Use of morin hydrate in the preparation of drugs against respiratory syncytial virus

Abstract

The application belongs to the field of medicine, and particularly relates to application of a mulberry pigment hydrate in preparation of an anti-respiratory syncytial virus drug. It has been verified that the mulberry pigment hydrate has obvious inhibitory effect on respiratory syncytial virus A2 and B strains. The mulberry pigment hydrate has relatively low cytotoxicity and high biological safety. The application provides a new scheme for treatment of the RSV virus and provides a theoretical basis for the application of the mulberry pigment hydrate in preparation of the anti-respiratory syncytial virus drug.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more particularly to the application of morin hydrate in the preparation of drugs against respiratory syncytial virus. Background Technology

[0002] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus in the family Paramyxoviridae. Its genome is approximately 15.2 kb in size and encodes 11 proteins, among which the membrane surface F and G proteins serve as important targets for neutralizing antibodies. Based on differences in the G protein antigen, RSV is classified into two subtypes, A and B, which alternate seasonally. It is one of the most important pathogens causing acute lower respiratory tract infections in infants and young children, and can also cause interstitial pneumonia and bronchiolitis. Furthermore, it can cause hospitalization and pneumonia-related deaths in the elderly and immunocompromised patients. Even though RSV has been a focus of attention for over 60 years, there is still a lack of effective vaccines and safe, highly effective treatments for this virus.

[0003] my country is a major producer of traditional Chinese medicine and natural drugs, possessing unique advantages for developing antiviral natural drugs. Morin, a light yellow pigment extracted from the bark of Moraceae plants such as the yellow mulberry tree and the mulberry-orange tree, as well as many traditional Chinese medicinal herbs, usually exists in hydrate form. It is slightly soluble in water but readily soluble in organic solvents such as methanol and ethanol, and can form stable complexes under acidic or alkaline conditions. Studies have shown that morin hydrate possesses anti-inflammatory, antioxidant, anti-tumor, anti-atherosclerotic, and blood sugar-lowering biological activities. This invention unexpectedly discovered that morin hydrate can inhibit RSV virus infection and replication, and holds promise for application in the development of drugs for the prevention or treatment of RSV infection, providing a new approach for the effective control of RSV. Summary of the Invention

[0004] To address the problems of the prior art, the main objective of this invention is to provide the application of morin hydrate in the preparation of anti-respiratory syncytial virus (RSV) drugs, wherein the morin hydrate has high biocompatibility and strong anti-RSV activity.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Application of morin hydrate in the preparation of drugs against respiratory syncytial virus.

[0007] Furthermore, the drug is a formulation prepared with morin hydrate as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients.

[0008] Furthermore, the morin hydrate is used as the sole active ingredient in the preparation of an anti-respiratory syncytial virus drug.

[0009] Furthermore, the content of the active ingredient in the formulation is ≥0.1 μM, preferably 0.1~100 μM, and more preferably 20~100 μM.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] Morin hydrate is derived from natural medicinal plants, is readily available, inexpensive, and has high safety profile; it also possesses strong resistance to respiratory syncytial virus (Strain A2 and Strain B). The method provided by this invention is simple to implement, features low dosage, minimal side effects, and readily available raw materials, making it suitable for industrial production. Attached Figure Description

[0012] The present invention will be further described below with reference to the accompanying drawings:

[0013] Figure 1 The graph shows the cytotoxic effects of different concentrations of morin hydrate and its antiviral effect on RSV-A2.

[0014] Figure 2 The graph shows the cytotoxic effects of different concentrations of morin hydrate and its antiviral effect on RSV-B.

[0015] Figure 3 This is a graph showing the weight changes of mice in the RSV-A2 challenge group, the RSV-A2 challenge + drug administration group, and the control group over 5 days.

[0016] Figure 4 This is a graph showing the viral load in the right lung of mice in the RSV-A2 challenge group, the RSV-A2 challenge + drug administration group, and the control group. Detailed Implementation

[0017] The technical content of the present invention will be described in detail below with reference to embodiments, but this is not intended to limit the scope of protection of the present invention. Unless otherwise stated, all raw materials used in the present invention are known products obtained by purchasing commercially available products.

[0018] The sources of some of the raw materials used in this invention are as follows:

[0019] Morin hydrate was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0020] The respiratory syncytial virus (RSV) strain A2 was provided by the Guangzhou National Laboratory.

[0021] The respiratory syncytial virus (RSV) strain RSV-B was provided by the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0022] Example 1:

[0023] In this embodiment, a cell culture screening method was used to detect the effect of morin hydrate on respiratory syncytial virus infection of Hela-2 cells and to quantify the anti-respiratory syncytial virus activity of the drug.

[0024] 1.1 Cell Culture

[0025] Hep-2 cells were seeded in 96-well plates containing 10% FBS DMEM medium (1×10⁻⁶ cells / well). 4 (each well contains 10 cells) and incubated overnight at 37°C in a 5% CO2 incubator.

[0026] 1.2 Preparation of mulberry pigment hydrates at different concentrations

[0027] The morin hydrate powder was dissolved in DMSO to prepare a 50 mM stock solution. The stock solution was then continuously diluted with DMEM solution containing 2% FBS to prepare eight gradient drug diluents (0.01, 0.1, 1, 10, 20, 50, 100, 250 μM), which were added to 96-well plates (100 μl / well). At the same time, an equal volume of control wells, i.e., the DMSO group only (final concentration 0.5%), was prepared. Each group had three replicates.

[0028] 1.3. Treatment of Hep-2 cells with different drug concentrations

[0029] Discard the culture medium in the 96-well plate containing Hep-2 cells using a pipette, add the prepared drug dilution, and incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0030] 1.4 Cytotoxicity Detection

[0031] The cytotoxic effects of different drug concentrations on cells were assessed using a cytotoxicity assay kit (CCK8 kit). The specific procedures are as follows:

[0032] Prepare the CCK8 detection solution according to the instructions: add 10 μl of CCK8 solution to 100 μl of sterile PBS solution per well, and mix well by pipetting. Remove the herbal dilution solution from the 96-well plate using a pipette, and add 100 μl of CCK8 detection solution to each well. Incubate the plate at 37°C in a 5% CO2 incubator for 1.5 hours. Place the plate in a microplate reader and gently shake for 1 minute, then measure the optical density (OD value) at 450 nm.

[0033] The formula for calculating cell viability is:

[0034] .

[0035] Example 2:

[0036] 2.1 Cell Culture

[0037] Hep-2 cells were seeded in 96-well plates containing 10% FBS DMEM medium (1×10⁻⁶ cells / well). 4 (each well contains 10 cells) and incubated overnight at 37°C in a 5% CO2 incubator.

[0038] 2.2 Preparation of drug diluents and virus mixtures at different concentrations

[0039] Morusin hydrate powder and ribavirin (RBV, positive control) were dissolved in DMSO to prepare a 50 mM stock solution. In the experiment, this stock solution was serially diluted with DMEM solution containing 2% FBS to prepare six gradient drug dilutions (0.2, 2, 20, 40, 100, 200 μM) and added to 96-well plates (50 μl / well). Then, 50 μl of RSV-A2 / B virus solution (for infecting Hep-2 cells at an MOI of 0.1) was added, and the mixture was gently pipetted to achieve final drug concentrations of 0.1, 1, 10, 20, 50, and 100 μM. Simultaneously, four control wells were prepared in equal volumes, including a virus-only group (final concentration 1 × 10⁻⁶). 3 PFU containing RSV-A2 / B virus solution), DMSO only (final concentration 0.5%), DMSO + virus group, and 2% DMEM blank group (cell control group), with three replicates for each group.

[0040] 2.3 Infection of Hep-2 cells with drug-virus mixtures of different concentrations

[0041] Discard the culture medium from the cell plate using a pipette, add the prepared drug and virus mixture separately, and incubate at 35°C in a 5% CO2 incubator for 6 days.

[0042] 2.4 Antiviral testing

[0043] The neutralizing ability of different concentrations of traditional Chinese medicine (TCM) against RSV was determined using a cell viability assay kit (CellTiter-Glo 2.0). Higher cell viability indicates a stronger neutralizing ability of that concentration of TCM against RSV. The specific procedure is as follows:

[0044] Aspirate the drug and virus mixture from the 96-well plate, wash twice with sterile PBS, and add 100 μl of 2% DMEM medium and an equal volume of CellTiter-Glo detection solution to each well. Incubate the plate at room temperature in the dark for 10 minutes, then mix thoroughly using a pipette and transfer 50 μl of the liquid to a clear white plate. Gently shake the white plate for 1 minute using a microplate reader and measure the chemiluminescence value.

[0045] The formula for the drug's inhibition rate against the virus is:

[0046] .

[0047] Experimental results are as follows Figure 1-2 As shown in the figure. It can be seen that morin hydrate has an effect on the CC of Hep-2 cells. 50 At concentrations above 250 μM, it showed no cytotoxicity against Hep-2 cells within the range of less than 100 μM. The inhibitory effect of morin hydrate on RSV-A2 was concentration-dependent, and its EC50... 50 =69.27μM, and the calculated SI > 3.61, indicating that morin hydrate has high biosafety. The inhibitory effect of morin hydrate on RSV-B is concentration-dependent, and its EC50 is... 50 =48.88μM, and the calculated SI>5.11 indicates that morin hydrate has high biocompatibility.

[0048] The above results indicate that morin hydrate has high cellular safety, meets the requirements for clinical candidate drugs, and its superior anti-RSV effect makes it a promising candidate for development as an anti-RSV drug.

[0049] Example 3:

[0050] The viral load of RSV-A2 in lung tissue was determined by quantitative real-time PCR (qPCR) to evaluate the ability of morin hydrate to inhibit RSV-A2 in vivo.

[0051] Healthy 6-8 week old Balb / c female mice were selected and randomly divided into experimental groups according to average body weight. The mice were divided into three groups: the A2 challenge experimental group, the A2 challenge plus drug administration group, and the control group. The specific experimental design is as follows:

[0052] A2 challenge experimental group: 30 μl nasal drops per mouse (4 × 10) 6 PFU RSV-A2 was used to anesthetize mice with isoflurane for about two minutes, and after weighing them, it was administered via nasal drops.

[0053] The treatment group: Mice were administered the drug powder by gavage 1 hour after being infected with RSV-A2. The drug powder was prepared in a 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. Mice were administered the drug powder by gavage for 4 consecutive days.

[0054] Control group: Mice in the control group were given a solution (10% DMSO + 40% PEG 300 + 5% Tween-80 + 45% Saline, v:v:v:v) by gavage for 4 consecutive days;

[0055] Mice were weighed daily. On the fourth day after gavage, the mice were euthanized, and their lungs were harvested. After weighing the right lung, 400 μl of PBS was added to the tube, and the tissue was homogenized. RNA was extracted from the tissue supernatant to determine the viral copy number.

[0056] Experimental results are as follows Figure 3-4 As shown in the figure, mice infected with RSV virus experienced a decrease in body weight. Neither the morin hydrate nor ribavirin treatments improved this decrease, indicating that RSV-A2 virus infection significantly impacted mouse health. The viral load in the lungs of mice in the morin hydrate and ribavirin treatment groups was significantly lower than that in the challenge group, demonstrating a statistically significant difference. This indicates that the treatments significantly reduced the viral load in mice and had a therapeutic effect on RSV infection. The viral load in the ribavirin treatment group was slightly lower than that in the morin hydrate treatment group, but the difference was not statistically significant.

[0057] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. Use of a morin hydrate as the sole active ingredient for the preparation of a medicament against respiratory syncytial virus, characterized in that: The respiratory syncytial virus is RSV-B, the EC of the anthocyanin hydrate on RSV-B is 48.88 μM. 50 48.88 μM.

2. Use according to claim 1, characterized in that, The drug is a preparation prepared by using morin hydrate as an active ingredient, together with pharmaceutically acceptable adjuvants or auxiliary ingredients.

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

4. Use according to claim 3, characterized in that, The content of the active ingredient in the preparation is 0.1-100 μM.

5. Use according to claim 4, characterized in that, The content of the active ingredient in the preparation is 20-100 μM.

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