Application of scutellarein in preparation of medicine for resisting respiratory syncytial virus

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of scutellarein in preparation of a medicine for resisting respiratory syncytial virus. Experiments prove that scutellarein has a good inhibition effect on respiratory syncytial viruses (A strain and B strain). Therefore, when being used as an active ingredient for preparing the medicine for resisting the respiratory syncytial virus, the compound has an important application value.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of baicalein in the preparation of anti-respiratory syncytial virus drugs. Background Art

[0002] Respiratory syncytial virus (RSV) belongs to the Paramyxoviridae family. It is a non-segmented, negative-strand RNA virus that is mainly transmitted through droplets and can easily cause repeated respiratory tract infections. It is the most common cause of hospitalization for severe acute lower respiratory tract infections in infants and young children, and the third most common cause of death in children, second only to pneumococcal pneumonia and Haemophilus influenzae type b infection. In severe cases, it can develop into fatal pneumonia. Due to the special characteristics of the virus and the mode of infection, the body's naturally acquired immunity is difficult to provide lasting and effective protection, which makes the current development of an effective vaccine difficult. The drugs currently used to treat RSV are ribavirin and palivizumab, but palivizumab is expensive, and ribavirin is a nucleoside antimetabolite with serious side effects. Therefore, it is necessary to develop new alternative antiviral molecules against respiratory syncytial virus.

[0003] Baicalein (6-hydroxyapigenin) is an active monomer component isolated from Chinese medicines with anti-inflammatory and analgesic properties such as Erigeron breviscapus, Chlorophytum corydalis, and Oroxylum indica. It is widely found in many traditional Chinese medicines such as Scutellaria baicalensis, Oroxylum indica, and Erigeron breviscapus. Studies have found that baicalein has a variety of biological activities, such as anti-inflammatory, antibacterial, anti-proliferative, anti-apoptotic, neuroprotective, and anti-cancer activities.

[0004] There is no report on the effect of baicalein against respiratory syncytial virus in the prior art. Summary of the invention

[0005] In order to solve the problems in the above background technology, the main purpose of the present invention is to provide the use of baicalein in the preparation of anti-respiratory syncytial virus drugs. The baicalein has a strong anti-respiratory syncytial virus effect and high biological safety.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] Application of baicalein in the preparation of anti-respiratory syncytial virus drugs.

[0008] Furthermore, the medicine contains a therapeutically effective amount of baicalein and a pharmaceutically acceptable carrier.

[0009] Furthermore, the scutellarin can be used as the sole active ingredient, or can be used as an active ingredient together with one, two or more other antiviral drugs.

[0010] Furthermore, the medicine also contains a pharmaceutically acceptable carrier.

[0011] Furthermore, the content of baicalein in the drug is ≥0.01 μM, preferably the content of baicalein is ≥10 μM.

[0012] Furthermore, the drug is a tablet, capsule, aerosol, pill, powder, solution, suspension, emulsion or granule.

[0013] The beneficial effects of the present invention are:

[0014] Baicalin comes from medicinal plants and has the advantages of simple acquisition, low cost and safe use. The present invention has confirmed through in vivo and in vitro experiments that baicalin has a strong anti-respiratory syncytial virus effect, and its SI value is greater than 4, which is higher than the safety range and has high biosafety. Therefore, baicalin has good application prospects in the treatment of RSV virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described below in conjunction with the accompanying drawings:

[0016] Figure 1 is a graph showing the cytotoxic effects of baicalein at different concentrations and its antiviral effects on RSV-A2;

[0017] Figure 2 is a graph showing the cytotoxic effects of baicalein at different concentrations and its antiviral effects on RSV-B;

[0018] Figure 3 This is a graph showing the weight changes of mice in the normal group, model group and treatment group over 5 days in Example 3;

[0019] Figure 4 This is a graph of the viral load in the right lung of mice in the normal group, model group and treatment group after 5 days in Example 3. DETAILED DESCRIPTION

[0020] The following examples further illustrate the technology of the present invention, but do not limit the protection scope of the present invention. Unless otherwise specified, the raw materials used in the present invention are all known products, which are obtained by purchasing commercial products.

[0021] The sources of some raw materials used in the present invention are as follows:

[0022] Scutellarin was purchased from MedChemExpress (MCE);

[0023] Respiratory syncytial virus RSV-A2 strain was provided by Guangzhou National Laboratory;

[0024] Respiratory syncytial virus RSV-B strain was provided by Wuhan Institute of Virology, Chinese Academy of Sciences.

[0025] Embodiment 1:

[0026] S1. Cell culture and plating

[0027] Human laryngeal cancer epithelial cells Hep-2 were seeded in 96-well plates containing 10% FBSDMEM medium, with 1×10 4 The cells were cultured overnight in a 37°C, 5% CO2 incubator.

[0028] S2. Prepare different concentrations of Chinese medicine dilutions and Chinese medicine virus mixtures

[0029] The baicalein powder was dissolved in DMSO to prepare a 50mM stock solution. In the experiment, the drug stock solution was continuously diluted into 8 gradients of Chinese medicine dilutions (0.01μM, 0.1μM, 1μM, 10μM, 20μM, 50μM, 100μM, 250μM) with DMEM solution containing 2% FBS, and added to a 96-well plate (100μl / well). At the same time, an equal volume control well was prepared, that is, only the DMSO group (final concentration 0.5%), and each group was repeated 3 times.

[0030] S3. Infection of Hep-2 cells with different concentrations of Chinese medicine

[0031] The culture supernatant in the 96-well plate was discarded, and the prepared drug dilutions were added respectively, and cultured in a 37°C, 5% CO2 incubator for 48 h.

[0032] S4. Drug cytotoxicity assay

[0033] After the drug has acted on the cells for 48 hours, a cytotoxicity detection kit (cck8 kit) is used to evaluate the toxicity of different concentrations of the drug on the cells. The optical density (OD value) of the solution is measured by an ELISA instrument, and the OD value is proportional to the activity of the cells. The specific operation is as follows:

[0034] Prepare the CCK8 detection solution according to the instructions, that is, add 10 μl / well of CCK8 solution to 100 μl / well of sterile PBS solution, pipette and mix well for use. Discard the drug dilution solution in the 96-well plate and add 100 μl of CCK8 detection solution to each well of the plate. Place the culture plate in a 37°C, 5% CO2 incubator and incubate for 1.5 hours. Place the culture plate in an ELISA reader and shake it slowly for 1 minute, measure the optical density (OD value) at 450nm, and calculate the cell survival rate. The results are as follows: Figure 1 shown.

[0035] The cell viability calculation formula is:

[0036]

[0037] Embodiment 2:

[0038] S1. Cell plating

[0039] Human laryngeal cancer epithelial cells Hep-2 were seeded in 96-well plates containing 10% FBSDMEM medium, with 1×10 4 The cells were cultured overnight in a 37°C, 5% CO2 incubator.

[0040] S2. Prepare different concentrations of Chinese medicine dilutions and Chinese medicine virus mixtures

[0041] The baicalein powder and the positive drug ribavirin were dissolved in DMSO to prepare a 50mM stock solution. In the experiment, the DMEM solution containing 2% FBS was used to continuously dilute the drug into 6 gradient dilutions (0.2μM, 2μM, 20μM, 40μM, 100μM, 200μM), and added to a 96-well plate (50μl / well), and then 50μl RSV-A2 strain or B strain virus solution (Hep-2 cells were infected with an infection multiplicity of MOI=0.1) was added, and the final concentration of the drug was 0.1μM, 1μM, 10μM, 20μM, 50μM, 100μM; at the same time, 4 groups of control wells were prepared with equal volumes, including a virus-only group (final concentration of 1×10 3 PFU of RSV virus solution), DMSO only group (final concentration 0.5%), DMSO + virus group, and 2% DMEM blank group (cell control group), each group was repeated 3 times.

[0042] S3. Infection of Hep-2 cells with different concentrations of Chinese medicine

[0043] The culture medium in the 96-well plate was discarded, and the prepared mixture of drugs and viruses was added respectively, and cultured in a 35°C, 5% CO2 incubator for 6 days.

[0044] S4. Antiviral testing

[0045] use 2.0Assay( The luminescence cell viability detection kit (CTG) is used to detect the neutralization ability of Chinese medicine of different concentrations against viruses. The more cells survive, the stronger the neutralization ability of Chinese medicine of this concentration against RSV, and the better the antiviral effect. Follow the instructions for operation, as follows:

[0046] Thaw CellTiter-Glo 2.0 Reagen at room temperature. Discard the culture medium in the 96-well plate, rinse twice with sterile PBS and replace with 2% DMEM culture medium, then add CTG detection solution (100μl / well), pipette to mix, and incubate on a shaker at room temperature for 10 minutes. Transfer 50ul / well to an opaque white well plate, place the culture plate in an ELISA reader and shake slowly for 2 minutes, then measure the luminescence value. Calculate the inhibition rate of the drug on the virus. The experimental results are as follows: Figure 2 shown.

[0047] The formula for the drug's inhibition rate on the virus is:

[0048]

[0049] S5. Drug selection index (SI)

[0050] SI is the safe range for judging the effect of drugs, and the selection index (SI) is CC 50 / EC 50 , the selected index is greater than 1 to be effective, and the larger the index, the larger the safety range.

[0051] Depend on Figure 1 and Figure 2 It can be seen that baicalein showed no cytotoxicity to Hep-2 cells at a concentration of up to 250 μM. At the same time, baicalein had an inhibitory effect on the replication of RSV-A2 and B strains, and it inhibited the EC 50 The values ​​were 60.61 and 47.11 μM, respectively. The selectivity index (SI) was greater than 4.12 and greater than 5.31, respectively, indicating that scutellarin has good anti-RSV activity and high safety in vitro.

[0052] Example 3: In vivo anti-RSV-A2 virus effect

[0053] Twelve female Balb / c mice aged 6-8 weeks were randomly divided into four groups: blank control group (Vehicle), RSV-A2 model group (RSV-A2), RSV+baicalein treatment group (RSV+baicalein), and RSV+ribavirin positive drug treatment group (RSV+RBV), with 3 mice in each group.

[0054] The experimental design was as follows: except for the Vehicle group, mice in the other three groups were intranasally instilled with 30 μl of RSV-A2 virus, 4×10 6 PFU / mouse. Mice were infected with RSV-A2 for 1h, and 4 groups of mice were gavaged. Specifically: ① RSV+scutellarin group was given 50 mg / kg scutellarin solution, ② RSV+RBV group was given 50 mg / kg ribavirin solution, ③ Vehicle group and RSV-A2 group were given equal volume solvent solution (10% DMSO+40% PEG 300+5% Tween-80+45% Saline, v:v:v:v). Gavage was continued for 4 days. During the experiment, the weight of mice was recorded every day. Figure 3 As shown. On the fifth day of the experiment, all mice were euthanized. The right lung tissue was weighed and placed in a grinding tube containing 400 μl PBS. After grinding with a homogenizer and centrifugation, the supernatant was taken to extract RNA and the viral copy number was determined by QPCR, as shown. Figure 4 shown.

[0055] Depend on Figure 3 It can be seen that the weight of mice in the model group RSV-A2 was significantly lower than that in the normal group on the third day after virus inhalation. Compared with the model group, the weight of mice infected with RSV-A2 in the baicalein group and ribavirin group increased significantly on the third day. The experimental results show that oral administration of baicalein and ribavirin can alleviate the weight loss of RSV-A2 infected mice.

[0056] Depend on Figure 4 It can be seen that compared with the normal group, the viral load in the lung tissue of the model group mice on the 4th day increased significantly. In contrast, compared with the model group, the viral load in the lung tissue of the baicalein group and the ribavirin group decreased (P<0.0001). This further proves that baicalein has a good anti-RSV-A2 virus effect in vivo.

[0057] The above embodiments are only preferred technical solutions of the present invention and should not be regarded as limiting the present invention. The protection scope of the present invention shall be the technical solutions recorded in the claims, including equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. Application of baicalin in the preparation of anti-respiratory syncytial virus drugs.

2. The use according to claim 1, characterized in that: The scutellarin can be used as the sole active ingredient, or can be used as an active ingredient together with one, two or more other antiviral drugs.

3. The use according to claim 2, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

4. The use according to claim 2, characterized in that: The content of baicalein in the medicine is ≥0.01 μM.

5. The use according to claim 2, characterized in that: The content of baicalein in the medicine is ≥10 μM.

6. The use according to claim 2, characterized in that: The medicine is in the form of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions or granules.

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