Application of licochalcone B in preparation of medicine for preventing and treating porcine epidemic diarrhea

Abstract

The invention discloses an application of licochalcone B (Lcochalcone B, LCB) in a medicine for preventing and treating porcine epidemic diarrhea, and belongs to the technical field of medicines. The antiviral effect of the licochalcone B on the porcine epidemic diarrhea virus (PEDV) at the level of Vero cells (African green monkey kidney cells) is researched through methods such as an indirect immunofluorescence experiment (IFA) and real-time fluorescent quantitative PCR (qRT-PCR), and experimental results show that the licochalcone B can remarkably inhibit proliferation of the porcine epidemic diarrhea virus. It is proved that licochalcone B has remarkable porcine epidemic diarrhea virus resisting activity, a brand-new effective candidate medicine is provided for prevention and treatment of porcine epidemic diarrhea, and the licochalcone B has good application prospects in the field of medicine for preventing and treating porcine epidemic diarrhea.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of glycyrrhizin B in drugs for the prevention and treatment of porcine epidemic diarrhea virus. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious enterovirus disease of pigs caused by porcine epidemic diarrhea virus (PEDV). Typical clinical manifestations include loss of appetite, watery diarrhea, severe dehydration, and vomiting. In particular, the mortality rate in piglets under two weeks of age is as high as 80-100%, causing huge economic losses to the livestock and poultry farming industry.

[0003] Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded, positive-sense RNA virus, belonging to the order Nidovirales, family Coronaviridae, and genus Alphacoronavirus. The virus particles are spherical or oval, with a layer of rod-shaped spikes on the surface, and a diameter of approximately 80-120 nm. The genome is about 28 kb long and mainly contains 7 open reading frames (ORFs), 5′ untranslated regions (UTRs), 3′ untranslated regions, and their connected poly(A) tail structures. The 7 ORFs primarily encode 4 structural proteins, 16 non-structural proteins, and 1 accessory protein.

[0004] In recent years, the high variability of PEDV strains has posed a huge challenge to vaccine development and prevention, resulting in low immunogenicity and limited preventive effect of traditional conventional vaccines. Therefore, the development and search for antiviral drugs to prevent and treat PED has become particularly important.

[0005] Licochalcone B (LCB) is a flavonoid compound extracted from licorice root. It belongs to the chalcone derivatives and its structure is shown below:

[0006]

[0007] Structural Formula I: Licochalcone B

[0008] Glycyrrhizin chalcone B possesses antiviral, anti-inflammatory, and antitumor effects. It can induce cell cycle arrest, significantly inhibiting tumor cell proliferation by arresting tumor cells in the S, G1, or G2 / M phases. CN202010994493.0 discloses the application of chalcone compounds in the treatment of coronavirus infection, and CN202510500169.1 discloses the application of glycyrrhizin chalcone in inhibiting coronavirus 3CL enzyme. However, there are no reports in the prior art that glycyrrhizin chalcone B can prevent or treat porcine epidemic diarrhea, nor are there any published related patents. Summary of the Invention

[0009] The purpose of this invention is to overcome the difficulties in the prevention and control of porcine epidemic diarrhea (PED) and to provide the application of glycyrrhizin B in the treatment of PED virus.

[0010] The first objective of this invention is to provide the use of glycyrrhizin B in the preparation of a medicament for the prevention and treatment of porcine epidemic diarrhea.

[0011] A second objective of this invention is to provide the use of glycyrrhizin B in the preparation of a medicament for the prevention and treatment of porcine epidemic diarrhea virus.

[0012] A third objective of this invention is to provide the use of glycyrrhizin B in the preparation of a medicament for inhibiting the expression of porcine epidemic diarrhea virus N protein.

[0013] A fourth objective of this invention is to provide the use of glycyrrhizin B in the preparation of a drug for inhibiting the expression of the N gene of porcine epidemic diarrhea virus.

[0014] To achieve the above objectives, the present invention is implemented through the following solution:

[0015] This invention marks the first discovery that glycyrrhizin chalcone B possesses significant antiviral activity against porcine epidemic diarrhea virus (PEDV). First, the toxicity of glycyrrhizin chalcone B to African green monkey kidney cells (Vero) was determined using the MTT assay to establish the maximum non-toxic dose. Then, indirect immunofluorescence assays (IFA) and real-time quantitative PCR (Real-time PCR) demonstrated that glycyrrhizin chalcone B effectively inhibits the infection and proliferation of PEDV, showing promise as a drug for the prevention and treatment of PEDV.

[0016] Therefore, this invention claims protection for the following:

[0017] Application of glycyrrhizin B in the preparation of drugs for the prevention and treatment of porcine epidemic diarrhea.

[0018] Application of glycyrrhizin B in the preparation of drugs for the prevention and treatment of porcine epidemic diarrhea virus.

[0019] Preferably, the prevention and control of porcine epidemic diarrhea virus (PEDV) is to inhibit PEDV infection.

[0020] Preferably, the prevention and control of porcine epidemic diarrhea virus (PEDV) involves inhibiting the proliferation of PEDV.

[0021] Application of glycyrrhizin B in the preparation of drugs that inhibit the expression of porcine epidemic diarrhea virus N protein.

[0022] Application of glycyrrhizin B in the preparation of drugs that inhibit the expression of the N gene of porcine epidemic diarrhea virus.

[0023] Preferably, the effective concentration of glycyrrhizin B is 7.5–30 μM.

[0024] More preferably, the effective concentration of glycyrrhizin B is 30 μM.

[0025] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention discloses that glycyrrhizin chalcone B has a significant antiviral effect against porcine epidemic diarrhea virus (PEDV). Its maximum non-toxic dose is 40 μM, and it can effectively inhibit the infection and proliferation of PEDV, making it a promising drug for the prevention and treatment of PED. Attached Figure Description

[0028] Figure 1 The results were obtained by measuring the toxicity of glycyrrhizin B to Vero cells using the MTT assay.

[0029] Figure 2 The results were obtained by using IFA to detect the inhibitory effect of different concentrations of glycyrrhizin B on the expression of PEDV N protein in Vero cells;

[0030] Figure 3 The results were obtained by using real-time quantitative PCR to determine the inhibitory effect of glycyrrhizin B on PEDV at the mRNA level. Detailed Implementation

[0031] The following description, in conjunction with specific embodiments, will provide a clearer picture of the present invention. However, these are merely exemplary and do not constitute any limitation on the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. The specific steps of the method of the present invention are as follows:

[0032] Example 1: Cytotoxicity assay of glycyrrhizin B on Vero cells

[0033] 1. Method:

[0034] 1.1 Preparation of glycyrrhizin B: Dissolve glycyrrhizin B standard in DMSO to prepare a stock solution, and store it at 4℃ for later use.

[0035] 1.2. Vero cells were seeded in 96-well cell culture plates. Once the cell density reached over 90%, the test drug was serially diluted with DMEM maintenance medium containing 2% FBS to create six different compound concentration groups: 5, 10, 20, 40, 80, and 160 μM. A blank control group and a control group containing 1% DMSO solvent were also included. Each group had six replicate wells, with 100 μL per well. The plates were incubated at 37°C in a 5% CP2 incubator. After 48 hours, the culture was terminated, the supernatant was discarded, and 0.5 mg / mL MTT solution diluted with DMEM maintenance medium was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator in the dark. Four hours later, the supernatant in each well was discarded, and 150 μL of dimethyl sulfoxide was added to each well. The wells were then shaken for 10 minutes to fully dissolve the formazan crystals. The OD value was measured at 490 nm using a multi-mode microplate reader to calculate cell viability. The half-maximal cytotoxic concentration (50% cytotoxic concentration, CC5) of glycyrrhizin B against Vero cells was calculated using GraphPad Prism 8.0 software. 50 ).

[0036] The calculation formula is as follows:

[0037]

[0038] like Figure 1 As shown, glycyrrhizin B has no toxicity to Vero cells at concentrations below 40 μM.

[0039] Example 2: Indirect immunofluorescence assay (IFA) was used to analyze the effect of different concentrations of glycyrrhizin B on the expression of PEDV N protein in Vero cells.

[0040] 2. Method:

[0041] Vero cells were seeded into 96-well cell culture plates. Once the Vero cell density in the 96-well plates reached over 90%, the cell suspension was gently aspirated. A blank control group, a negative control group, a positive drug group, and three concentrations of glycyrrhizin B were set up, with two replicates for each group. PEDV virus solution was serially diluted to 100 TCID using 2% FBSDMEM maintenance medium. 50Except for the blank control group which was replaced with 2% FBS DMEM maintenance medium, each well of the other control groups was inoculated with 100 μL of PEDV virus solution and incubated at 37°C in a 5% CO2 incubator. After 2 hours, the supernatant was discarded, and the cells were washed twice with sterile PBS. Glycyrrhizin chalcone B was diluted with DMEM maintenance medium containing 2% FBS to different concentrations of 7.5, 15, and 30 μM, with 100 μL per well, and incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0042] Indirect immunofluorescence: Terminate culture, gently aspirate cell fluid, add universal tissue fixative (100 μL per well), incubate at room temperature for 15 min, discard fixative, and wash three times with PBS; add 0.3% Triton X-100 solution (100 μL per well), incubate at room temperature for 10 min, discard supernatant, and wash three times with PBS; add 5% BSA (50 μL per well), incubate at room temperature for 1 h, discard supernatant, and wash once with PBS; add Anti-PEDV-N Antibody (1:500 dilution) (50 μL per well), incubate overnight at 4°C, recover primary antibody, wash three times with PBS, and add Alexa Fluor 488-labeled Goat Anti-Mouse. 50 μL of lgG (H&L) (1:500 dilution) was added to each well and incubated at room temperature in the dark for 1 h. The mixture was then washed three times with PBS. 50 μL of DAPI solution (final concentration 300 nM) was added to each well and incubated at room temperature in the dark for 10 min. The supernatant was discarded and the mixture was washed three times with PBS. The mixture was observed and photographed using an inverted fluorescence microscope. Green fluorescence represents PEDVN protein and blue fluorescence represents the cell nucleus.

[0043] like Figure 2 As shown, glycyrrhizin B inhibits PEDV proliferation in a dose-dependent manner at the protein expression level.

[0044] Example 3: Real-time quantitative PCR determination of the effect of glycyrrhizin B on PEDV mRNA gene expression in Vero cells.

[0045] 3. Method:

[0046] Vero cells were seeded into 12-well plates. Once the cell density reached over 90%, the cell suspension was gently aspirated. A blank control group, a negative control group, a positive drug group, and a glycyrrhizin-chalcone B group were established. PEDV virus solution was serially diluted to 100 TCID using 2% FBSDMEM maintenance medium. 50Except for the blank control group which was replaced with 2% FBSDMEM maintenance medium, each well of the other cells was inoculated with 1 mL of PEDV virus solution and incubated at 37°C in a 5% CO2 incubator. After 2 hours, the supernatant was discarded, and the cells were washed twice with sterile PBS. Glycyrrhizin chalcone B was diluted to 30 μM with DMEM maintenance medium containing 2% FBS, and 1 mL was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 48 hours. The cell samples were then subjected to three freeze-thaw cycles at -80°C and 4°C and stored at -80°C for later use.

[0047] Total RNA was extracted following the instructions of the Servicebio Animal Tissue / Cell Total RNA Extraction Kit (Servicebio). The RNA was then reverse transcribed into cDNA using the StarScript II RT Kit (GenStar).

[0048] Using cDNA obtained from reverse transcription as a template and GAPDH as an internal reference gene, qRT-PCR (Bio-Rad CFX96 qRT-PCR system) was performed using SYBR Green I (Servicebio 2×Universal Blue SYBR Green qPCR Master Mix) as a fluorescent dye.

[0049] Table 1 Specific primer sequences

[0050]

[0051] Reaction conditions: Activate the hot-start enzyme at 95℃ for 30s; denature at 95℃ for 15s, anneal at 55℃ for 10s, extend at 72℃ for 10s, for 40 cycles; collect fluorescence signals at this step.

[0052] like Figure 3 As shown, glycyrrhizin B at a concentration of 30 μM significantly inhibited the expression of PEDV mRNA in Vero cells.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of glycyrrhizin B in the preparation of drugs for the prevention and treatment of porcine epidemic diarrhea virus.

2. The application according to claim 1, characterized in that, The prevention and control of porcine epidemic diarrhea virus (PEDV) involves inhibiting PEDV infection.

3. The application according to claim 1, characterized in that, The prevention and control of porcine epidemic diarrhea virus (PEDV) involves inhibiting the proliferation of PEDV.

4. The application according to any one of claims 1 to 3, characterized in that, The effective concentration of glycyrrhizin B is 7.5–30 μM.

5. The application according to claim 4, characterized in that, The effective concentration of glycyrrhizin B is 30 μM.

6. The application according to any one of claims 1 to 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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