Application of mulberone H in preparation of medicine for treating or preventing porcine epidemic diarrhea virus infection

Abstract

The invention discloses an application of mulberone H in preparation of a medicine for treating or preventing PEDV (Porcine Epidemic Diarrhea Virus) infection. In-vitro test results prove that the mulberone H significantly inhibits the enzyme digestion activity of PEDV 3C-like protease, and further verifies that the mulberone H inhibits PEDV infected cells, so that the mulberone H can be used for preparing the medicine for preventing and treating PEDV. Besides, mulberone H, as a natural substance separated from mulberries, can be used as a feed additive for a long time so as to achieve the purpose of preventing epidemic diseases, does not cause generation of drug-resistant strains after long-term use, and does not have other toxic or side effects. The defects of immune failure possibly caused by vaccination, virus variation caused by use of antiviral drugs and the like are overcome.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pig virus prevention and treatment, and particularly relates to application of Kuwanon H in preparation of a medicine for treating or preventing porcine epidemic diarrhea virus (PEDV) infection. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) mainly harms piglets, and the mortality rate of piglets infected within 7 days can reach 100%, which is an important factor affecting the survival rate of piglets and pig production. Porcine epidemic diarrhea virus (PEDV) is the main cause of PED. After PEDV enters the body through the digestive tract, it can enter the cell to replicate and proliferate by combining with the receptor, and antagonize the natural immune response of the host, causing immune suppression of the body, resulting in high morbidity and mortality of newborn piglets.

[0003] PEDV infection has caused a large number of newborn piglets to die, resulting in huge economic losses and seriously hindering the development of pig breeding industry. Vaccines are currently the main means of preventing PEDV infection, but vaccine immunization only has the effect of preventing the occurrence of viral diseases and cannot provide protection for piglets that have been infected and have developed the disease. Therefore, relying solely on vaccines can easily lead to immunization failure, and large-scale PEDV infection diseases still occur from time to time.

[0004] In view of the problems of existing vaccines in preventing and treating PEDV, developing a medicine with anti-PEDV will have broad application prospects. Kuwanon H is a tetrahydroxy flavone isolated from mulberry trees, which has been found to have the activity of inhibiting alpha-glucosidase and tyrosinase. The molecular formula of Kuwanon H is C 45 H 44 O 11 , and the molecular weight is 760.82. In recent years, it has been found that Kuwanon H can exert an anti-melanoma effect in vivo and in vitro by inducing endoplasmic reticulum stress and autophagy pathways, indicating that the molecule has anti-melanoma activity. So far, there has been no report on the antiviral effect of Kuwanon H. SUMMARY

[0005] The PEDV encoded replicase polyprotein (Replicase polyprotein 1a, pp1a, Replicase polyprotein 1ab, pp1ab) must be cleaved by viral proteases to form mature non-structural proteins, i.e. NSP1-NSP16 sixteen non-structural proteins produced under the cleavage of NSP3 encoded papain-like protease and NSP5 encoded 3C-like protease. Among them, the papain-like protease is responsible for cleaving the proteins between NSP1-NSP4, and the 3C-like protease is responsible for cleaving the proteins between NSP5-NSP16. In addition, the 3C-like protease can also inhibit the production of interferon by host cells by cleaving NEMO protein, which is closely related to the immune escape of PEDV. Since the mature non-structural proteins play a crucial role in the process of viral transcription, replication and anti-host cell defense mechanism, the 3C-like protease is essential for the process of PEDV infection, and anti-PEDV drugs can be developed based on the 3C-like protease target.

[0006] Therefore, the purpose of the present application is to provide a drug or method for inhibiting the enzymatic activity of PEDV 3C-like protease, so as to achieve the effect of inhibiting PEDV.

[0007] In order to achieve or at least partially achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] The application of mulberrypeptone H in the preparation of a medicine for treating or preventing porcine epidemic diarrhea virus infection.

[0009] Compared with the prior art, the above technical solution has at least the following beneficial effects:

[0010] 1. The mulberrypeptone H provided by the present application is a natural substance isolated from mulberry trees, which can be used as a feed additive for long-term use to achieve the purpose of preventing epidemic diseases. Long-term use will not lead to the emergence of drug-resistant virus strains, nor will it have other toxic side effects, overcoming the shortcomings of possible immunization failure and virus variation caused by the use of antiviral drugs.

[0011] 2. The in vitro test results prove that mulberrypeptone H significantly inhibits the enzymatic activity of PEDV 3C-like protease, and further verifies that mulberrypeptone H inhibits PEDV infected cells, and therefore can be used for the preparation of drugs for preventing and treating PED. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The gel electrophoresis detection results of prokaryotic expression and purification of PEDV 3C-like protease provided in Example 1 of the present application.

[0013] Figure 2The detection results of the activity of PEDV 3C-like protease of different concentrations provided for Example 1 of the present application.

[0014] Figure 3 The schematic diagram of the structure of the reporter plasmid provided for Example 2 of the present application.

[0015] Figure 4 The Western blot detection results provided for Example 2 of the present application.

[0016] Figure 5 The detection results of the cleavage activity of PEDV 3C-like protease provided for Example 2 of the present application.

[0017] Figure 6 The verification results of the effect of morusins H on the extracellular inhibition of PEDV 3C-like protease activity provided for Example 3 of the present application.

[0018] Figure 7 The analysis of the mode of action of morusins H and PEDV 3C-like protease provided for Example 3 of the present application.

[0019] Figure 8 The cytotoxicity test results of morusins H provided for Example 4 of the present application, wherein Figure 8 A is the toxicity test results of Vero cells, Figure 8 B is the toxicity test results of LLC-PK1 cells.

[0020] Figure 9 The test results of morusins H inhibiting the infection of Vero cells by DR13-GFP strain provided for Example 5 of the present application; wherein Figure 9 A, 9B respectively show the influence results of morusins H on the expression of M gene and N gene of PEDV DR13-GFP strain, Figure 9 C shows the influence results of morusins H on the titer of DR13-GFP, Figure 9 D is the fluorescence microscope observation results.

[0021] Figure 10 The test results of morusins H inhibiting the infection of Vero cells by PEDV YN strain provided for Example 6 of the present application; wherein Figure 10 A, 10B respectively are the influence results of morusins H on the expression of M gene and N gene of PEDV YN strain, Figure 10 C shows the influence results of morusins H on the titer of PEDV YN strain, Figure 10 D is the indirect immunofluorescence test results for evaluating the influence of morusins H on the number of cells infected by YN strain.

[0022] Figure 11Results of Kuwanon H inhibiting YN strain infecting LLC-PK1 cells provided in Example 7 of the present application; wherein Figure 11 A, 11B are results of Kuwanon H affecting expression of M gene and N gene of PEDV YN strain, respectively, Figure 11 C is a result of Kuwanon H affecting titer of PEDV YN strain.

[0023] Figure 12 Results of Kuwanon H inhibiting 3C-like protease activity in cells provided in Example 8 of the present application, wherein RLU1: luciferase activity; RLU2: sea cucumber luciferase activity. DETAILED DESCRIPTION

[0024] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below with reference to examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0025] Those skilled in the art can understand that, unless specifically stated otherwise, "the", "this", "the aforementioned" used in the text of the present application can also include the plural form. It should be further understood that the phrase "comprising" used in the specification of the present application means that the features, steps, operations exist, but does not exclude the existence or addition of one or more other features, integers, steps.

[0026] Those skilled in the art can understand that, unless specific experimental steps or conditions are indicated in the examples, the operation or conditions can be carried out according to the conventional experimental steps described in the literature in the art; and the raw materials or instruments and equipment not indicated by the manufacturer are all conventional products that can be obtained by purchase.

[0027] Those skilled in the art can understand that, unless otherwise stated in the present application, when the examples give a numerical range, each numerical range and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application are consistent with the understanding of the prior art by those skilled in the art and the description of the present application. Any method, equipment and material of the prior art similar or equivalent to the method, equipment and material described in the examples of the present application can also be used to realize the present application.

[0028] The concentration values in the present application, unless otherwise specified, are mass fractions, such as "1% PSF" means 1% PSF by mass.

[0029] "Kuwanon H" in the present application is a tetrahydroxy flavone isolated from mulberry, and the molecular formula of Kuwanon H is C 45 H 44 O11 Molecular weight is 760.82. In the prior art, mulberrone H has been found to have the activity of inhibiting alpha-glucosidase and tyrosinase, and can exert an anti-melanoma effect in vivo and in vitro by inducing endoplasmic reticulum stress and autophagy pathway, but there is no report on the application of mulberrone H in anti-PEDV virus.

[0030] The embodiment of the present application finds that mulberrone H significantly inhibits the enzymatic activity of PEDV 3C-like protease through in vitro test, and further verifies that mulberrone H can inhibit the infection of PEDV to cells, and the mulberrone H can be used for preparing anti-PEDV drugs.

[0031] Based on this, the embodiment of the present application provides the application of mulberrone H in the preparation of drugs for treating or preventing porcine epidemic diarrhea virus infection.

[0032] In some embodiments, the drug is mulberrone H. The mulberrone H is directly used as a drug as an active ingredient.

[0033] In some embodiments, the drug is mulberrone H and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient is a non-active substance for ensuring the safety, stability, effectiveness of the preparation or improving its properties.

[0034] In some embodiments, the dosage form of the drug includes at least one of oral liquid, granules, tablets, and powder. Different dosage forms need to select different excipients for preparation.

[0035] In some embodiments, the drug can be used as a feed additive added to pig feed.

[0036] The technical solutions and technical effects of the present application are described in more detail below through more specific embodiments.

[0037] Example 1: Establishment of 3C-like protease activity FRET system

[0038] 1. Materials and methods

[0039] In this embodiment, according to the site of PEDV 3C-like protease cleavage of PEDV replicase polyprotein ppla and pplab, a modified polypeptide Dabcyl-YNSTLQ↓AGLRKM-E-Edans is synthesized in Kings River Biological Company. In the intact case, the fluorescence of the C-terminal modification group Edans is quenched by the N-terminal Dabcyl group due to FRET effect, and in the presence of 3C-like protease, the polypeptide is cleaved, the FRET system is destroyed, and the fluorescence signal of E-Edans is released.

[0040] To optimize the 3C-like protease activity detection system, the enzyme digestion reaction system was prepared as 10 μM modified polypeptide and different concentrations (1-80 μg / mL) of 3C-like protease, the total volume of the reaction system was 200 μL, the buffer was 20 mM Tris / HCl solution (pH 7.5), and the multifunctional enzyme marker was used at 37°C, 340 nm excitation light, and the emission intensity at 480 nm was continuously detected to determine the optimal protein addition amount.

[0041] 2. Test results

[0042] The PEDV 3C-like protease with a 6×his tag was expressed using a prokaryotic expression system, and the expression results were detected by gel electrophoresis, as shown in Figure 1 From Figure 1 , it can be seen that the protein can be expressed in the supernatant with a protein size of about 35 kD; the target protein in the supernatant was purified using a Ni-NTA nickel column, and the results showed that the protein can be effectively purified.

[0043] In the FRET system for detecting the enzyme digestion activity of PEDV 3C-like protease, different concentrations of purified PEDV 3C-like protease were added, as shown in Figure 2 , with the increase of the concentration of the added protease and the increase of the reaction time, the fluorescence signal of the system was continuously enhanced, indicating that the purified protein can effectively cut the FRET substrate, and the cutting activity is dose-dependent. The above test results prove that the established FRET system can effectively detect the activity of PEDV 3C-like protease outside the cell, and can be used to verify the inhibitory effect of 3C-like protease inhibitors on the protease.

[0044] Example 2: Establishment of luciferase reporter system for detecting PEDV 3C-like protease activity

[0045] 1. Materials and methods

[0046] 1.1 Main test materials

[0047] 293T cells (laboratory preserved cell strain), lipofectamine 2000 transfection reagent (Sai Mei), Promega dual luciferase detection kit (Puluo Meige).

[0048] 1.2 Test method

[0049] (1) In order to verify that the screened molecules also have 3C-like protease inhibitor activity in cells, as Figure 3As shown, the PEDV 3C-like protease cleavage substrate polypeptide (Cleave peptides, the amino acid sequence is: YNSTLQAGLRKM (SEQ ID NO. 1)) is fused with the luciferase to express, a reporter plasmid is constructed, and due to the introduction of the intein DnaE, the luciferase protein expressed after the reporter plasmid transfecting cells exists in the form of a circular protein in a natural state, there is a large steric hindrance between the circular proteins, and it is difficult for them to approach each other to avoid self-activation; only when the 3C-like protease is expressed in cells, the cleavage sequence can be recognized and cleaved, the steric hindrance is reduced after cleavage, the luciferase activity is restored, and thus the cleavage activity of the 3C-like protease can be detected.

[0050] (2) The constructed reporter plasmid, the renilla luciferase plasmid (pRL-TK) and the plasmid expressing the 3C-like protease (pCAGGS-NSP5) are co-transfected into 293T cells, the maintenance solution is replaced 6 hours after transfection, and 24 hours after transfection, it is found by Western blot detection that after transfection of pCAGGS-NSP5, Western blot and Promega dual luciferase detection kit are used to detect whether the circularized protein can be cleaved.

[0051] 2. Test results

[0052] As Figure 4 shown, the Western blot test results show that the PEDV 3C-like protease (HA-NSP5) can be effectively expressed in cells after transfection of pCAGGS-NSP5, and the cyclized luciferase (cyclized-luc) and the cleaved luciferase (cleaved-luc) can be detected, indicating that the PEDV 3C-like protease (HA-NSP5) can recognize and cleave the cyclized luciferase in cells. At the same time, the activities of the firefly luciferase and the renilla luciferase are detected by using the dual luciferase detection kit, and it is found that the activity of the firefly luciferase after transfection of pCAGGS-NSP5 is significantly higher than that of the control group, as Figure 5 shown. The above results show that the system can effectively detect the activity of the PEDV 3C-like protease in cells, and can be used to verify the inhibitory effect of the 3C-like protease inhibitor on the protease in cells.

[0053] Example 3 Verification of inhibition of the cleavage activity of PEDV 3C-like protease by Sangpiketon H in cells

[0054] 1. Materials and methods

[0055] 1.1 Test materials

[0056] Sangpiketon H: purchased from Shanghai Yuan Ye Biology, purity > 98%.

[0057] 1.2 Test method

[0058] The verification method of mulberrone H for inhibiting the activity of PEDV 3C-like protease outside cells is as follows:

[0059] According to the detection method optimized in Example 1, the test group and the control group were set up respectively, and 20 μg / mL of PEDV 3C-like protease was selected as the working concentration. Different concentrations of mulberrone H were added to the FRET reaction system in the test group, and no mulberrone H was added in the control group. The fluorescence intensity was measured at 340 nm excitation light at 37°C at the initial reaction (0 min) and the end of the reaction (90 min) respectively. According to the fluorescence intensity detection results, the inhibitory effect of mulberrone H on the cleavage activity of PEDV 3C-like protease under each concentration condition was calculated. The calculation formula is:

[0060] Inhibition rate (%) = (1 - F s / F d ) x 100%, wherein F s is the difference between the fluorescence intensity at the end of the reaction (90 min) and the initial reaction (0 min) in the test group, and F d is the difference between the fluorescence intensity at the end of the reaction (90 min) and the initial reaction (0 min) in the control group.

[0061] 2. Test results

[0062] As shown in Figure 6 , different concentrations of mulberrone H were added to the system for detecting the cleavage activity of PEDV 3C-like protease. With the increase of the concentration of mulberrone H, the inhibition rate of mulberrone H on the cleavage activity of PEDV 3C-like protease was continuously enhanced, indicating that mulberrone H could effectively inhibit the cleavage of PEDV 3C-like protease on the substrate outside the cells. Through molecular docking analysis, it was found that mulberrone H could effectively dock to the active center of PEDV 3C-like protease and form interaction with 6 amino acid sites (THR47, Phe19, His162, Gln163, Glu165 and Gln191) near the active center, as shown in Figure 7 , it is speculated that mulberrone H may inhibit the cleavage activity of the enzyme by interacting with the cleavage activity center of PEDV 3C-like protease. The above test results confirm that mulberrone H is an effective inhibitor of PEDV 3C-like protease.

[0063] Example 4 Cell toxicity test of mulberrone H

[0064] 1. Materials and methods

[0065] 1.1 Test materials

[0066] Moracenone H: purchased from Shanghai Yuan Ye Bio, purity > 98%.

[0067] Cell lines: Vero (African green monkey kidney cells) and LLC-PK1 (porcine kidney cells) are laboratory preserved cell strains.

[0068] CCK8 kit: purchased from Novi Biological Technology Co., Ltd.

[0069] 1.2 Test method

[0070] This example sets up the test group and the blank control group, wherein the test group is to add cell culture medium (DMEM containing 2% fetal bovine serum) containing different concentrations of moracenone H into the 96-well plate full of single-layer Vero and LLC-PK1 cells, each concentration is set with 8 repeats, each repeat is 1 well, each well is 100 μL, and is placed in 37℃, 5% CO2, and cultured for 24h. The blank control group does not add moracenone H; discard the culture solution, wash twice with PBS, add 100 μL of 10% CCK-8 solution (1% PSF and 10% CCK-8 are added to the DMEM culture medium) to each well, at the same time set up the negative control group (add 10% CCK-8 culture solution to the blank well), and place it in the incubator for 2h, then use the enzyme label instrument to measure the OD value of each treatment group at a wavelength of 450nm. The cell viability calculation formula is as follows:

[0071] Cell viability (%) = (test group OD value-negative control group OD value) / (blank control group OD value-negative control group OD value) x 100%.

[0072] 2. Test results

[0073] Figure 8 The results of the cytotoxicity test of moracenone H are shown in the table below: Figure 8 A is the toxicity test result of Vero cells, Figure 8 B is the toxicity test result of LLC-PK1 cells, from the results in the figure, when the concentration of moracenone H is not higher than 12.5 μM, the activity of Vero and LLC-PK1 cells is higher than 90%. In the subsequent antiviral application examples, moracenone H with a concentration of 12.5 μM or less is selected for testing.

[0074] Example 5: Moracenone H inhibits PEDV DR13-GFP strain infection of Vero cells

[0075] This example verifies the effect of moracenone H on inhibiting PEDV DR13-GFP strain infection of Vero cells.

[0076] 1. Materials and methods

[0077] 1.1 Test materials

[0078] Moracenone H: purchased from Shanghai Yuan Ye Bioengineering, purity > 98%.

[0079] DR13-GFP strain (the ORF3 gene of DR13 strain is replaced by GFP gene through reverse genetics technology).

[0080] 1.2 Test method

[0081] Vero cells were infected with PEDV DR13-GFP strain (MOI = 0.001), and different concentrations of Moracenone H were added at the same time of infection; samples were collected 36 h after infection, and RT-PCR, TCID 50 The effect of Moracenone H on DR13-GFP in vitro infection was evaluated by determination and fluorescence microscope observation.

[0082] 2. Test results

[0083] Figure 9 The test results of Moracenone H inhibiting DR13-GFP strain infecting Vero cells, wherein Figure 9 A and 9B respectively show the results of Moracenone H affecting the expression of M gene and N gene of PEDV DR13-GFP strain, and it can be seen from the figure that Moracenone H inhibits the expression of PEDV M and N genes in DR13-GFP infected Vero cells in a dose-dependent manner; Figure 9 C shows the results of Moracenone H affecting the titer of DR13-GFP, and TCID 50 The test results show that Moracenone H can reduce the titer of the virus, and presents obvious dose-dependent manner; Figure 9 D is the result of fluorescence microscope observation, and it can be seen from the figure that Moracenone H can significantly reduce the number of DR13-GFP infected cells, and presents obvious dose-dependent manner. The above test results confirm that Moracenone H can effectively inhibit DR13-GFP strain infecting Vero cells.

[0084] Example 6: Moracenone H inhibits PEDV YN strain infecting Vero cells

[0085] This example verifies the effect of Moracenone H on inhibiting PEDV YN strain infecting Vero cells.

[0086] 1. Materials and methods

[0087] 1.1 Test materials

[0088] Moracenone H: purchased from Shanghai Yuan Ye Bioengineering, purity > 98%.

[0089] PEDV strain: PEDV YN strain is a virus strain isolated by the laboratory (GenBank accession No. KT021228).

[0090] Cell line: Vero cells are cell strains preserved by the laboratory.

[0091] 1.2 Test method

[0092] Vero cells were infected with PEDV YN strain (MOI = 0.001), and different concentrations of Morusin H were added at the same time. Samples were collected 24 h after infection, and RT-PCR, TCID 50 The effect of Morusin H on the in vitro infection of YN strain was evaluated by RT-PCR, TCID

[0093] 2. Test results

[0094] Figure 10 The test results of Morusin H inhibiting PEDV YN strain infection in Vero cells, wherein Figure 10 A, 10B are the results of the effect of Morusin H on the expression of M gene and N gene of PEDV YN strain, respectively. As can be seen from the figure, Morusin H inhibits the expression of PEDV M and N genes in YN strain infected Vero cells in a dose-dependent manner; Figure 10 C shows the results of the effect of Morusin H on the titer of PEDV YN strain, TCID 50 The test results show that Morusin H can reduce the titer of YN strain, and presents obvious dose-dependent manner; Figure 10 D is the result of the effect of Morusin H on the number of YN strain infected cells evaluated by indirect immunofluorescence test. As can be seen from the figure, Morusin H can significantly reduce the number of YN strain infected cells, and presents obvious dose-dependent manner. The above test results confirm that Morusin H can effectively inhibit YN strain infection in Vero cells.

[0095] Example 7: Morusin H inhibits PEDV YN strain infection in LLC-PK1 cells

[0096] This example verifies the effect of Morusin H on inhibiting PEDV YN strain infection in LLC-PK1 cells.

[0097] 1. Materials and methods

[0098] 1.1 Test materials

[0099] Morusin H: purchased from Shanghai Yuan Ye Biology, purity > 98%.

[0100] PEDV strain: PEDV YN strain is a virus strain isolated by the laboratory (GenBank accession No. KT021228).

[0101] LLC-PK1 cells are cell strains preserved by the laboratory.

[0102] 1.2 Test method

[0103] LLC-PK1 cells were infected with PEDV YN strain (MOI = 0.01), and different concentrations of Morusin H were added at the same time. Samples were collected 24 h after infection, and the effect of Morusin H on in vitro infection of YN strain was evaluated using RT-PCR and TCID50 determination.

[0104] 2. Test results

[0105] Figure 11 Results showing that Morusin H inhibits the infection of YN strain in LLC-PK1 cells, wherein Figure 11 A and 11B are the results of the effect of Morusin H on the expression of M gene and N gene of PEDV YN strain, respectively. As can be seen from the figure, Morusin H inhibits the expression of PEDV M and N genes in YN strain infected Vero cells in a dose-dependent manner; Figure 11 C is the effect of Morusin H on the titer of PEDV YN strain. As can be seen from the figure, Morusin H can reduce the titer of YN strain, and presents obvious dose-dependent. The above test results confirm that Morusin H can reduce the titer of YN strain, and presents obvious dose-dependent. Further prove that Morusin H can effectively inhibit the infection of YN strain in LLC-PK1 cells.

[0106] The test results of Examples 5, 6 and 7 prove that Morusin H can effectively inhibit the in vitro infection of PEDV.

[0107] Example 8: Inhibition of the enzymatic activity of PEDV 3C-like protease by Morusin H in cells

[0108] This example verifies the effect of Morusin H on the inhibition of the enzymatic activity of PEDV 3C-like protease in cells.

[0109] 1. Materials and methods

[0110] 1.1 Test materials

[0111] Cell line: 293T cells (human embryonic kidney cells) are cell strains preserved by the laboratory

[0112] Promega Dual-Luciferase Kit is purchased from Promega.

[0113] 1.2 Test method

[0114] pCAGGS-NSP5 and reporter plasmid 358DnaE-PEDV and pRL-TK were co-transfected into 293T cells, and the culture medium containing different concentrations of morusin H was replaced 6h after transfection. The cleavage activity of 3C-like protease was detected 24h after transfection using the promega dual luciferase kit.

[0115] 2. Test results

[0116] Figure 12 The results of intracellular inhibition of 3C-like protease activity by morusin H are shown, where RLU1: luciferase activity; RLU2: sea cucumber luciferase activity. As can be seen from the figure, morusin H can inhibit the cleavage of PEDV 3C-like protease on its substrate in cells, and it is obviously dose-dependent.

[0117] In summary, the embodiments of the present application demonstrate that morusin H can effectively inhibit the activity of 3C-like protease, thereby exerting an anti-PEDV in vitro infection effect, further demonstrating that morusin H can be used to prepare an anti-PEDV drug for prevention or treatment.

[0118] The above has described the present application in detail, and the principles and implementation modes of the present application have been described by applying specific examples. The above description of the embodiments is only for the purpose of helping to understand the present application and the core idea. It should be pointed out that, for ordinary skilled persons in the technical field, some improvements and modifications can be made to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. Use of mulberone H in the preparation of a drug for treating or preventing porcine epidemic diarrhea virus infection.

2. The use according to claim 1, characterized in that The drug is mulberton H.

3. The use according to claim 1, characterized in that The medicine is mulberton H and pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that The dosage form of the drug includes at least one of oral solution, granules, tablets, and powder.

5. The use according to claim 1, characterized in that The medicine can be used as a feed additive and added into pig feed.

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