Use of punicalin in the preparation of a medicament for the treatment of respiratory syncytial virus

Abstract

The application discloses application of Punicalin in preparation of a medicine for resisting respiratory syncytial virus. The respiratory syncytial virus is an RNA virus, and is the most important virus body causing acute lower respiratory tract infection of children under 5 years old in the world. It is found that the Punicalin can effectively inhibit replication of the respiratory syncytial virus and effectively resist respiratory syncytial virus infection. Therefore, the Punicalin has the prospect of being developed into the medicine for resisting the respiratory syncytial virus.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and relates to a new use of a known compound, in particular to application of Punicalin in preparation of a medicine for resisting respiratory syncytial virus. BACKGROUND

[0002] Respiratory syncytial virus (RSV) is an RNA virus, which is mainly spread through droplets and contact, and is the most important virus body causing acute lower respiratory tract infection in children under 5 years old in the world, and is also an important factor for hospitalization and even death of infants.

[0003] At present, there is no report of Punicalin for resisting respiratory syncytial virus. SUMMARY

[0004] The application aims to provide application of Punicalin in preparation of a medicine for resisting respiratory syncytial virus.

[0005] The above object of the application is achieved by the following technical scheme:

[0006] Application of Punicalin in preparation of a medicine for resisting respiratory syncytial virus.

[0007] Further, the medicine takes Punicalin as an active ingredient, and further contains a pharmaceutically acceptable carrier, and is prepared into a pharmaceutically acceptable dosage form.

[0008] Still further, the carrier includes solid, liquid and semi-solid carriers.

[0009] Still further, the dosage form includes tablets, capsules, injections and drops.

[0010] Beneficial effects:

[0011] It is found in the application that Punicalin can effectively inhibit replication of respiratory syncytial virus and effectively resist respiratory syncytial virus infection. Therefore, Punicalin has the prospect of being developed into a medicine for resisting respiratory syncytial virus. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Effects of different concentrations of Punicalin on A549 cell viability;

[0013] Figure 2 Effects of Punicalin on RSV viral load in A549 cells. DETAILED DESCRIPTION

[0014] The substantial content of the present application will be specifically introduced below in combination with examples, but the protection scope of the present application is not limited by this.

[0015] I. Experimental materials

[0016] 1. Instruments

[0017] Beckman ALLegra 64R high-speed refrigerated centrifuge (Beckman, USA); Revco UXF ultra-low temperature refrigerator (Thermo Fisher, USA); CPA225D electronic balance (Sartorius, Germany); DHG-9071A constant temperature oven (Jinghong, China); protein nucleic acid analyzer, PCR reverse transcription instrument (Eppendorf, Germany); Quantstudio7Flex fluorescence quantitative PCR instrument (Life technologies, USA).

[0018] 2. Reagents

[0019] Phosphate buffer solution (PBS) was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; Punicalin (CAS number: 65995-64-4) was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.; Anhydrous ethanol, dimethylbenzene were purchased from Nanjing Chemical Reagent Co., Ltd.; HiScript III RT SuperMix for qPCR (+gDNA wiper), ChamQ Universal SYBR qPCR MasterMix, FastPure Cell / Tissue Total RNA Isolation Kit V2 were purchased from Nanjing Novozyme Biotech Co., Ltd.; SYBR Green dye was purchased from Bio-Rad Company, USA; CCK8 assay kit was purchased from APExBIO company; Human RSV strain A2 virus strain was purchased from Wuhan Institute of Virology; Streptavidin, glycine, dimethyl sulfoxide DMSO were purchased from aladdin company.

[0020] II. Experimental methods

[0021] 1. Cell culture and virus amplification

[0022] Human lung adenocarcinoma A549 epithelial cell line (CCL-185) and human Hep-2 cell line (CRL-9609) were purchased from ATCC (Manassas, USA). Cells were cultured in DMEM medium with the addition of 10% heat-inactivated fetal bovine serum, and cultured in a 37°C incubator with 5% CO2. The virus was initially propagated in Hep-2 cells. Virus titer was detected by TCID50 method. TCID50 was calculated by Reed-Muench method.

[0023] 2. Drug toxicity test

[0024] A549 cells were distributed at a rate of 1 × 10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates and incubated for 24 hours. The original medium was then replaced with medium containing 0–10 μM Punicalin. After the 24-hour incubation period, 10 μL of a solution containing 1 mg / mL of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide was added to each well. The reaction was allowed to continue for 1 hour. The absorbance of the samples was measured at 570 nm using a microplate reader (TECAN, Switzerland).

[0025] 3. In vitro inhibition of RSV by drugs

[0026] A549 cells were used at a rate of 1×10 5 Cells were transferred to 24-well plates at a density of [number] cells / well. After cell adhesion, the cells were infected with RSV (MOI=1) and 2 μM Punicalin was added simultaneously. In the presence of the virus, the cell culture medium was replaced with DMEM containing 2% FBS, and cells were collected after 24 hours. A mock group without Punicalin was set up as a control.

[0027] 4. Real-time fluorescence quantitative PCR experiment

[0028] The collected cells were lysed with Trizol, and RNA was extracted using an RNA extraction kit (RC101, Vazyme, Nanjing, China). RNA concentration was quantified using a protein and nucleic acid analyzer. Total RNA was reverse transcribed into cDNA according to the kit instructions, and the RSV-nucleoprotein (N) gene transcription level was detected using SYBR Green II real-time quantitative PCR. Table 1 shows the primer sequences. The real-time quantitative PCR reaction conditions (two-step method) were: 95℃ pre-denaturation for 15 s, 95℃ denaturation for 5 s, and 60℃ annealing for 34 s, for 40 cycles. Analysis was performed using QuantStudio Real-time PCR Software (ABI, USA). The measured viral RNA is plotted as a percentage of the control on a linear scale in each graph.

[0029] Table 1 Primer sequences

[0030]

[0031] 4. Data Analysis

[0032] Data processing and plotting were performed using the statistical software Graphpad Prism 8, and data were expressed as Mean ± SD. Multiple groups of data were analyzed by non-parametric test (Kruskal-Wallis test), and P < 0.05 was considered statistically significant.

[0033] III. Experimental results

[0034] 1. Cytotoxicity experiment

[0035] We first evaluated the cytotoxicity of Punicalin by exposing A549 cells to different concentrations of Punicalin for 24 hours under experimental conditions. To determine the cytotoxic effect, we used CCK8 assay and calculated the CC50 value. The CCK-8 results showed that there was no obvious cytotoxicity of Punicalin in the range of 0-2.5 uM ( Figure 1 ).

[0036] According to the above results, we selected the drug concentration of 2uM without obvious toxicity to host cells for subsequent experiments.

[0037] 2. Evaluation of the ability of Punicalin to inhibit viral replication

[0038] We evaluated the ability of Punicalin to inhibit respiratory syncytial virus in vitro using A549 cells. First, A549 cell monolayers were exposed to Punicalin while being infected with RSV (MOI = 1) for 24 hours. RT-qPCR was used to detect the expression of RSV-N gene to determine the antiviral effect of Punicalin. The results showed that compared with the positive control group, the expression of RSV-N gene was significantly reduced by 2uM of Punicalin, with an inhibition rate of nearly 70%, indicating that Punicalin at this concentration can effectively inhibit the replication of RSV virus ( Figure 2 ). This result suggests that Punicalin has anti-RSV infection activity and is an effective small molecule compound against respiratory syncytial virus.

[0039] The above examples serve to specifically introduce the essential content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

Claims

1. Use of punicalin for the preparation of a medicament against respiratory syncytial virus.

2. Use according to claim 1, wherein said medicament comprises punicalin as an active ingredient, in combination with a pharmaceutically acceptable carrier, in a pharmaceutically acceptable dosage form.

3. Use according to claim 2, wherein said carrier comprises solid, liquid and semi-solid carriers.

4. Use according to claim 2, wherein said dosage form comprises tablets, capsules, injections and drops.

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