CN113288930A

CN113288930A - In-vivo and in-vitro anti-RSV effective part of liquorice and preparation method and application thereof

Info

Publication number: CN113288930A
Application number: CN202110449035.3A
Authority: CN
Inventors: 崔清华; 田景振; 李忠原; 李保宏
Original assignee: Shandong University of Traditional Chinese Medicine
Current assignee: Shandong University of Traditional Chinese Medicine
Priority date: 2021-04-25
Filing date: 2021-04-25
Publication date: 2021-08-24

Abstract

The application discloses an in vivo and in vitro anti-RSV effective part of liquorice and a preparation method and application thereof, belonging to the technical field of medicines. The effective part of the liquorice for resisting RSV in vivo and in vitro is an n-butyl alcohol part obtained from a liquorice crude extract; the crude extract of Glycyrrhrizae radix is obtained by extracting Glycyrrhrizae radix with water. The licorice effective part of the in vivo and in vitro anti-RSV has better inhibiting effect on the adsorption stage and the replication stage of the RSV; can improve lung tissue damage caused by RSV, obviously reduce the lung index of mice, obviously reduce the expression levels of TLR4, NF-k B, TNF-alpha and keap1 in the serum of the mice, and obviously increase the expression levels of Nrf2 and IFN-beta in the serum of the mice.

Description

In-vivo and in-vitro anti-RSV effective part of liquorice and preparation method and application thereof

Technical Field

The application relates to an in vivo and in vitro anti-RSV effective part of liquorice, a preparation method and application thereof, belonging to the technical field of medicines.

Background

Respiratory Syncytial Virus (RSV) infection is a common disease in pediatric clinics, with infants being the major sick population and almost all children under 2 years of age having RSV viral infection. RSV is highly contagious, can be transmitted directly through human and human body fluids, can survive on body surfaces, is easily contagious when exposed to contaminated objects, and is frequently subject to recurrent infections because RSV virus infection does not produce life-long immunity.

At present, no specific medicine for treating RSV infection exists at home and abroad, and only ribavirin and palivizumab are approved to be used internationally, but the effect is controversial, and especially ribavirin has a teratogenic effect. Therefore, the development of safe RSV infection treatment drugs becomes a key.

Disclosure of Invention

In order to solve the problems, the liquorice effective part for resisting RSV in vivo and in vitro is provided, and has better inhibiting effect on the adsorption stage and the replication stage of the RSV; can improve lung tissue damage caused by RSV, obviously reduce the lung index of mice, obviously reduce the expression levels of TLR4, NF-k B, TNF-alpha and keap1 in the serum of the mice, and obviously increase the expression levels of Nrf2 and IFN-beta in the serum of the mice.

A Glycyrrhrizae radix effective fraction for resisting RSV in vivo and in vitro is obtained from n-butanol fraction of Glycyrrhrizae radix crude extract; the crude extract of Glycyrrhrizae radix is obtained by extracting Glycyrrhrizae radix with water.

Preferably, the effective part of the liquorice is an n-butyl alcohol part obtained by sequentially extracting the liquorice crude extract with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol.

According to another aspect of the present application, there is provided a method for preparing an effective fraction of licorice for in vitro and in vivo anti-RSV, comprising the steps of:

(1) adding water into Glycyrrhrizae radix, soaking, heating, reflux extracting, and concentrating to obtain Glycyrrhrizae radix water extractive solution;

(2) sequentially extracting the licorice water extract with petroleum ether, dichloromethane, ethyl acetate and n-butanol, and retaining n-butanol extract to obtain the licorice effective component.

Preferably, the step (2) includes:

adding petroleum ether into the licorice aqueous extract, wherein the volume ratio of the petroleum ether to the licorice aqueous extract is 1:1, extracting with petroleum ether for 2-3 times until the petroleum ether layer is colorless, separating, and retaining a water phase as a first water phase;

adding dichloromethane into the first aqueous phase, wherein the volume ratio of the dichloromethane to the first aqueous phase is 1:1, extracting with dichloromethane for 2-3 times until the dichloromethane layer is colorless, separating, and retaining the aqueous phase as a second aqueous phase;

adding ethyl acetate into the second water phase, wherein the volume ratio of the ethyl acetate to the second water phase is 1:1, extracting with ethyl acetate for 2-3 times until the ethyl acetate layer is colorless, separating, and retaining the water phase as a third water phase;

adding n-butanol into the third water phase at a volume ratio of 1:1, extracting with n-butanol for 2-3 times until the n-butanol layer is colorless, separating, and retaining the n-butanol layer to obtain the effective component of Glycyrrhrizae radix.

Preferably, the step (1) includes:

cleaning Glycyrrhrizae radix, adding 12 times of water, soaking for 30min, heating and reflux extracting for 90min twice, mixing the two extractive solutions, and concentrating to obtain Glycyrrhrizae radix water extract.

Preferably, the n-butanol layer in step (2) is concentrated and dried under reduced pressure to a dry powder.

Preferably, the n-butanol in step (2) is a water-saturated n-butanol solution.

According to still another aspect of the application, the application of the liquorice effective part for resisting RSV in vivo and in vitro in preparing the medicament for preventing and/or treating RSV is provided.

Preferably, the application of the effective part of the liquorice in preparing the medicament for preventing and/or treating respiratory system injury caused by RSV; the respiratory system injury is selected from the group consisting of respiratory system injury caused by inflammatory response and oxidative stress.

Preferably, the active part of the liquorice has the anti-RSV activity of CC₅₀At 2170. mu.g/mL, EC ₅₀60 μ g/mL, and a therapeutic index TI of 36.22.

Preferably, the licorice effective part has an inhibitory effect on the adsorption stage and the replication stage in the RSV replication cycle.

Preferably, when the effective part of the liquorice is used for resisting RSV in vitro, the addition time is-2-2 h and 8-12h, and the effect of inhibiting RSV is achieved.

Preferably, the addition time is between-2 and 2 hours, so that the virulence of the virus can be reduced by 96.47 percent, and the addition time is between 8 and 12 hours, so that the virulence of the virus can be reduced by 82.12 percent.

In the present application, "CC₅₀", means half the lethal dose;

“EC₅₀", means half the effective amount;

"DMEM" is a medium containing various amino acids and glucose.

"TI" refers to the therapeutic index, calculated as TI ═ CC₅₀/EC₅₀。

Benefits of the present application include, but are not limited to:

1. the licorice effective part for resisting RSV in vivo and in vitro has better inhibiting effect on the adsorption stage and the replication stage of RSV; can improve lung tissue damage caused by RSV, obviously reduce the lung index of mice, obviously reduce the expression levels of TLR4, NF-k B, TNF-alpha and keap1 in the serum of the mice, and obviously increase the expression levels of Nrf2 and IFN-beta in the serum of the mice.

2. According to the preparation method of the effective part of the liquorice for resisting RSV in vivo and in vitro, the process is simple and convenient, the operation is easy, and the popularization is facilitated.

3. According to the application of the liquorice effective part for resisting RSV in vivo and in vitro, the liquorice effective part has better effect in preventing and/or treating respiratory system injury infected by RSV and has small side effect.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is a graph showing the anti-RSV action at the n-butanol site according to example 1 of the present application;

FIG. 2 is a graph showing the anti-RSV effect of glycyrrhizic acid according to example 1 of the present application;

FIG. 3 is a graph showing the anti-RSV action of glycyrrhetinic acid according to example 1 of the present application;

FIG. 4 is a graph showing the anti-RSV effect of monoammonium glycyrrhizinate according to example 1 of the present application;

FIG. 5 is a graph of the anti-RSV effect of ribavirin in accordance with example 1 of the present application;

FIG. 6 is a time course experiment of ribavirin as it relates to example 2 of the present application during RSV infection;

FIG. 7 is a temporal experiment of the n-butanol fraction involved in example 2 of the present application during RSV infection;

fig. 8 is a lung tissue structure diagram of a mouse according to example 3 of the present application, in which a is a lung tissue structure diagram of a mouse in a model group, B is a lung tissue structure diagram of a mouse in a normal control group, C is a lung tissue structure diagram of a mouse in an n-butanol group, and D is a lung tissue structure diagram of a mouse in a ribavirin group;

FIG. 9 is a graph of the effect of the n-butanol fraction involved in example 3 of the present application on the lung index of RSV infected mice;

FIG. 10 shows the effect of n-butanol fraction on the expression levels of TLR4, NF- κ B, TNF- α, and IFN- β in serum of RSV infected mice according to example 3 of the present application;

FIG. 11 shows the effect of the n-butanol fraction on the levels of keap1 and Nrf2 expression in the serum of RSV-infected mice according to example 3 of the present application.

Detailed Description

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

Unless otherwise specified, the materials in the examples of the present application were all purchased commercially, wherein:

1. medicine and main reagent

Licorice root, purchased from Zhongqiang Chinese herbal pieces Limited (origin: Xinjiang Hotan, lot # 191101);

ribavirin, available from Beijing Soilebao Tech Co., Ltd;

fetal bovine serum (Gibco, USA, batch No. 42A 0378K);

DMEM cell culture fluid (Gibco, USA, batch: 8120010);

penicillin mixed solution (Biosharp, lot: 70011000);

phosphate buffer (Gibco, USA, batch: 8120150);

0.25% EDTA pancreatin (Gibco, USA, batch: 1951208);

dimethyl sulfoxide (DMSO, Amresco, USA, lot number 821D 035);

thiazole blue dye liquor (VWR, batch No. 1636C 097);

nrf2 kit (CUSABIO, batch: B06011498);

NF-. kappa.B kit (CUSABIO, batch No. B07011499);

keap1 kit (CUSABIO, lot: A16019408);

TLR4 kit (enzyme-linked, batch No.: 12/2020);

TNF-alpha kit (enzyme-linked, batch: 12/2020);

IFN- β kits (enzyme-linked, batch: 12/2020).

2. Host cell and virus seed:

human laryngeal carcinoma epithelial cells (Hep2 cells) were purchased from shanghai shengbo biomedical science and technology ltd;

respiratory Syncytial Virus (RSV) is provided by the institute of basic medicine, institute of medical sciences, shandong province.

3. Experimental animals:

40 BALB/c female mice of 4 weeks old, 12-14 g of body mass, purchased from Beijing Wittingle laboratory animal technology Limited, and the certification number: SCXK (Jing) 2016-; license number used by experimental unit: SYXK (lu) 20170022.

4. The instrument comprises the following steps:

microplate reader (Molecular Device, Spectra Max M5);

biosafety cabinets (Shanghai force science instruments, Inc., HFsafe-1200 TE);

CO₂cell constant temperature incubator (Shanghai Li Shen scientific instruments Co., Ltd., HF 90);

TDD5M bench balance centrifuge (changsha mundane instruments ltd);

model DZF-6050 vacuum drying oven (Shanghai Bingfeng industries, Ltd.);

a freeze drier (Christ, Germany, ALPHA1-4LD plus);

CKX-31 inverted microscope (Olympus).

The effective part of the liquorice used for resisting RSV in vivo and in vitro in the embodiment of the application is an n-butanol part obtained from a liquorice crude extract; the crude extract of Glycyrrhrizae radix is water extract of Glycyrrhrizae radix obtained by water extraction;

further, the effective part of the liquorice is n-butyl alcohol extract obtained by sequentially extracting water extract of the liquorice with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol.

The preparation method of the licorice effective part for resisting RSV in vivo and in vitro comprises the following steps:

The specific preparation method of the effective part of the liquorice comprises the following steps:

(1) cleaning 200g of licorice root, adding water 12 times of the weight of the licorice root, soaking for 30min, heating and refluxing for 90min, extracting twice, combining the two extracting solutions, centrifuging, filtering and concentrating to obtain a licorice root water extracting solution;

(2) adding petroleum ether into Glycyrrhrizae radix water extractive solution at a volume ratio of petroleum ether to Glycyrrhrizae radix water extractive solution of 1:1, extracting with petroleum ether for 2-3 times until petroleum ether layer is colorless, separating, and retaining water phase as first water phase;

adding dichloromethane into the first water phase, wherein the volume ratio of the dichloromethane to the first water phase is 1:1, extracting with dichloromethane for 2-3 times until the dichloromethane layer is colorless, separating, and retaining the water phase as a second water phase;

adding n-butanol into the third water phase at a volume ratio of 1:1, extracting with n-butanol for 2-3 times until the n-butanol layer is colorless, separating, and retaining the n-butanol layer;

the n-butanol layer was concentrated and dried under reduced pressure to a dry powder.

In step (2), the n-butanol used is a water-saturated n-butanol solution.

Example 1 in vitro anti-RSV study of Glycyrrhiza effective fractions

1.1 sample preparation:

preparation of a test solution: the dry powder was prepared as a 6mg/mL solution in DMEM cell culture containing 2% FBS.

Preparation of control solutions: preparing 1mM of glycyrrhizic acid solution, glycyrrhetinic acid solution, monoammonium glycyrrhizinate solution and ribavirin solution from 1% of DMSO and 2% of FBS in DMEM cell culture solution.

1.2 culture and recovery of cells

The preserved cells were removed from the liquid nitrogen tank and rapidly thawed at 37 ℃. Transferring the cell suspension to a sterile centrifuge tube in a biological safety cabinet, centrifuging (1000r, 3min), discarding the supernatant, adding new cell culture solution, blowing to remove cells, transferring the suspension to a cell culture dish, and introducing CO₂Culturing in constant temperature incubator (37 deg.C, 5% CO)₂And the relative humidity is 75%), changing the liquid after 6h, and continuously culturing for 1:3 subculture when 80% of monolayer cells are combined.

1.3 amplification of viruses

When the cells are converged to about 70%, the original culture solution is discarded, and cell fragments are removed by PBS (phosphate buffer solution) washing. 0.2mL of virus solution was addedRSV, cells were exposed to the virus by gentle shaking of the culture dish. In CO₂Culturing in a constant temperature incubator, and shaking the culture dish every 15 min. After two hours, the culture solution is discarded, after washing, 10mL of DMEM cell culture solution containing 2% FBS is added, the pathological changes are observed by microscopic examination every day, and the culture is stopped after 48 hours. And (3) sealing the virome culture dish, repeatedly freezing and thawing for 3-4 times, transferring the virus liquid into a sterile centrifuge tube, centrifuging (3000r for 3min), taking supernatant, subpackaging, and storing at-80 ℃ for later use.

1.4 determination of viral virulence

1×10⁴Cells/well were seeded in 96-well plates at 37 ℃ with 5% CO₂And culturing for 12 h. The virus solution obtained in 1.3 was diluted continuously by 10-fold to obtain 10^-1～10^-88 dilutions were added to the cells grown in monolayers and incubated for 48h, repeating 6 wells for each virus dilution. Observing and recording the infection condition by a CPE method, and calculating the half infection concentration TCID of RSV according to a Reed-Muench formula₅₀。

Results of virus virulence assay: the RSV takes Hep2 cells as host cells, and the RSV virus has half infection concentration TCID₅₀Is 3.4X 10^-7。

1.5 cytotoxicity assays of drugs

1×10⁴Cells/well were seeded in 96-well plates at 37 ℃ with 5% CO₂After culturing for 12h, diluting the sample solution prepared in 1.1 according to 2-fold ratio to 8 concentrations, sequentially inoculating the diluted sample solution into cells, wherein each concentration is 3 multiple wells, a cell control well and a blank control well are arranged, culturing for 48h in an incubator, observing and recording by using a CPE method, detecting the activity of the cells by using an MTT method, detecting the OD value of absorbance at 490nm of an enzyme labeling instrument, and calculating the CC of the medicine according to the Reed-Muench formula₅₀。

1.6 determination of anti-RSV Activity of drugs

1×10⁴Cells/well were seeded in 96-well plates at 37 ℃ with 5% CO₂After incubation for 12h, the n-butanol fraction was diluted 2-fold with 8 concentrations of the test solution and inoculated into cells using 100TCID₅₀Virus per well infected cells, 3 wells per concentration were replicated and a cell control group, a virus control group, and a normal control group were set. Culturing in an incubator for 48h, observing and recording by using a CPE method,detecting cell activity by MTT method, detecting absorbance OD value at 490nm of enzyme labeling instrument, and calculating EC of medicine according to Reed-Muench formula₅₀。

The results of the cytotoxicity assay of 1.5 drug, the results of the assay of 1.6 drug against RSV, and the therapeutic index TI values for each drug against RSV are shown in fig. 1-5.

Wherein n-butanol fraction (also called Glycyrrhrizae radix effective fraction) is used for treating CC of Hep2 cell₅₀2170 μ g/mL glycyrrhizic acid, monoammonium glycyrrhizinate, and CC of ribavirin for Hep2 cells₅₀Are all made of>500 μ M Glycyrrhetinic acid CC for Hep2 cells₅₀The value was 51.25. mu.M; EC of n-butanol fraction for RSV ₅₀60 μ g/mL, a TI value of 36.22, an EC50 value of ribavirin for RSV of 36.12 μ M, a TI value of>13.84, it can be seen that the n-butanol part has better effect on resisting RSV than ribavirin, and glycyrrhizic acid, glycyrrhetinic acid and monoammonium glycyrrhizinate have a certain effect on resisting RSV but have no obvious effect.

Example 2 time-lapse study to investigate the effect phase on the RSV replication cycle

To investigate the n-butanol site effect at a particular stage of the RSV replication cycle, a time-addition experiment was performed with Hep2 cells at 8X 10 hours prior to the experiment⁴Each cell was plated in a 24-well plate, and the cells were infected at 0h with a virus amount of 3 MOI at 4 ℃ for 2 hours, followed by washing the cells three times with cold PBS and adding a new culture solution to the cells for culture in a cell incubator. The n-butanol fraction (0.3mg/mL) and the positive drug ribavirin (100. mu.M) were added at-2, 0, 2, 4, 6, 8, 12, 16h, respectively, and the supernatants were collected at 20h and assayed for viral virulence according to the assay of 1.4 in example 1.

The test results are shown in fig. 6 and 7. Referring to FIG. 6, the positive control drug ribavirin has a good inhibition effect on RSV in 6-10 h, mainly in the virus replication stage, and has no inhibition effect on RSV in-2-6 h; referring to fig. 7, the n-butanol fraction has a good inhibitory effect on RSV in-2-2 hours and 8-12 hours, the addition time is-2-2 hours, the virus virulence can be reduced by 96.47%, the addition time is 8-12 hours, the virus virulence can be reduced by 82.12%, the inhibitory effect on the virus is gradually reduced after 12 hours, the n-butanol fraction mainly has a good inhibitory effect on the adsorption and replication stages of the virus, and has no inhibitory effect on the penetration stage of the virus, and the n-butanol fraction has a certain drug prevention effect on RSV compared with ribavirin.

Example 3 in vivo anti-RSV action and mechanism of action at n-butanol site

3.1 establishment and administration treatment of RSV-infected mouse model

Dividing BALB/c female mice of 4 weeks old into 4 groups of 10 mice, wherein the groups are respectively a normal control group, a model group and a positive drug (ribavirin 36 mg.Kg)^-1·d^-1) Group, n-butanol fraction (30 mg. Kg)^-1·d^-1) And (4) grouping. After the mice are adaptively raised for 3 days, each mouse is subjected to intragastric administration of 0.1 mL/mouse every day, and normal control groups and model groups are subjected to intragastric administration of physiological saline with the same volume. On the first day of administration, RSV nasal drop test was conducted, ether anesthesia was conducted, and 100. mu.L/virus (RSV titer 3.4X 10) was administered nasally^-7TCID₅₀/mL) was dripped into the nose for 2 consecutive days, and a normal control group was dripped into the nose with an equal volume of control culture solution.

3.2 sample treatment and index detection

The medicine is administered for 4 days according to a preset scheme, after administration for 1 hour on the fifth day, eyeballs take blood, the necks of the eyeballs are removed, mice are dissected, lung tissues are taken and weighed, lung indexes are calculated, 4% formaldehyde is used for fixing, and the lung tissues are dehydrated, transparent, waxed, embedded, sliced and dried to obtain HE-stained pathological sections for observing lung lesions. Blood taken out from eyeballs is placed in an EP tube for overnight at 4 ℃, centrifuged for 20min at the rotating speed of 3000r/min at 4 ℃, supernatant fluid is taken, and the contents of keap1, Nrf2, NF-kappa B, TNF-alpha, IFN-beta and TLR4 in the serum of mice are measured by an ELISA method.

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

3.3 Effect of n-butanol fraction on pathological forms such as inflammatory infiltration of lung tissue of RSV-infected mice

And observing pathological morphological change of lung tissue caused by RSV infected mice by using an HE staining method. The results are shown in FIG. 8.

Normal control mice (fig. 8B) had clear alveolar structures, no exudate was seen in the alveolar spaces, and no inflammatory cell infiltration was seen; in the model group mice (fig. 8A), alveolar walls were thickened, most alveolar spaces became small or almost invisible, and significant bleeding was observed in the alveolar spaces and in the pulmonary interstitium with inflammatory cell infiltration; the n-butanol fraction group (fig. 8C) improves alveolar wall hyperplasia, alveolar morphology tends to be normal, alveolar hemorrhage is reduced, and inflammatory cell infiltration is reduced; the alveolar wall of ribavirin group (fig. 8D) was improved compared to the model group, alveolar cavity and interstitial bleeding were improved, and inflammatory cell infiltration was reduced. The n-butanol part can obviously improve the lung tissue pathological change condition.

3.4 Effect of n-butanol fraction on pulmonary index of RSV infected mice

Referring to fig. 9, compared with the normal control group, the lung index of the model group is significantly increased (P <0.01), indicating that the model construction of the RSV-infected mouse is successful, the lung index of the n-butanol fraction and ribavirin group is significantly decreased (P <0.05), and the action effect of the n-butanol fraction and ribavirin is not much different. The n-butanol part can inhibit mouse viral pneumonia caused by RSV infection. (FIG. 9, Note: comparison of # P <0.05, # P <0.01 with the normal control group; comparison of # P <0.05, # P <0.01 with the model group)

3.5 Effect of n-butanol fraction on inflammation and immune response-related factors in RSV infected mice

Referring to fig. 10, the n-butanol fraction group and ribavirin group both showed significantly reduced (P <0.05) expression levels of NF- κ B P105 protein compared to the model group, and the effect of ribavirin group was more significant than that of n-butanol fraction group; compared with the model group, the n-butanol part group can obviously reduce (P <0.05) the level of TLR4 in the serum of the mouse, and the ribavirin group has poor effect; compared with the model group, the n-butanol fraction group and the ribavirin group can obviously (P <0.05) reduce the level of TNF-alpha in the serum of the mouse; the n-butanol moiety group significantly increased (P <0.05) IFN- β levels in mouse serum compared to the model group. (FIG. 10, Note: comparison of # P <0.05, # P <0.01 with the normal control group; comparison of # P <0.05, # P <0.01 with the model group)

3.6 Effect of n-butanol site on oxidative stress pathway of RSV infected mice

Referring to fig. 11, compared with the model group, both the n-butanol fraction group and the ribavirin group significantly increased (P <0.01) the Nrf2 level in the serum of the mice, and the ribavirin group was more effective than the n-butanol fraction group; the n-butanol fraction group significantly reduced the amount of keap1 in the mouse serum compared to the model group. (FIG. 11, Note: comparison of # P <0.05, # P <0.01 with the normal control group; comparison of # P <0.05, # P <0.01 with the model group)

According to the experimental data, after the RSV virus invades the body, oxidative stress injury of tissues and a series of inflammatory injuries can be caused. TLR4 is a bridge linking innate immunity and inflammation, TLR4 of a host can recognize F protein of RSV, and further activates NF-kB pathway in TLR4 signaling, thereby inducing expression of inflammatory factors (such as TNF-alpha, IL-1 beta, IL-6 and IL-8) and promoting inflammatory response. The interferon is a cell factor with broad-spectrum antiviral effect generated by the body, and G protein of RSV can inhibit the expression of IFN-beta so as to promote the immune escape of RSV. The Nrf2 can be used as a potential target for treating respiratory virus diseases, and the Nrf2 is an important target for resisting oxidative stress damage of an organism; under physiological conditions, Nrf2 and keap1 are coupled in cytoplasm, and Nrf2 dissociates from cytoplasm into nucleus under stress condition and promotes expression of antioxidant genes. Functional interaction exists between the Nrf2 and NF-kB, the activity of the NF-kB is intensified by the reduction of Nrf2, and the inflammation is promoted to be relieved by the activation of Nrf 2; the n-butyl alcohol part in vivo anti-RSV experimental result shows that the n-butyl alcohol part can improve the expression level of IFN-beta in serum of mice infected with RSV and inhibit the immune escape of RSV; through inhibiting TLR 4/NF-kB pathway and reducing TNF-alpha level, lung inflammatory reaction caused by RSV is inhibited; and can reduce the oxidative stress level of mice through a keap1/Nrf2 channel so as to improve lung tissue injury of the mice caused by RSV.

The application carries out certain research on the in-vivo and in-vitro anti-RSV function of the n-butyl alcohol part of the liquorice through the aspects of virus inhibition and killing, inflammatory reaction, immunoregulation and oxidative stress, and lays a certain foundation for the subsequent deep antiviral action mechanism research of the liquorice and the search of an active substance foundation.

The above description is only an example of the present application, and the protection scope of the present application is not limited by these specific examples, but is defined by the claims of the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.

Claims

1. An effective part of liquorice for resisting RSV in vivo and in vitro is characterized in that the effective part of liquorice is an n-butyl alcohol part obtained from a liquorice crude extract; the crude extract of Glycyrrhrizae radix is obtained by extracting Glycyrrhrizae radix with water.

2. The licorice effective fraction for resisting RSV in vivo and in vitro according to claim 1, wherein the licorice effective fraction is an n-butanol fraction obtained by sequentially extracting the crude licorice extract with petroleum ether, dichloromethane, ethyl acetate and n-butanol.

3. A method for preparing an effective part of liquorice for resisting RSV in vivo and in vitro is characterized by comprising the following steps:

4. The method according to claim 3, wherein the step (2) comprises:

5. An application of the effective part of liquorice root for resisting RSV in vivo and in vitro in preparing the medicine for preventing and/or treating RSV.

6. The use according to claim 5, wherein the licorice effective fraction is used for preparing a medicament for preventing and/or treating respiratory system damage caused by RSV; the respiratory system injury is selected from the group consisting of respiratory system injury caused by inflammatory response and oxidative stress.

7. The use of claim 5 or 6, wherein the anti-RSV activity of the licorice effective fraction is CC₅₀At 2170. mu.g/mL, EC₅₀60 μ g/mL, and a therapeutic index TI of 36.22.

8. The use according to claim 5 or 6, wherein the licorice effective fraction has an inhibitory effect on the adsorption phase and the replication phase in the RSV replication cycle.

9. The use of claim 5 or 6, wherein the effective part of licorice has an inhibitory effect on RSV within-2 h and 8-12h when used for in vitro anti-RSV; the addition time is-2-2 h, the virulence of the virus can be reduced by 96.47%, and the addition time is 8-12h, the virulence of the virus can be reduced by 82.12%.