Application of morin hydrate in preparation of medicine for resisting respiratory syncytial virus

Abstract

The invention belongs to the field of medicines, and particularly relates to application of morin hydrate in preparation of a medicine for resisting respiratory syncytial virus. Through verification, the morin hydrate has an obvious inhibition effect on the respiratory syncytial virus A2 strain and the respiratory syncytial virus B strain. The morin hydrate has low toxicity to cells, and has high biological safety. The invention provides a new scheme for the treatment of the RSV virus, and provides a theoretical basis for the application of the morin hydrate in the preparation of the anti-respiratory syncytial virus drug.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of morin hydrate in preparing anti-respiratory syncytial virus drugs. Background Art

[0002] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus of the family Paramyxoviridae. Its genome size is about 15.2kb and encodes 11 proteins, of which the membrane surface F protein and G protein can serve as important targets for neutralizing antibodies. According to the difference in RSV surface G protein antigens, it is divided into two subtypes, A and B, which appear alternately in epidemic seasons. It is one of the most important pathogens causing acute lower respiratory tract infections in infants and young children. It can also cause interstitial pneumonia and bronchiolitis, and can also cause hospitalization and pneumonia death in the elderly and immunocompromised patients. Even though RSV respiratory syncytial virus has attracted much attention as early as 60 years ago, there is still a lack of effective vaccines and safe and efficient therapeutic drugs for the virus.

[0003] my country is a country with a large population of traditional Chinese medicine and natural medicines, and has unique advantages in developing antiviral natural medicines. Morin is a light yellow pigment extracted from the bark of Moraceae plants such as yellow mulberry wood and mulberry orange tree and many Chinese herbal medicines. It usually exists in the form of hydrates, is slightly soluble in water, and is easily soluble in organic solvents such as methanol and ethanol. It can form stable complexes under acidic or alkaline conditions. Studies have shown that morin hydrate has biological activities such as anti-inflammatory, antioxidant, anti-tumor, anti-atherosclerosis, and lowering blood sugar. The present invention unexpectedly discovered that morin hydrate can inhibit the infection and replication of RSV virus, and is expected to be used in the development of drugs for preventing or treating RSV infection, providing a new solution for the effective prevention and control of RSV. Summary of the invention

[0004] In order to solve the problems of the prior art, the main purpose of the present invention is to provide the use of morin hydrate in the preparation of anti-respiratory syncytial virus drugs, wherein the morin hydrate has high biosafety and strong anti-respiratory syncytial virus effect.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is: Application of morin hydrate in the preparation of anti-respiratory syncytial virus drugs.

[0006] Furthermore, the drug is a preparation prepared with morin hydrate as an active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients.

[0007] Furthermore, the morin hydrate is used as the sole active ingredient in the preparation of an anti-respiratory syncytial virus drug.

[0008] Furthermore, the content of active ingredient in the preparation is ≧0.1 μM, preferably 0.1-100 μM, more preferably 20-100 μM.

[0009] Compared with the prior art, the present invention has the following beneficial effects: Morin hydrate comes from natural medicinal plants, is easy to obtain, has low cost, and is highly safe to use; it has strong resistance to respiratory syncytial virus (strain A2 and strain B). The method provided by the present invention is simple to implement, has the characteristics of small dosage, small toxic and side effects, and easy availability of raw materials, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The present invention will be further described below in conjunction with the accompanying drawings: Figure 1 is a graph showing the cytotoxic effects of morin hydrate at different concentrations and its antiviral effects on RSV-A2; Figure 2 is a graph showing the cytotoxic effects of morin hydrate at different concentrations and its antiviral effects on RSV-B; Figure 3 This is a graph showing the 5-day weight changes of mice in the RSV-A2 challenge group, RSV-A2 challenge + drug administration group, and control group; Figure 4 This is a graph of the viral load in the right lungs of mice in the RSV-A2 challenge group, RSV-A2 challenge + medication group, and control group. DETAILED DESCRIPTION

[0011] The technical content of the present invention is specifically described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the raw materials used in the present invention are all known products, obtained by purchasing commercially available products.

[0012] The sources of some raw materials used in the present invention are as follows: Morin hydrate was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; Respiratory syncytial virus RSV-A2 strain was provided by Guangzhou National Laboratory; Respiratory syncytial virus RSV-B strain was provided by Wuhan Institute of Virology, Chinese Academy of Sciences.

[0013] Embodiment 1: In this example, a cell culture screening method was used to detect the effect of morin hydrate on Hela-2 cells infected with respiratory syncytial virus, and to quantify the anti-respiratory syncytial virus activity of the drug.

[0014] 1.1 Cell culture Hep-2 cells were seeded in 96-well plates (1×10 4The cells were cultured overnight in a 37°C, 5% CO2 incubator.

[0015] 1.2. Preparation of different concentrations of mulberry pigment hydrate The morin hydrate powder was dissolved in DMSO to prepare a 50 mM stock solution, and the drug stock solution was continuously diluted with DMEM solution containing 2% FBS into 8 gradient drug dilutions (0.01, 0.1, 1, 10, 20, 50, 100, 250 μM), added to a 96-well plate (100 μl / well), and at the same time, an equal volume of control wells was prepared, i.e., only DMSO group (final concentration 0.5%), and three replicate wells were set for each group.

[0016] 1.3 Effects of different drug concentrations on Hep-2 cells Use a pipette to discard the culture medium in the 96-well plate containing Hep-2 cells, add the prepared drug dilutions respectively, and culture in a 37°C, 5% CO2 incubator for 48 hours.

[0017] 1.4 Cytotoxicity assay The cytotoxicity detection kit (CCK8 kit) was used to evaluate the toxicity of different concentrations of drugs on cells. The specific operation is as follows: 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. Use a pipette to remove the Chinese medicine dilution in the 96-well plate, and add 100 μl of CCK8 detection solution to each well in the plate. Place the culture plate in a 37°C, 5% CO2 incubator for 1.5 hours. Place the culture plate in an ELISA reader and shake it slowly for 1 minute, then measure the optical density (OD value) at 450 nm.

[0018] The cell viability calculation formula is:

[0019] .

[0020] Embodiment 2: 2.1 Cell culture Hep-2 cells were seeded in 96-well plates (1×10 4 The cells were cultured overnight in a 37°C, 5% CO2 incubator.

[0021] 2.2. Preparation of drug dilutions and virus mixtures of different concentrations Morin hydrate powder and ribavirin (RBV, positive control drug) were dissolved in DMSO to prepare a 50mM stock solution. In the experiment, DMEM solution containing 2% FBS was used to continuously dilute the drug into 6 gradient dilutions (0.2, 2, 20, 40, 100, 200μM) and added to a 96-well plate (50μl / well). Then, 50μl RSV-A2 / B virus solution (Hep-2 cells were infected with an infection multiplicity of MOI=0.1) was added and gently pipetted to mix, so that the final concentration of the drug was: 0.1, 1, 10, 20, 50, 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-A2 / B virus solution), DMSO only group (final concentration 0.5%), DMSO+virus group, and 2% DMEM blank group (cell control group), with three replicate wells in each group.

[0022] 2.3. Infection of Hep-2 cells with drug-virus mixtures of different concentrations Use a pipette to discard the culture medium in the cell plate, add the prepared mixture of drugs and viruses, and culture in a 35°C, 5% CO2 incubator for 6 days.

[0023] 2.4 Antiviral Detection A cell viability test kit (CellTiter-Glo 2.0) was used to determine the neutralization ability of different concentrations of Chinese medicine against viruses. The more cells survived, the stronger the neutralization ability of the Chinese medicine at that concentration against RSV. The specific operation is as follows: The drug and virus mixture in the 96-well plate was aspirated, and the plate was washed twice with sterile PBS. 100 μl of 2% DMEM culture medium and an equal volume of CellTiter-Glo detection solution were added to each well of the plate. After the culture plate was incubated at room temperature and away from light for 10 minutes, 50 μl of liquid was transferred to a transparent white well plate by blowing and aspirating with a dispenser. The white well plate was shaken slowly for 1 minute with the aid of an ELISA reader, and the chemiluminescence value was measured.

[0024] The formula for the drug's inhibition rate on the virus is: .

[0025] The experimental results are as follows Figure 1-2 As shown in Figure 2, it can be seen that morin hydrate has an effect on the CC of Hep-2 cells. 50 >250μM, and had no cytotoxicity to Hep-2 cells within the range of less than 100μM. The inhibitory effect of morin hydrate on RSV-A2 was concentration-dependent, and its EC 50=69.27μM, and the calculated SI>3.61 indicates that the biosafety of morin hydrate is relatively high. The inhibitory effect of morin hydrate on RSV-B is concentration-dependent, and its EC 50 =48.88μM, and the calculated SI>5.11, indicating that the biosafety of morin hydrate is relatively high.

[0026] The above results indicate that morin hydrate has high cellular safety and meets the requirements of clinical candidate drugs. Its superior anti-RSV effect makes it a promising candidate for development as an anti-RSV virus drug.

[0027] Embodiment 3: Fluorescence quantitative PCR (Q-PCR) was used to measure the RSV-A2 viral load in lung tissue and to evaluate the ability of morin hydrate to inhibit RSV-A2 in vivo.

[0028] Healthy 6-8 week old Balb / c female mice were selected and randomly divided into three groups according to their average body weight: A2 challenge experimental group, A2 challenge plus drug group, and control group. The specific experimental design is as follows: A2 challenge experimental group: 30 μl 4×10 6 PFU of RSV-A2, anesthetize mice with isoflurane for about two minutes, weigh them, and then drip into the nose; Drug administration group: mice were gavaged 1 hour after being infected with RSV-A2. The drug powder was solvent (10% DMSO + 40% PEG 300 + 5% Tween-80 + 45% Saline, v:v:v:v). The dosage of morin hydrate and RBV was 50 mg / kg. The mice were gavaged for 4 consecutive days. Control group: Mice in the control group were given solvent (10% DMSO + 40% PEG 300 + 5% Tween-80 + 45% Saline, v:v:v:v) by gavage for 4 consecutive days; The weight of mice was recorded every day, and mice were euthanized on the 4th day after gavage, and lungs were removed. After weighing the right lung, 400 μl PBS was added to the tube, and the lungs were ground with a grinder, and RNA was extracted from the tissue supernatant to determine the number of viral copies.

[0029] The experimental results are as follows Figure 3-4As shown. It can be seen that the weight of mice decreased after being infected with RSV virus, while the groups treated with mulberry pigment hydrate and ribavirin failed to improve the weight loss of mice, indicating that RSV-A2 virus infection had a significant impact on the health of mice. The viral load in the lungs of mice in the mulberry pigment hydrate and ribavirin administration groups was lower than that in the challenge group, and there was a statistical difference, indicating that the administration group can significantly reduce the viral load in mice and has a therapeutic effect on RSV infection. The viral load of the ribavirin administration group was slightly lower than that of the mulberry pigment hydrate administration group, but there was no statistical difference.

[0030] 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 mulberry pigment hydrate in the preparation of anti-respiratory syncytial virus drugs.

2. The use according to claim 1, characterized in that: The medicine is a preparation prepared by taking morin hydrate as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

3. The use according to claim 2, characterized in that: The morin hydrate is used as the only active ingredient for preparing an anti-respiratory syncytial virus drug.

4. The use according to claim 2, characterized in that: The content of active ingredient in the preparation is ≧0.1 μM.

5. The use according to claim 4, characterized in that: The content of active ingredients in the preparation is 0.1-100 μM.

6. The use according to claim 5, characterized in that: The content of active ingredients in the preparation is 20-100 μM.

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