduced (P is less than 0.05 or 0.01). The medicine provided by the invention is prompted to have the obvious effect of reducing the fever and body temperature of rabbits caused by typhoid fever and paratyphoid fever.

4.5 Effect of the inventive Agents on Permeability of capillary vessels in Abdominal Cavity of mice

4.5.1 Experimental methods

50 ICR mice are selected, the weight of the ICR mice is 18-22 g, and the ICR mice are half female and half male. The weight of the medicine is randomly divided into 5 groups, namely a model group, a positive control group and a small, medium and large dosage group of the medicine. The medicine is administrated by gastric perfusion, the model control group is administrated with 10 mL/kg of normal saline, the positive medicine control group is administrated with 0.1 g/kg of aspirin, and the small, medium and large dosage groups of the medicine are respectively administrated with 2.4 g of crude drug/kg, 4.8 g of crude drug/kg and 9.6 g of crude drug/kg. The preparation is administered 1 time daily for 3 days. 45 min after the last administration, 0.5% Evans blue 0.1mL/10 g body weight is injected into each rat tail vein, then 0.6% acetic acid (0.2 mL/mouse) is injected into the abdominal cavity immediately, 20 min later, the cervical vertebra is removed, the mouse is killed, the abdominal cavity is opened, 10 mL of normal saline is injected for washing, washing liquid is sucked out, 3000 r/min is centrifuged for 10min, supernatant is taken, and the Optical Density (OD) value (OD value indirectly represents permeability) is measured at 590 nm by using a UV 1600-proportion double-beam ultraviolet-visible spectrophotometer. Data to

s denotes that t-test was performed between groups and inhibition was calculated. Inhibition = (absorbance of model control group-absorbance of drug use group)/absorbance of model control group × 100%.

4.5.2 results of the experiment

The results are shown in Table 20.

TABLE 20 Effect of the drugs of the present invention on the permeability of the capillaries in the peritoneal cavity of mice: (

s）

Note: t-test, compare to model group,. P < 0.05; p <0.01

As can be seen from Table 20, the OD value of the positive control group is significantly reduced (P < 0.01) and the inhibition rate is 48.93% compared with the model control group; compared with the model control group, the middle and large dose groups of the medicament have obviously reduced OD values (P <0.05 or 0.01), and the inhibition rates are 38.52% and 44.73% respectively. The medicine of the present invention has high medicine dosage, can reduce the permeability of mouse abdominal cavity capillary and has certain antiphlogistic effect.

4.6 Effect of the inventive drug on swelling of mouse auricle caused by Paraxylene

4.6.1 Experimental methods

50 ICR mice are selected, the mice are SPF-grade, half male and half female, and the weight is 18-22 g. The weight of the animal was randomly divided into 5 groups, i.e., a model control group, a positive control group, and small, medium, and large dose groups of the drug of the present invention. The medicine is administrated by gastric perfusion, the model control group is administrated with 10 mL/kg of normal saline, the positive medicine control group is administrated with 0.1 g/kg of aspirin, and the small, medium and large dosage groups of the medicine are respectively administrated with 2.4 g of crude drug/kg, 4.8 g of crude drug/kg and 9.6 g of crude drug/kg. 45 min after administration, coating dimethylbenzene on the right ear of each mouse for about 30 mu L, killing the animals after 20 min, punching the ear plates on the same positions of the left ear and the right ear by using a 9 mm puncher, weighing the ear plates by using a ten-thousandth balance, and taking the weight difference of the two ear plates as the swelling degree of auricles. And the swelling inhibition rate was calculated. Swelling inhibition rate = (blank control group swelling degree-drug group swelling degree)/blank control group swelling degree × 100%.

4.6.2 results of the experiment

The results are shown in Table 21.

TABLE 21 Effect of the drugs of the present invention on swelling of mouse ear by xylene (n =10,. + -. S)

Group of	Dosage (g/kg)	Animal number (only)	Difference between two ear weights	Inhibition ratio (%)
					Model control group	-	10	16.1±3.82	-
Positive drug control group	0.1	10	7.3±2.81**	54.7
					Small dose group of the present invention	2.4	10	13.3±2.60	17.3
Medium-dose group of the medicament of the invention	4.8	10	11.4±3.89*	29.6
					Large dose group of the present invention	9.6	10	10.5±3.20**	35.0

Note: t-test, P <0.05 compared to model control; p <0.01

As can be seen from the results in table 21, the difference between the weight of the left and right ear pieces was significantly reduced (P < 0.01) in the positive control (aspirin) group compared to the model control group, and the swelling inhibition rate was 54.7%; compared with the model control group, the weight difference of the left and right auricles of the traditional Chinese medicine is obviously reduced (P is less than 0.05 or 0.01), and the swelling inhibition rates are 29.6 percent and 35.0 percent. The invention shows that the medicament of the invention can inhibit mouse auricle swelling caused by dimethylbenzene in medium and large dosage, and has certain anti-inflammatory effect.

4.7 Effect of the inventive drug on acetic acid-induced writhing pain in mice

4.7.1 Experimental methods

50 ICR mice are selected, the weight of the ICR mice is 18-22 g, and the ICR mice are half female and half male. The weight of the medicine is randomly divided into 5 groups, namely a model control group, a positive medicine control group and small, medium and large dosage groups. The medicine is administrated by gastric perfusion, 10 mL/kg of physiological saline of a model control group, 100 mg/kg of aspirin of a positive group, 2.4 g of crude drug/kg, 4.8 g of crude drug/kg, 9.6 g of crude drug/kg of the normal saline solution of the medicine respectively given to small, medium and large dosage groups of the medicine, and 10 mL/kg of administration volume. 30 min after administration, 0.6% acetic acid 0.2 mL/mouse was intraperitoneally injected, and the writhing frequency of each mouse was immediately recorded within 15min, and the writhing reaction inhibition rate was calculated.

The inhibition rate of writhing reaction = (average writhing number of control group-average writhing number of administration group)/average writhing number of control group × 100%.

4.7.2 results of the experiment

The results are shown in Table 22.

Table 22 effect of the drugs of the invention on acetic acid-induced writhing pain in mice (n =10,

±S）

note: t-test, P <0.05 compared to model control; p <0.01

As can be seen from table 22, the number of writhing times within 15min after administration is significantly reduced (P is less than 0.05) in the positive control group (aspirin group) compared with the model control group, and the writhing inhibition rate is 65.67%; compared with a model control group, the drug large-dose group of the invention has the advantages that the drug large-dose group is obviously reduced (P is less than 0.05), and the writhing inhibition rate is 63.43 percent. The large dosage of the medicine can reduce the times of body writhing pain of mice caused by acetic acid and has certain analgesic effect.

4.8 analgesic action of the inventive drug on mice (Hot plate method)

4.8.1 Experimental methods

Screening qualified mice: 80 KM mice are taken, the mice are SPF-grade, the weight is 18-22 g, and the mice are female. Each time 1 was placed on a hot plate, the time (seconds) required for a mouse to sit on the hot plate until licking occurred was taken as the pain threshold for that mouse. The time of the foot after licking is less than 5 seconds or more than 30 seconds or is discarded by skipper. Dividing qualified mice into 5 groups at random according to body weight, repeatedly measuring normal pain threshold values (interval of 5 min), and taking the two normal pain threshold values as the pain threshold values of the mice before administration.

50 qualified ICR mice are taken and randomly divided into 5 groups according to the weight, namely a model control group and a positive drug control group, and the drugs of the invention are small, medium and large dose groups. The normal saline solution of the model contrast group is perfused with 10 mL/kg of stomach physiological saline, the positive drug contrast group is injected with 5 mg/kg of morphine in the abdominal cavity, and the small, medium and large dosage groups of the drug of the invention are separately perfused with 2.4 g of crude drug/kg, 4.8 g of crude drug/kg and 9.6 g of crude drug/kg of the normal saline solution of the drug of the invention. The pain threshold of the mice was measured 15, 30, 60 min after administration. If there is no reaction for 60 seconds, the mice are taken out to avoid scalding, and the pain threshold is calculated as 60 seconds.

4.8.2 results of the experiment

See table 23.

Table 23 analgesic effect of the present drug on mice (hot plate method) (n =10,

±S）

note: t-test, P <0.05, P <0.01, compared to model control group

The results in table 23 show that there was no significant difference in pain thresholds for the groups before administration (P > 0.05); compared with the model control group, the pain threshold values of the positive drug control group at 30 min, 60 min and 90 min after administration are obviously increased (P is less than 0.01); compared with a model control group, the pain threshold value of the traditional Chinese medicine is obviously increased (P is less than 0.05) 60 and 90 min after the traditional Chinese medicine is administrated to a dose group; compared with the model control group, the pain threshold of the drug high-dose group is obviously increased (P is less than 0.05) in 30, 60 and 90 min.

Conclusion of the experiment

The medicament can inhibit and kill common pathogenic bacteria and viruses of respiratory tract infection in vitro, block the infection of the viruses to cells and inhibit the proliferation of the viruses in the cells; can inhibit the infection of bacteria and viruses in mice and reduce the lung inflammation caused by the infection of bacteria and viruses; the composition has obvious antipyretic effect on rabbit fever models caused by typhoid fever and paratyphoid fever; the anti-inflammatory drug has a remarkable anti-inflammatory effect on a mouse peritoneal capillary permeability model and has a certain anti-inflammatory effect on a mouse auricle swelling model caused by xylene; and has certain analgesic effect. The pharmacodynamic results support the functional indications of the medicament, and provide pharmacological basis for further clinical trial and research.

The function and the main treatment of the medicine of the invention are as follows: dispel wind, relieve exterior syndrome, clear heat and remove toxicity. Can be used for treating respiratory tract infection with wind-heat syndrome. The symptoms include fever, aversion to cold, sore throat and dry throat, or red swelling and pain of tonsillitis in throat, nasal obstruction, yellow and turbid nasal discharge, distending pain of head, cough, sticky or yellow phlegm, dry mouth and thirst and the like.

The specification of the medicine of the invention is as follows: the medicine oral liquid of the invention is 10 mL per bottle, and each milliliter contains 0.96 g of traditional Chinese medicine raw materials; the weight of each bag of the medicine granule is 10 g, and each gram of the medicine granule contains 0.96 g of traditional Chinese medicine raw materials; each tablet of the medicine tablet of the invention weighs 0.5 g, and each gram contains 4.8 g of traditional Chinese medicine raw materials; the weight of each capsule is 0.4 g, and each gram contains 6 g of traditional Chinese medicine raw materials.

The application and dosage of the medicine of the invention are as follows: the oral liquid of the medicine is taken orally 1 bottle at a time and 2 times a day; the medicine granules are orally taken 1 bag at a time and 2 times a day; the medicine tablet is orally taken, 4 tablets are taken at a time, and 2 times a day; the medicine capsule of the invention is orally taken 4 capsules at a time and 2 times a day.

The effective period of the medicament of the invention is as follows: and 24 months.

Claims

1. An oral medicine for treating respiratory tract infection is characterized in that the medicine is prepared by the following traditional Chinese medicine raw materials according to the weight portion by a conventional preparation method:

10-30 parts of fructus forsythiae and 5-25 parts of lonicera confusa

5-20 parts of schizonepeta, 5-20 parts of mulberry leaves

5-25 parts of radix bupleuri and 5-20 parts of radix scutellariae

10-25 parts of green Chinese cabbage and 5-20 parts of fructus viticis

5-20 parts of burdock and 1-10 parts of liquorice.

2. The oral medicine for treating respiratory tract infection according to claim 1, wherein the weight parts of the raw materials of the traditional Chinese medicines are as follows:

10-15 parts of fructus forsythiae and 8-15 parts of lonicera confusa

5-10 parts of schizonepeta, 5-10 parts of mulberry leaves

8-15 parts of radix bupleuri and 5-10 parts of scutellaria baicalensis

10-15 parts of green Chinese cabbage and 5-10 parts of fructus viticis

5-10 parts of burdock and 1-5 parts of liquorice.

3. The oral medicine for treating respiratory tract infection according to claim 1, wherein the weight parts of the raw materials of the traditional Chinese medicines are as follows:

13 parts of fructus forsythiae and 11 parts of lonicera confusa

9 parts of schizonepeta and 9 parts of mulberry leaves

11 parts of radix bupleuri and 9 parts of radix scutellariae

13 parts of folium isatidis and 9 parts of fructus viticis

9 parts of burdock fruit and 3 parts of liquorice.

4. An oral medicament for the treatment of respiratory infections according to any of claims 1 to 3, characterized in that: the oral medicine is oral liquid, granules, tablets or capsules in the pharmaceutics.