CN111358929A

CN111358929A - New application of small bupleurum particles in inhibiting influenza A virus

Info

Publication number: CN111358929A
Application number: CN202010408145.0A
Authority: CN
Inventors: 郑如文; 刘宏; 司徒文辉; 江志强; 毕聪; 张俊华; 何逸禧; 杜海泳
Original assignee: Guangzhou Baiyunshan Guanghua Pharmacy Co ltd
Current assignee: Guangzhou Baiyunshan Guanghua Pharmacy Co ltd
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2020-07-03

Abstract

The invention relates to a new application of bupleurum particles in preventing and treating influenza A virus. The invention discovers for the first time that the bupleurum particles can obviously inhibit the subtype of the influenza A virus H3N2, improve the lung injury caused by the subtype of the influenza A virus H3N2, especially have obvious scavenging effect on excessive free radicals caused by the subtype of the influenza A virus H3N2 and present dose-effect relationship characteristics. The invention provides a new application of small bupleurum particles in inhibiting influenza A virus H3N2 and pneumonia and excessive free radicals caused by the influenza A virus.

Description

New application of small bupleurum particles in inhibiting influenza A virus

Technical Field

The invention belongs to the field of Chinese patent medicines, and particularly relates to a new application of a Chinese medicinal composition, namely small bupleurum particles, in inhibiting influenza A virus.

Background

Influenza viruses belong to the family of orthomyxoviridae and are classified into types a, b and c. Among them, influenza a has high pathogenicity to humans, has caused pandemics all over the world many times, causing tens of millions of people to die. Influenza A viruses are divided into many subtypes according to the difference between H and N antigens, H can be divided into 18 subtypes (H1-H18), and N has 11 subtypes (N1-N11). Of these, only H1N1, H2N2, H3N2 primarily infect humans, and many other subtypes of natural hosts are diverse avian and animal species. Once the avian influenza viruses with high pathogenicity such as H5N1, H7N7, H7N9, H9N2 and the like have the capability of transmitting the avian influenza viruses from person to person due to mutation, the avian influenza viruses between people can be caused to be epidemic, and the avian influenza viruses have great potential threat to human beings. Human infection with influenza virus can have systemic symptoms and local symptoms such as cough, nasal obstruction, watery nasal discharge and the like to different degrees, and is easy to have the risk of complication and even death for the old, children and people with potential health problems. Recent studies have shown that influenza virus induces oxidative stress in host cells upon infection of the body, producing large amounts of Reactive Oxygen Species (ROS), thereby causing lung inflammatory lung tissue damage.

A radical is a molecule, atom, ion or group of atoms with unpaired electrons, which actively takes electrons from other atoms or molecules in order to stabilize itself. If the balance between the production and scavenging of free radicals is broken, the free radicals can cause damage to the body. Influenza virus invaders induce the production of a large number of oxygen radicals, such as: hydrogen peroxide, hydroxyl radicals, singlet oxygen, and the like. Meanwhile, macrophages are activated to generate a large amount of Nitric Oxide (NO) free radicals to react to generate peroxynitrite. The two substances jointly cause the increase of the oxidative stress level, cause the local damage of the organism and the reduction of the whole body immune response function, enhance the virus replication capacity, and also cause the lipid of the biological membrane to be damaged by peroxidation, thereby causing the damage of the lung tissue. It follows that free radicals are a major contributor to body injury following infection of the body by influenza virus. Therefore, the compound can eliminate excessive free radicals caused by influenza virus and has important significance for protecting the body and treating the influenza.

Xiaochaihu decoction is one of eight famous prescriptions in traditional Chinese medicine, and is a representative prescription of Shaoyang disease in Shang Han miscellaneous diseases treatise on Sheng Zhang Zhong Jing (medical science). The bupleurum tenue granule is a granular Chinese patent medicine preparation which is developed according to the first family of the Xiaochaihu decoction in Guangzhou Baiyunshanguanghua pharmacy, the active ingredients of the preparation comprise seven traditional Chinese medicines of bupleurum tenue, scutellaria baicalensis, ginger processed pinellia tuber, codonopsis pilosula, ginger, liquorice and Chinese date, the preparation has the effects of relieving exterior syndrome, dissipating heat, soothing liver and harmonizing stomach, and is used for treating exogenous diseases and the syndrome of pathogen attacking Shaoyang. Since the market, the bupleurum tenue granules have good curative effect, are approved by a plurality of doctors and patients, and have good market competitiveness and social benefit. In 2020 Xiaochaihu granules are listed in "Kangdong New type Chinese medicinal treatment scheme for coronavirus pneumonia in Guangdong province (trial second edition). The latest clinical research shows that lopinavir, ritonavir (kresoxim-methyl) and arbidol for treating the novel coronavirus pneumonia are not superior to a control group in the aspects of improving clinical symptoms and accelerating virus removal, and the curative effect is not obvious. The pneumonia disease is a complex disease with multi-system disorder, the effect of treating the disease by actively attacking virus is not good, and the advantages of the traditional Chinese medicine are utilized, so that the better curative effect can be obtained by combining the defensive treatment of anti-inflammation, anti-virus or other mechanisms during the infection resistance. At present, the bupleurum tenue decoction has been reported to have an inhibiting effect on influenza A virus H1N1 subtype and H7N9 subtype, but whether the problems are related to the clearance of free radicals and how the specific mechanism is, the problems are not clear. It is not clear whether the therapeutic agent is effective against other subtypes and which subtypes do have effects and can be clinically used.

Disclosure of Invention

The invention aims to provide a new application of a traditional Chinese medicine composition, namely small bupleurum particles, in inhibiting influenza A viruses.

The invention discovers the new application of the bupleurum particles in inhibiting the influenza A virus H3N2 subtype and free radical damage caused by the influenza A virus for the first time, and particularly solves the problem of removing free radicals in the treatment of the influenza A virus H3N2 subtype infection.

An application of a traditional Chinese medicine composition in preparing a medicine for preventing and treating influenza A virus H3N2 subtype infection is provided, wherein the traditional Chinese medicine composition comprises the following active components in parts by weight: 150 plus or minus 5 parts of radix bupleuri, 56 plus or minus 5 parts of radix scutellariae, 56 plus or minus 3 parts of ginger processed pinellia, 56 plus or minus 3 parts of radix codonopsitis, 56 plus or minus 3 parts of ginger, 56 plus or minus 3 parts of liquorice and 56 plus or minus 3 parts of Chinese date.

The invention also aims to provide application of the Chinese medicinal composition, namely the small bupleurum particles, in preparing medicaments for eliminating excessive free radicals caused by infection of influenza A virus H3N2 subtype.

The application of a traditional Chinese medicine composition in preparing a medicine for eliminating excessive free radicals caused by infection of influenza A virus H3N2 subtype comprises the following active components in parts by weight: 150 plus or minus 5 parts of radix bupleuri, 56 plus or minus 5 parts of radix scutellariae, 56 plus or minus 3 parts of ginger processed pinellia, 56 plus or minus 3 parts of radix codonopsitis, 56 plus or minus 3 parts of ginger, 56 plus or minus 3 parts of liquorice and 56 plus or minus 3 parts of Chinese date.

The invention also aims to provide application of the traditional Chinese medicine composition in preparing a medicine for treating pneumonia caused by influenza A virus H3N2 subtype.

The application of a traditional Chinese medicine composition in preparing a medicine for treating pneumonia caused by influenza A virus H3N2 subtype comprises the following active components in parts by weight: 150 plus or minus 5 parts of radix bupleuri, 56 plus or minus 5 parts of radix scutellariae, 56 plus or minus 3 parts of ginger processed pinellia, 56 plus or minus 3 parts of radix codonopsitis, 56 plus or minus 3 parts of ginger, 56 plus or minus 3 parts of liquorice and 56 plus or minus 3 parts of Chinese date.

In one embodiment, the active ingredients of the traditional Chinese medicine composition comprise the following components in parts by weight: 150 parts of radix bupleuri, 56 parts of scutellaria baicalensis, 56 parts of ginger processed pinellia, 56 parts of codonopsis pilosula, 56 parts of ginger, 56 parts of liquorice and 56 parts of Chinese dates.

In a preferred embodiment, the dosage form of the traditional Chinese medicine composition is granules.

Through in vitro antiviral experiments and in vivo mouse influenza model experiments, the invention discovers for the first time that a traditional Chinese medicine composition (comprising bupleurum particles) can obviously inhibit H3N2 subtype influenza A virus, improve lung injury (such as lung index, lung wet-dry specific gravity and the like) caused by H3N2 subtype influenza A virus, especially has obvious scavenging effect (obviously regulating and controlling CAT, SOD, GSH-px, MDA, NO, MPO, TNF-a, IL-6, ROS, superoxide anion and the like) on excessive free radicals caused by H3N2 subtype influenza A virus, and presents dose-effect relationship characteristics. The invention provides a new application of small bupleurum particles in inhibiting influenza A virus H3N2 and pneumonia and excessive free radicals caused by the influenza A virus.

Drawings

FIG. 1, FIG. 2 and FIG. 3 are histograms of the effect of the small bupleurum particles on the lung index, the lung wet-dry ratio and the inhibition rate of the H2N2 subtype, the H3N2 subtype and the H5N1 subtype infected mouse pneumonia model in example 2 of the present invention, respectively. FIG. 4, FIG. 5, FIG. 6 and FIG. 7 are histograms of the effects of small bupleurum particles on H2N2 subtype, H3N2 subtype and H5N1 subtype infected model mice serum TNF-a, IL-6, ROS and superoxide anion in example 3 of the present invention, respectively.

FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12 and FIG. 13 are histograms of the effect of small bupleurum particles on H2N2 subtype, H3N2 subtype and H5N1 subtype infection model mouse lung tissues CAT, SOD, GSH-px, MDA, NO and MPO, respectively, in example 3 of the present invention.

Detailed Description

The following examples of the present invention are experimental methods without specifying specific conditions, generally according to conventional conditions, or according to conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device that comprises a list of steps is not limited to only those steps or components listed, but may alternatively include other steps or components not listed, or inherent to such process, method, product, or device.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The active ingredients of the bupleurum tenue granules of the invention are: 150 plus or minus 5 parts of radix bupleuri, 56 plus or minus 5 parts of radix scutellariae, 56 plus or minus 3 parts of ginger processed pinellia, 56 plus or minus 3 parts of radix codonopsitis, 56 plus or minus 3 parts of ginger, 56 plus or minus 3 parts of liquorice and 56 plus or minus 3 parts of Chinese date.

The bupleurum tenue granules in the following embodiments are sold by Guangzhou Baiyunshan Guanghua pharmacy, and the active ingredients of the bupleurum tenue granules specifically comprise: 150g of radix bupleuri, 56g of radix scutellariae, 56g of ginger processed pinellia, 56g of radix codonopsitis, 56g of ginger, 56g of liquorice and 56g of Chinese date, and the preparation is granules. Since the adjuvant sucrose can cause false positive results in the antibacterial test, this study used bupleurum tenue granules (sugarless version) for the subsequent tests.

Example 1: in vitro H2N2 subtype, H3N2 subtype and H5N1 subtype inhibition experiment of influenza A virus

1. Materials:

1.1 test drugs: xiaochaihu granules (sugar-free, Guangzhou Baiyunshan Guanghua pharmaceutical GmbH).

1.2 control drugs: ribavirin.

1.3 Virus strains: influenza a viruses (H2N2 subtype, H3N2 subtype, H5N1 subtype). Continuously passaging the virus for 2 times by using a MDCK cell monolayer, harvesting a cell culture solution, detecting BSL-3 (three-level in a biological safety laboratory), subpackaging, and freezing in a refrigerator at the temperature of-80 ℃ for later use.

1.4 cell model: dog kidney MDCK cells undergo apoptosis upon challenge with influenza a virus.

The above medicines are dissolved fully by dimethyl sulfoxide (DMSO), and then stored at-20 ℃ for later use.

2. The method comprises the following steps:

2.1 determination of viral virulence

100 μ l of 10-fold serial diluted virus solution was serially inoculated into 96-well plates with a monolayer of MDCK cells, each containing 8 multiple wells, and cell control was performed. 37 ℃ and 5% CO₂The virus was cultured in an incubator, and cytopathic effect (CPE) was observed day by day with an inverted microscope for 3 days continuously, and detection was carried out by MTT method. The absorbance OD was measured at a wavelength of 570nm in a microplate reader, and the half-infective dose of the virus (TCID50) was calculated by the Reed-Muench method.

And (3) measuring results: influenza A viruses (H2N2 subtype, H3N2 subtype and H5N1 subtype) have virus virulence TCID50 of 10 in MDCK cells^-5.7/0.1mL、10^-5.3/0.1mL、10^-4.9/0.1mL。

2.2 measurement of cytotoxicity of Xiaochaihu granules

A96-well cell culture plate with MDCK cells grown into a monolayer is taken, culture solution is poured out, the 96-well cell culture plate is washed 2 times by PBS, serum-free DMEM culture medium is added to continuously dilute (1600 mu g/mL-50 mu g/mL) small bupleurum particle liquid medicine in a series of 2 times, 100 mu l/well is provided, 4 multiple wells are arranged at each concentration, and 4 normal cell control wells are arranged. 37 ℃ and 5% CO₂Culturing in virus incubator for 72h, detecting by MTT method, and measuring absorbance of each experimental group and cell control group at 570nm wavelength. The viability ratio of the cells in each test well was calculated with reference to the control well. Cell TC50 was calculated according to the Reed-Muench method, and the whole experiment was repeated 3 times and averaged.

As a result: the cell half toxicity concentration TC50 of the bupleurum tenue particles is more than 1150 mu g/mL.

2.3 Activity test of Small Bupleurum particles against influenza A Virus

Collecting 96-well cell culture plate with MDCK cells grown into monolayer, decanting the culture solution, washing with PBS 2 times, adding 100TCID50 virus solution at a concentration of 100 μ l/well, 37 deg.C, and 5% CO₂Adsorbing for 2 h. Adding serum-free DMEM mediumSerially diluting 2 times (200 μ g/mL-12.5 μ g/mL) radix bupleuri granule liquid medicine, and setting virus control group, cell control group and positive medicine group. At 37 deg.C, 5% CO₂The inhibition rate of the drug on the virus is calculated according to the following formula of (percent) virus inhibition rate is × 100% (drug treatment group OD mean value-virus control group OD mean value)/(cell control group OD mean value-virus control group OD mean value) and (percent) the inhibition rate of the drug on the CPE caused by the influenza A virus (H2N2 subtype, H3N2 subtype and H5N1 subtype) and the IC50, and the whole experiment is repeated for 3 times to obtain the average value.

As a result: according to MTT results, calculated by a Reed-Muench method, the suppression rate of bupleurum tenue granules on CPE of H3N2 subtype infected cells exceeds 60 percent, and the suppression rate on CPE of H2N2 subtype and H5N1 subtype infected cells is about 30 percent; the inhibition of the ribavirin group on CPE of the H2N2 subtype, H3N2 subtype and H5N1 subtype infected cells is over 40 percent; the small bupleurum particles can obviously inhibit the proliferation of H3N2 subtype viruses in cells. Wherein, the IC50 of the bupleurum tenue particles to influenza A viruses (H2N2 subtype, H3N2 subtype and H5N1 subtype) is respectively 45.6 mug/mL, 10.4 mug/mL and 39.8 mug/mL, and the IC50 of the positive medicament ribavirin to the influenza A viruses (H2N2 subtype, H3N2 subtype and H5N1 subtype) is respectively 17.3 mug/mL, 18.6 mug/mL and 20.1 mug/mL.

And (4) conclusion: in MDCK cells, the bupleurum tenue particles have obvious inhibitory action (superior to ribavirin) on influenza A virus H3N2 subtype, namely antiviral effect; the bupleurum tenue granules have no obvious inhibition effect (weaker than ribavirin) on influenza A virus H2N2 subtype and H5N1 subtype, namely have no antiviral effect.

Example 2: small bupleurum particles for in vivo experiment of influenza A virus H2N2 subtype, H3N2 subtype and H5N1 subtype infected mouse pneumonia model

1. Materials:

1.2 control drugs: and (3) tamiflu.

1.3 Virus: influenza H2N2 subtype, H3N2 subtype, H5N1 subtype virus A/Hong Kong/1968 mouse lung adapted strain. Continuously passaging the virus for 2 times by using a MDCK cell monolayer, harvesting a cell culture solution, detecting BSL-3 (three-level in a biological safety laboratory), subpackaging, and freezing in a refrigerator at the temperature of-80 ℃ for later use.

1.4 animals:

the Kunming mouse has the weight of 18-20 g, and 160 male mice are provided by SYXK (Guangdong) 2019-. The experiment is started one week after the experimental animals are adapted to the new environment, and an appropriate method is adopted to reduce the harm to the animals in the experimental process.

2. The method comprises the following steps:

randomly dividing mice into a blank control group, a model group H2N2, a model group H3N2, a model group H5N1, a Tamiflu drug group H2N2, a Tamiflu drug group H3N2, a Tamiflu drug group H5N1, a small bupleurum particle low dose group H2N2(5.4g/kg, which is a human clinical equivalent dose, other corresponding groups are the same), a small bupleurum particle medium dose group H2N2(10.8g/kg, other corresponding groups are the same), a small bupleurum particle high dose group H2N2(21.6g/kg, other corresponding groups are the same), a small bupleurum particle low dose group H3N2, a small bupleurum particle medium dose group H3N2, a small bupleurum particle high dose group H3N2, a small bupleurum particle low dose group H5N1, a small bupleurum particle medium dose group H5N1, a small bupleurum particle high dose group H5N1, a total of 16 groups of 10 mice were lightly anaesthetized with ether and 25 μ L of 50LD was used per mouse for the corresponding virus subtype, except for the blank control group.₅₀The virus drops are used for nasal infection, and the blank control group is used for nasal dropping with the same amount of physiological saline. 1h after infection, the blank control group and the model group are subjected to intragastric perfusion by using physiological saline with the same volume, and the low, medium and high dose experimental groups are subjected to intragastric perfusion by using the bupleurum tenue particle aqueous solution once a day for 5 consecutive days.

On the 6 th day after infection, mice are killed by dissection, the mice are fasted for more than 12h before killing by dissection, the mice are taken out after axillary artery exsanguination and sacrifice, the mice are placed in a culture dish containing 9g/L of physiological saline, the surface blood is cleaned, the surface water is sucked by absorbent paper, then the mice are placed on tin foil and weighed together to be wet mass, the mice are marked and placed in a 65 ℃ oven to be dried for 48h, the dry mass is weighed after the mass is not changed, the lung wet-dry ratio and the lung index inhibition rate are calculated, the calculation formula is that the lung wet-dry ratio is lung wet mass (g)/dry lung mass (g), the lung index is lung mass (g)/weight (g) is × 100%, the lung index inhibition rate is lung index inhibition rate (model control group average lung index-experiment group average lung index)/model control group average lung index × 100%, the data processing and statistical method is that the data are expressed by average +/-standard deviation, single factor analysis is adopted for comparison, and SPSS 22.0 software is adopted for processing the data.

3. Results and conclusions:

the greater the lung index value to lung wet-dry ratio, the more severe the pneumonia. The results show (table 1, fig. 1-fig. 3) that small bupleurum particles can significantly reduce the lung index to lung wet-dry ratio of influenza a virus subtype H3N2 infected mice and exhibit a dose-dependent effect; the bupleurum tenue particles have no obvious effect on the lung index and the lung wet-dry ratio of H2N2 subtype and H5N1 subtype infected mice. Namely, the bupleurum tenue particles are found to be capable of inhibiting pneumonia damage caused by influenza A virus H3N2 subtype.

TABLE 1 Effect of Xiaochaihu granules on pulmonary index and pulmonary wet-dry ratio of H2N2 subtype, H3N2 subtype and H5N1 subtype infected mouse model pneumonia (N10)

Note: compared with the blank control group, # P <0.05, # P < 0.01; p <0.05, P <0.01, compared to model control group

Example 3: experiment for eliminating excessive free radicals in mice pneumonia model infected with influenza A virus H2N2 subtype, H3N2 subtype and H5N1 subtype by using bupleurum particles

1. Materials:

1.2 control drugs: and (3) tamiflu.

3 viruses: influenza H2N2 subtype, H3N2 subtype, H5N1 subtype virus A/Hong Kong/1968 mouse lung adapted strain. Continuously passaging the virus for 2 times by using a MDCK cell monolayer, harvesting a cell culture solution, detecting BSL-3 (three-level in a biological safety laboratory), subpackaging, and freezing in a refrigerator at the temperature of-80 ℃ for later use.

1.4. Animals:

the Kunming mouse has the weight of 18-20 g and is male, 160 mice, provided by the Guangdong provincial medical experimental animal center, SYXK (Guangdong) 2019-. The experiment is started one week after the experimental animals are adapted to the new environment, and an appropriate method is adopted to reduce the harm to the animals in the experimental process.

2. The method comprises the following steps:

randomly dividing mice into a blank control group, a model group H2N2, a model group H3N2, a model group H5N1, a Tamiflu drug group H2N2, a Tamiflu drug group H3N2, a Tamiflu drug group H5N1, a small bupleurum particle low dose group H2N2(5.4g/kg, which is a human clinical equivalent dose, other corresponding groups are the same), a small bupleurum particle medium dose group H2N2(10.8g/kg, other corresponding groups are the same), a small bupleurum particle high dose group H2N2(21.6g/kg, other corresponding groups are the same), a small bupleurum particle low dose group H3N2, a small bupleurum particle medium dose group H3N2, a small bupleurum particle high dose group H3N2, a small bupleurum particle low dose group H5N1, a small bupleurum particle medium dose group H5N1, a small bupleurum particle high dose group H5N1, a total group H16, a small bupleurum particle medium dose group H5N1, a total group H3N 1, a small bupleurum particle medium dose group H5N 50, a small bupleurum particle, the blank control group was nasally instilled with an equal amount of physiological saline. 1h after infection, the blank control group and the model group are subjected to intragastric perfusion by using physiological saline with the same volume, and the low, medium and high dose experimental groups are subjected to intragastric perfusion by using the bupleurum tenue particle aqueous solution once a day for 5 consecutive days.

On day 6 after infection, the mouse eyeballs were bled, centrifuged at 3500r/min at 4 ℃ for 15min to separate serum, and stored at-20 ℃ for future use. Detecting the content of TNF-a, ROS and IL-6 in serum and resisting superoxide anion (O) according to the kit specification₂-) units of activity.

Taking out the whole lung after killing the animal, placing the lung in a homogenizer, adding normal saline, grinding to prepare homogenate, centrifuging for 20min at the temperature of 4 ℃ at 2500r/min, taking supernatant, and storing at the temperature of minus 20 ℃ for later use. Detecting the contents of CAT, SOD, GSH-px, MDA, NO and MPO in lung tissues according to the kit specification.

The experimental data were statistically analyzed using SPSS 22.0 software, the data were expressed as mean. + -. standard deviation, the differences between groups were analyzed using one-way variance, and p <0.05 was statistically significant.

3. Results and conclusions:

TNF-a and IL-6 can induce lung endothelial cell activation, leukocyte migration, granulocyte degranulation, capillary leakage and the like, activate damaged granulocytes, endothelial cells, blood platelets and the like, further release oxygen free radicals, lysosomal enzymes and the like, and induce and aggravate inflammatory response. The higher the level of MDA, the higher the degree of lipid peroxidation; high levels of NO, ROS and MPO cause cytotoxic effects, mediating immune damage. The results show (table 2, table 3, fig. 4-fig. 7, fig. 8-fig. 13) that after mice are infected with influenza a virus subtype H3N2, the administration of bupleurum tenue particles can obviously inhibit the expression of TNF-a and IL-6, inhibit the release of oxygen free radicals, simultaneously reduce the contents of MDA, NO, ROS and MPO, reduce the degree of lipid peroxidation and reduce the levels of various inflammatory factors, thereby reducing inflammatory reaction; after mice are infected with influenza A virus H2N2 subtype and H5N1 subtype, the administration of the small bupleurum particles has no obvious inhibition effect on the inflammatory reaction of organisms.

The higher the unit of activity of the superoxide anion, the more superoxide anion (O) is eliminated₂-) the stronger the ability; CAT, SOD and GSH-px are important antioxidant enzymes of the organism, can clear free radicals and relieve oxidative damage. The results show (table 2, table 3), after mice are infected with influenza A virus H3N2 subtype, the activity of CAT, SOD and GSH-px and the capability of resisting superoxide anion are obviously improved by the administration of the bupleurum particles, the capability of eliminating free radicals is enhanced, and the lung tissue damage is reduced; after mice are infected with influenza A virus H2N2 subtype and H5N1 subtype, the administration of the small bupleurum particles has no obvious effect of eliminating excessive free radicals of organisms.

TABLE 2 Effect of Xiaochaihu granules on H2N2 subtype, H3N2 subtype and H5N1 subtype infection model mice serum TNF-a, IL-6, ROS, anti-superoxide anion (N10)

Group of	TNF-a(pg/ml)	IL-6(pg/ml)	ROS(U/ml)	Anti-superoxide anion (U/L)
					Blank control group	2.06±0.18	8.94±0.75	100.20±8.81	220.56±18.29
Model group H2N2	3.56±0.31##	21.97±0.99##	328.31±19.81##	142.33±8.18##
					Tamiflu drug group H2N2	2.36±0.16**	12.01±0.92**	169.64±12.08**	190.25±13.47**
Xiaochaihu granules H2N 2-low	3.47±0.35	20.56±1.96	319.11±17.14	151.21±14.95
					Xiaochaihu granules H2N 2-middle	3.39±0.39	19.66±1.25	311.99±12.58	152.34±16.27
Xiaochaihu granules H2N 2-Gao	3.22±0.51	19.21±1.33	313.02±14.66	159.12±18.62
					Model group H3N2	3.37±0.29##	20.31±0.73##	337.55±19.62##	150.36±9.20##
Tamiflu drug group H3N2	2.17±0.18**	11.79±0.87**	150.80±10.04**	183.15±10.43**
					Xiaochaihu granules H3N 2-low	2.28±0.2**	16.14±1.22**	246.63±21.40**	197.19±17.96**
Xiaochaihu granules H3N 2-Zhongzhong	2.21±0.17**	13.69±0.99**	180.74±13.41**	213.58±18.85**
					Xiaochaihu granules H3N 2-Gao	2.19±0.19**	12.37±0.90**	137.98±9.42**	217.22±20.63**
Model group H5N1	3.76±0.23##	18.39±1.17##	353.22±23.69##	155.31±11.39##
					Tamiflu drug group H5N1	2.25±0.21**	12.56±1.03**	158.63±15.63**	178.29±11.08**
Xiaochaihu granules H5N 1-low	3.42±0.38	17.25±1.02	336.59±18.24	161.21±17.45
					Xiaochaihu granules H5N 1-Zhongzhong	3.34±0.29	16.89±1.22	329.34±12.36	158.39±16.55
Xiaochaihu granules H5N 1-Gao	3.18±0.47	17.21±0.98	313.29±15.33	162.33±15.92

Note: compared with the blank control group, # P <0.05, # P < 0.01; p <0.05 compared to model control,

**P<0.01

TABLE 3 action of Xiaochaihu granules on H2N2 subtype, H3N2 subtype and H5N1 subtype infection model mouse lung tissue CAT, SOD, GSH-px, MDA, NO and MPO (N10)

And (4) conclusion: the experimental data show that the bupleurum tenue particles can effectively relieve the peroxidation injury of respiratory tracts and lungs caused by the H3N2 subtype of influenza A virus, increase the capability of organism in clearing free radicals and achieve the effect of treating pneumonia caused by virus infection in the process of inhibiting the H3N2 subtype of influenza A virus and the free radical injury caused by the subtype of influenza A virus.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. An application of a traditional Chinese medicine composition in preparing a medicine for preventing and treating influenza A virus H3N2 subtype infection is characterized in that the traditional Chinese medicine composition comprises the following active components in parts by weight: 150 plus or minus 5 parts of radix bupleuri, 56 plus or minus 5 parts of radix scutellariae, 56 plus or minus 3 parts of ginger processed pinellia, 56 plus or minus 3 parts of radix codonopsitis, 56 plus or minus 3 parts of ginger, 56 plus or minus 3 parts of liquorice and 56 plus or minus 3 parts of Chinese date.

2. The use of claim 1, wherein the active ingredients of the Chinese medicinal composition comprise, in parts by weight: 150 parts of radix bupleuri, 56 parts of scutellaria baicalensis, 56 parts of ginger processed pinellia, 56 parts of codonopsis pilosula, 56 parts of ginger, 56 parts of liquorice and 56 parts of Chinese dates.

3. The use of claim 1 or 2, wherein the dosage form of the Chinese medicinal composition is granules.

4. The application of a traditional Chinese medicine composition in preparing a medicine for eliminating excessive free radicals caused by infection of influenza A virus H3N2 subtype comprises the following active components in parts by weight: 150 plus or minus 5 parts of radix bupleuri, 56 plus or minus 5 parts of radix scutellariae, 56 plus or minus 3 parts of ginger processed pinellia, 56 plus or minus 3 parts of radix codonopsitis, 56 plus or minus 3 parts of ginger, 56 plus or minus 3 parts of liquorice and 56 plus or minus 3 parts of Chinese date.

5. The use of claim 4, wherein the active ingredients of the Chinese medicinal composition comprise, in parts by weight: 150 parts of radix bupleuri, 56 parts of scutellaria baicalensis, 56 parts of ginger processed pinellia, 56 parts of codonopsis pilosula, 56 parts of ginger, 56 parts of liquorice and 56 parts of Chinese dates.

6. The use of claim 4 or 5, wherein the dosage form of the Chinese medicinal composition is granules.

7. The application of a traditional Chinese medicine composition in preparing a medicine for treating pneumonia caused by influenza A virus H3N2 subtype comprises the following active components in parts by weight: 150 plus or minus 5 parts of radix bupleuri, 56 plus or minus 5 parts of radix scutellariae, 56 plus or minus 3 parts of ginger processed pinellia, 56 plus or minus 3 parts of radix codonopsitis, 56 plus or minus 3 parts of ginger, 56 plus or minus 3 parts of liquorice and 56 plus or minus 3 parts of Chinese date.

8. The use of claim 7, wherein the active ingredients of the Chinese medicinal composition comprise, in parts by weight: 150 parts of radix bupleuri, 56 parts of scutellaria baicalensis, 56 parts of ginger processed pinellia, 56 parts of codonopsis pilosula, 56 parts of ginger, 56 parts of liquorice and 56 parts of Chinese dates.

9. The use of claim 7, wherein the Chinese medicinal composition is XIAOCHAIHU granule.