Application of procyanidine A1 in preparation of medicine for inhibiting porcine epidemic diarrhea virus

Abstract

The invention belongs to the technical field of antiviral drugs, and in particular relates to a novel application of procyanidine A1 in preparation of a drug for inhibiting porcine epidemic diarrhea virus (PEDV). The porcine epidemic diarrhea (PED) is a high-contact intestinal infectious disease caused by the PEDV (porcine epidemic diarrhea virus), so that huge economic loss is caused to the global pig breeding industry. Currently, PEDV serving as an RNA virus is extremely easy to mutate, so that the protection effect of the existing vaccine is limited, and a special-effect antiviral therapeutic drug is lacked clinically. It is found for the first time that the natural compound procyanidine A1 has remarkable inhibitory activity on the porcine epidemic diarrhea virus and can effectively inhibit replication of the virus in cells. In-vitro experiments prove that the procyanidine A1 has no obvious toxicity to cells at an effective antiviral concentration and has high safety. The invention provides a brand new and effective drug choice for prevention and control of PEDV, and has important application value and wide market prospect.

Description

Technical Field

[0001] This invention belongs to the field of antiviral drug preparation, specifically relating to the application of a natural compound, proanthocyanidin A1, in the preparation of a drug for inhibiting porcine epidemic diarrhea virus. Background Technology

[0002] Proanthocyanidin A1 (PCA1) is a naturally occurring type A proanthocyanidin dimer belonging to the polyphenol flavonoid family. It is widely found in various plants, such as lychee seeds, peanut skins, cinnamon, and cranberries. Existing studies have shown that proanthocyanidin family compounds have a variety of biological activities, the most prominent of which is their strong antioxidant capacity. In addition, they have also been reported to have a variety of pharmacological effects, such as anti-inflammatory, immunomodulatory, antitumor, and antibacterial effects (Zeng Y, Zhao L, Wang K, et al. A-type proanthocyanidins: Sources, structure, bioactivity, processing, nutrition, and potential applications. Compr Rev Food Sci FoodSaf. 2024; 23(3):e13352. doi:10.1111 / 1541-4337.13352).

[0003] In the field of antiviral research, studies have shown that certain type A proanthocyanidins have inhibitory effects on a variety of human and animal viruses, such as influenza virus, herpes simplex virus, respiratory syncytial virus, and even porcine reproductive and respiratory syndrome virus (PRRSV) (Sibille G, Mannino G, Frasson I, et al. The Novel A-Type Proanthocyanidin-Rich Phytocomplex SP4). TM Acts as a Broad-Spectrum Antiviral Agent against Human Respiratory Viruses. Int J Mol Sci. 2024; 25(13):7370. Published 2024 Jul 5. doi:10.3390 / ijms25137370). However, prior to the disclosure of this invention, no literature or patent has reported that proanthocyanidin A1 has inhibitory activity against porcine epidemic diarrhea virus (PEDV), and its application in this field remains blank. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art, namely, to provide a novel drug application to address the current predicament of lacking effective treatments for porcine epidemic diarrhea virus (PEDV) and the poor protective effect of vaccines.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides the use of proanthocyanidin A1 in the preparation of medicaments for the prevention or treatment of PEDV infection.

[0007] This invention, through in-depth research, has for the first time discovered that proanthocyanidin A1 can efficiently and dose-dependently inhibit the replication of PEDV in host cells in vitro, and has extremely low cytotoxicity at effective concentrations, showing excellent therapeutic potential. Attached Figure Description

[0008] Figure 1 Figure showing the cytotoxicity test results of proanthocyanidin A1 on Vero cells;

[0009] Figure 2 Figure 1 shows the experimental results of proanthocyanidin A1 inhibiting the replication of porcine epidemic diarrhea virus in vitro. In figure A, the expression level of viral PEDV-N protein was observed by Western blot, and in figure B, the expression of viral protein was observed by immunofluorescence staining. Detailed Implementation

[0010] The following embodiments are used to further illustrate the present invention, but are not intended to limit the scope of the invention. Any simple modifications or equivalent substitutions made to the present invention are within the scope of protection of the present invention.

[0011] The proanthocyanidin A1 (PCA1) used in these examples was purchased from MedChemExpress, with a purity greater than 99% as determined by high-performance liquid chromatography (HPLC). The cell line used was African green monkey kidney cells (Vero, ATCCCCL-81), a commonly used cell model for in vitro PEDV studies. The porcine epidemic diarrhea virus (PEDV) strain used was the CV777 standard strain. The positive control drug ribavirin was purchased from MedChemExpress.

[0012] Example 1

[0013] This embodiment aims to evaluate the safety of proanthocyanidin A1 in Vero cells within the effective antiviral concentration range. The experiment followed standard cytotoxicity testing procedures, employing methods consistent with routine safety evaluations of veterinary drugs.

[0014] Vero cells in logarithmic growth phase were injected with 1×10⁻⁶ cells.4 Cells were seeded at a density of [number] cells / well in 96-well cell culture plates and incubated at 37°C in 5% CO2 for 24 hours until a monolayer formed. The original culture medium was discarded, and maintenance culture medium (DMEM supplemented with 2% fetal bovine serum) containing different concentrations of proanthocyanidin A1 was added. The final concentrations of proanthocyanidin A1 were set at 25, 37.5, 50, 75, 100, 125, and 150 μg / mL, with six replicates for each concentration. A control group without the drug was also included.

[0015] After culturing the cell plates under the same conditions for another 48 hours, cell viability was assessed using the CCK-8 assay (Cell Counting Kit-8). 10 μL of CCK-8 solution was added to each well, and incubation was continued for 2 hours. The absorbance (OD) value of each well was measured at 450 nm using a microplate reader. The cell viability was calculated using the formula: Cell viability (%) = (OD value of experimental wells / OD value of control wells) × 100%.

[0016] Experimental results are as follows Figure 1 As shown, even at concentrations as high as 150 μg / mL, the cell viability of the proanthocyanidin A1 treatment group remained above 95%, with no significant difference compared to the control group, indicating that it had no significant cytotoxicity to Vero cells. Based on the experimental data, the 50% cytotoxic concentration (CC) of proanthocyanidin A1 was calculated. 50 The concentration was greater than 150 μg / mL. This result provides a basis for the selection of concentration in subsequent antiviral activity experiments and preliminarily demonstrates its potential safety as a drug.

[0017] Example 2

[0018] This embodiment aims to verify the inhibitory effect of proanthocyanidin A1 on PEDV replication and quantify its activity. The experimental design followed standard antiviral drug screening procedures.

[0019] Vero cells were seeded in 24-well plates and cultured until 80%-90% confluence. After washing the cells with serum-free medium, they were inoculated with PEDV virus solution (MOI = 0.1) and incubated at 37°C for 1 hour for adsorption. After adsorption, the virus solution was discarded, and the cells were washed three times with PBS to remove unadsorbed virus particles. Subsequently, maintenance culture medium containing different concentrations of proanthocyanidin A1 (10, 20, 30 μg / mL), ribavirin (10 μM), or proanthocyanidin B2 (30 μg / mL) was added. The cell culture plates were then incubated at 37°C in a 5% CO2 incubator for another 24 hours.

[0020] Viral inhibition was detected by real-time Western blot and immunofluorescence assay (IFA) 24 hours after infection.

[0021] 1. Western blot detection of viral genome replication: Cells from each group were collected, washed twice with pre-chilled PBS, and lysed on ice for 15 minutes with RIPA lysis buffer (containing protease inhibitors). Cell lysates were collected, centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentrations in each group were determined using a BCA kit. Equal amounts of protein (e.g., 30 μg per well) were subjected to SDS-PAGE gel electrophoresis, and then the protein was transferred to a PVDF membrane. After blocking with TBST buffer containing 5% skim milk powder at room temperature for 1 hour, mouse anti-PEDVN protein monoclonal antibody and mouse anti-β-actin (internal control) antibody were added, and incubated overnight at 4°C. The next day, the membrane was washed with TBST, and horseradish peroxidase (HRP)-labeled secondary antibody was added, and incubated at room temperature for 1 hour. After washing, the membrane was developed using an ECL chemiluminescence kit, and images were acquired using a gel imaging system. Results are shown below. Figure 2 As shown in Figure A, compared with the virus control group, the expression level of PEDVN protein in the proanthocyanidin A1 treatment group was significantly reduced in a dose-dependent manner. The expression of the internal control protein β-actin showed no significant difference among the groups, indicating that the loading amount was consistent. These results strongly confirm that proanthocyanidin A1 can effectively inhibit the replication of porcine epidemic diarrhea virus at the protein level.

[0022] 2. Immunofluorescence assay (IFA) for viral protein expression: Cells in 24-well plates were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.2% Triton X-100 for 10 minutes. PEDVN protein monoclonal antibody was used as the primary antibody (incubated at 37°C for 1 hour), and FITC-labeled goat anti-mouse IgG was used as the secondary antibody (incubated at 37°C for 1 hour). Finally, the cell nuclei were stained with DAPI. The cells were observed and photographed under a fluorescence microscope. Results are shown below. Figure 2 As shown in Figure B, the virus control group exhibited abundant bright green fluorescent signals, indicating high viral protein expression. With increasing proanthocyanidin A1 concentration, both the number of green fluorescent cells and the fluorescence intensity decreased significantly, directly demonstrating the inhibitory effect of proanthocyanidin A1 on PEDV. However, proanthocyanidin B2 showed no significant inhibitory effect on PEDV.

[0023] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of proanthocyanidin A1 in the preparation of drugs for the prevention or treatment of porcine epidemic diarrhea virus (PEDV) infection.

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