Use of arctin in preparation of a medicine for inhibiting replication and / or release of porcine epidemic diarrhea virus

Abstract

The application belongs to the technical field of veterinary medicine, and specifically discloses application of atractylin in preparation of a medicine for inhibiting replication and / or release of a porcine epidemic diarrhea virus (PEDV). The atractylin is added into DMEM culture medium according to different final concentrations, and the antiviral effect of the atractylin on the PEDV is studied. The results show that the atractylin does not produce cytotoxicity at a concentration of 200 μM and a treatment time of 24 h, and has good antiviral effect on the PEDV at a concentration of 10-100 μM. The atractylin raw material is easy to obtain, the extraction method is simple, the purity reaches 98.5%, the atractylin has good antiviral effect on the PEDV, is safe and low-toxic, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of veterinary medicine technology, and particularly relates to the application of atractylodes in the preparation of drugs that inhibit the replication and / or release of porcine epidemic diarrhea virus (PEDV). Background Technology

[0002] Coronaviruses belong to the order Nidovirales, family Coronaviridae, and genus Coronavirus. They are enveloped, linear, single-stranded, positive-sense RNA viruses. Viral particles range in diameter from 60 to 200 nm, are spherical or elliptical in shape, and exhibit pleomorphism. Coronaviruses infect only vertebrates, such as humans, mice, pigs, cats, dogs, chickens, cattle, and poultry. Porcine epidemic diarrhea (PED) is a common disease in large-scale pig farms, and infection with Porcine epidemic diarrhea virus (PEDV) is the main cause. PEDV is a coronavirus that specifically infects pigs. It was first successfully isolated in the UK and Belgium in 1978. Since 2010, PEDV has repeatedly circulated in my country, indicating that current vaccines and antiviral drugs are ineffective in preventing infections caused by newly mutated, highly pathogenic PEDV strains. PEDV can infect pigs at all stages, but it has the greatest impact on suckling piglets, causing severe enteritis, watery diarrhea, vomiting, and dehydration. The mortality rate in piglets under 3 days old is as high as 100%. PEDV spreads rapidly and has a high infection rate, seriously affecting the growth and development of pigs and causing huge economic losses to the pig farming industry.

[0003] Currently, treatment and control methods for PEDV are relatively limited. Traditional prevention and control methods mainly rely on biosafety measures, vaccines, and antiviral drugs. However, due to the variability and drug resistance development of PEDV, traditional vaccines and drugs may show limited effectiveness in some cases. Therefore, there is an urgent need to find new and highly effective antiviral drugs to effectively control the spread of PEDV and the epidemic.

[0004] Atractylodesin is a natural product (molecular formula C). 13 H 10 O (molecular weight: 182.2179) belongs to the sitosterol class of compounds and is mainly found in the rhizome of Atractylodes lancea. It is believed to have a variety of pharmacological activities, including anti-inflammatory, antioxidant, antibacterial, and antitumor effects. Summary of the Invention

[0005] Given the relatively limited treatment and control methods for PEDV in existing technologies and the insufficient development and utilization of atractylodes in the pharmaceutical field, the purpose of this invention is to provide the application of atractylodes in the preparation of drugs that inhibit PEDV replication and / or release.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides the use of atractylodesin in the preparation of a drug for inhibiting PEDV replication and / or release. Atractylodesin can efficiently inhibit PEDV replication and / or release, and further, the drug is used in pigs at various stages.

[0008] Furthermore, the sole active ingredient of the drug is atractylodesin;

[0009] Furthermore, the drug is administered via the gastrointestinal tract or via injection.

[0010] Furthermore, the drug is used on suckling piglets.

[0011] Furthermore, the drug is used on suckling piglets under 7 days old.

[0012] Furthermore, a 20 μM concentration of atractylodes lancea showed the best inhibitory effect on the release of porcine epidemic diarrhea virus (PEDV) and was therefore the preferred choice; treatment with a 200 μM concentration of atractylodes lancea for 24 hours showed no cytotoxicity.

[0013] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0014] This invention applied atractylodesin to Vero cells and found that atractylodesin significantly inhibited the replication and release of PEDV. Quantitative real-time PCR detection confirmed the inhibitory effect of atractylodesin on PEDV replication and release in cells. Results showed that atractylodesin at a concentration of 200 μM for 48 h had only a slight effect on the morphology of Vero cells, while lower concentrations and shorter treatment times had no significant effect on the morphology of Vero cells; indicating that atractylodesin has a wide safe concentration range and high safety. Furthermore, it was found that atractylodesin at concentrations between 10 μM and 100 μM significantly inhibited the expression level of PEDV N protein, with 20 μM atractylodesin showing the best inhibitory effect on PEDV release. During use, the dosage can be appropriately adjusted within this range to reduce the potential burden of drug metabolism. Attached Figure Description

[0015] Figure 1 The effect of different concentrations of atractylodesin on Vero cell morphology;

[0016] Figure 2 To detect the cytotoxicity of different concentrations of atractylodesin to Vero cells;

[0017] Figure 3 To detect the inhibitory effect of different concentrations of atractylodesin on PEDV using quantitative real-time polymerase chain reaction (PCR);

[0018] Figure 4To detect the inhibitory effect of different concentrations of atractylodesin on PEDV using Western blot;

[0019] Figure 5 , 6 To detect the inhibitory effect of different concentrations of atractylodesin on PEDV using immunofluorescence staining;

[0020] Figure 7 The effect of 20 μM atractylodesin on PEDV adhesion;

[0021] Figure 8 The effect of 20 μM atractylodesin on PEDV internalization;

[0022] Figure 9 The effect of 20 μM atractylodesin on PEDV release. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the applicant will now describe the invention in detail with reference to the embodiments, but these descriptions should not be construed as limiting the scope of protection claimed by this invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0025] In the following examples: the purity of atractylodesin was ≥98.5%; the PEDV was PEDV CV777 strain, which is the G1a subtype and came from Professor He Qigai's research group at Huazhong Agricultural University.

[0026] Example 1

[0027] Effects of atractylodesin on Vero cell morphology

[0028] Take Vero cells in the logarithmic growth phase, digest them with trypsin, and then divide them into groups of 5 × 10⁻⁶ cells per well. 5 (1 mL per well) cells were seeded into a 12-well plate and cultured for 24 h. The culture medium was then discarded and replaced with the following culture medium containing atractylodesin: atractylodesin was diluted to 0.1 μM, 1 μM, 10 μM, 100 μM and 200 μM with cell culture medium and treated for 24 and 48 hours respectively. Bright-field images were then taken using a microscope.

[0029] The cell culture medium was DMEM, containing 10% FBS and 1% penicillin-streptomycin antibiotics.

[0030] Figure 1 The results showed that when the concentration of atractylodesin was 200 μM and the treatment time was 48 h, atractylodesin had only a slight effect on the morphology of Vero cells, while other concentrations and treatment times had no significant effect on the morphology of Vero cells.

[0031] Example 2

[0032] Cytotoxicity assay of atractylodesin in Vero cells

[0033] Take Vero cells in the logarithmic growth phase, digest them with trypsin, and then divide them into doses of 1 × 10⁻⁶ per well. 5 100 μL of cells per well were seeded into 96-well plates and cultured for 24 h. The culture medium was then discarded and replaced with the following medium containing atractylodesin: atractylodesin was diluted to 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, and 200 μM using cell culture medium, and the cells were treated for 24 and 48 h, respectively. Cytotoxicity was detected using a CCK8 assay kit.

[0034] The cell culture medium was DMEM, containing 10% FBS and 1% penicillin-streptomycin antibiotics.

[0035] Figure 2 The results showed that atractylodesin did not have significant cytotoxic effects on Vero cells even at the highest concentration of 200 μM and the longest treatment time of 48 h.

[0036] Example 3

[0037] Atractylodesin inhibits PEDV replication.

[0038] Take Vero cells in the logarithmic growth phase, digest them with trypsin, and then divide them into groups of 5 × 10⁻⁶ cells per well. 5 1 mL of cells per well were seeded into a 12-well plate and cultured for 24 h. The culture medium was then discarded, and the cells were washed three times with PBS (0.01 M, pH 7.4, the same below, not repeated). The culture medium was then replaced with a virus maintenance medium containing different concentrations of atractylodesin without serum. The virus maintenance medium contained PEDV with an MOI of 0.01. After culturing for 24 h, the cells were collected, RNA was extracted, and the mRNA level of PEDV was detected by real-time PCR.

[0039] The virus maintenance solution was DMEM, containing 1 μg / mL porcine trypsin (Gibco 27250018 trypsin (1:250), the same below, not described in detail) and 1% penicillin-streptomycin double antibody.

[0040] Figure 3 The results showed that as the concentration of atractylodesin gradually increased from 10 μM to 100 μM, it had a significant inhibitory effect on PEDV.

[0041] Example 4

[0042] Take Vero cells in the logarithmic growth phase, digest them with trypsin, and then divide them into doses of 1 × 10⁻⁶ per well. 6(2 mL per well) cells were seeded into a 6-well plate and cultured for 24 h. The culture medium was discarded, and the cells were washed three times with PBS. The medium was then replaced with a virus maintenance medium containing different concentrations of atractylodesin without serum. The virus maintenance medium contained PEDV with an MOI of 0.01. After culturing for 24 h, the cells were collected, proteins were extracted, and the protein level of PEDV was detected by Western blot.

[0043] The virus maintenance solution was DMEM, containing 1 μg / mL porcine trypsin and 1% penicillin-streptomycin antibiotics.

[0044] Figure 4 The results showed that as the concentration of atractylodesin gradually increased from 10 μM to 100 μM, it significantly inhibited the expression level of PEDV N protein.

[0045] Example 5

[0046] Place sterile coverslips in the 24-well plate, at a density of 2.5 × 10⁻⁶ cm⁻¹ per well. 5 500 μL of cells per well were seeded into a 24-well plate and cultured for 24 h. The culture medium was discarded, and the cells were washed three times with PBS. The medium was then replaced with a virus maintenance medium containing different concentrations of atractylodesin without serum. The virus maintenance medium contained PEDV with an MOI of 0.01. After culturing for 24 h, the cells were collected, fixed on coverslips, and immunofluorescence staining was performed using anti-PEDV-N protein and anti-dsRNA antibodies. The cells were then photographed using a fluorescence microscope.

[0047] The virus maintenance solution was DMEM, containing 1 μg / mL porcine trypsin and 1% penicillin-streptomycin antibiotics.

[0048] Figure 5 , 6 The results showed that as the concentration of atractylodesin gradually increased from 10 μM to 50 μM, its inhibitory effect on the N protein and dsRNA of PEDV also increased in a dose-dependent manner.

[0049] Example 6

[0050] Take Vero cells in the logarithmic growth phase, digest them with trypsin, and then divide them into doses of 1 × 10⁻⁶ per well. 6 2 mL of cells per well were seeded into a 6-well plate and cultured for 24 h. The culture medium was discarded, and the cells were washed three times with PBS. The medium was then replaced with serum-free virus maintenance medium. The cells were pretreated with dimethyl sulfoxide solution containing 20 μM atractylodesin at 4 °C for 2 h. PEDV with an MOI of 0.01 was then added and allowed to adsorb at 4 °C for 1 h. RNA was then extracted, and the mRNA level of PEDV was detected by real-time PCR.

[0051] The virus maintenance solution was DMEM, containing 1 μg / mL porcine trypsin and 1% penicillin-streptomycin antibiotics.

[0052] Figure 7 The results showed that 20 μM atractylodesin did not affect the adhesion of PEDV.

[0053] Example 7

[0054] Take Vero cells in the logarithmic growth phase, digest them with trypsin, and then divide them into doses of 1 × 10⁻⁶ per well. 6 2 mL of cells per well were seeded into a 6-well plate and cultured for 24 h. The culture medium was discarded, and the cells were washed three times with PBS. The medium was then replaced with serum-free virus maintenance medium. The cells were pretreated with dimethyl sulfoxide solution containing 20 μM atractylodesin at 4 °C for 2 h. PEDV with an MOI of 0.1 was then added and allowed to adsorb at 4 °C for 1 h. The cells were then cultured in a cell culture incubator for 6 h. RNA was extracted and the mRNA level of PEDV was detected by real-time PCR.

[0055] The virus maintenance solution was DMEM, containing 1 μg / mL porcine trypsin and 1% penicillin-streptomycin antibiotics.

[0056] Figure 8 The results showed that 20 μM atractylodes slightly inhibited the internalization of PEDV.

[0057] Example 8

[0058] Take Vero cells in the logarithmic growth phase, digest them with trypsin, and then divide them into doses of 1 × 10⁻⁶ per well. 6 (2 mL per well) cells were seeded into 6-well plates and cultured for 24 h. The culture medium was discarded, and the cells were washed three times with PBS. The medium was then replaced with serum-free virus maintenance medium containing PEDV with an MOI of 0.01 and incubated for 1 h, followed by three washes with PBS. The same procedure was repeated in the control group, where the virus maintenance medium was replaced with serum-free medium containing no atractylodes and no PEDV and incubated for 1 h, followed by three washes with PBS. Similarly, in the treatment group, the virus maintenance medium was replaced with serum-free medium containing 20 μM atractylodes and PEDV with an MOI of 0.01 and incubated for 24 h. The supernatant was then collected. TCID45 was then used to... 50 Methods for detecting viral titers.

[0059] The virus maintenance solution was DMEM, containing 1 μg / mL porcine trypsin and 1% penicillin-streptomycin antibiotics.

[0060] Figure 9 The results showed that 20 μM atractylodesin had the best effect in inhibiting PEDV release.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made 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. Use of atractylenolide in the preparation of a medicament for inhibiting the replication and / or release of porcine epidemic diarrhea virus (PEDV); The porcine epidemic diarrhea virus is of G1a subtype.

2. Use according to claim 1, characterized in that: The only active ingredient of the medicament is atractylenolide.

3. Use according to claim 1, characterized in that: The dosage form of the medicament is a trans-gastrointestinal administration dosage form or an injection.

4. Use according to claim 1, characterized in that: The medicament is used for pigs at various stages.

5. Use according to claim 4, characterized in that: The medicament is used for suckling piglets.

6. Use according to claim 5, characterized in that: The medicament is used for suckling piglets within 7 days of age.

7. Use according to any one of claims 1 to 5, characterized in that: 20 μM is the concentration of atractylenolide for inhibiting the porcine epidemic diarrhea virus.

8. Use according to any one of claims 1 to 5, characterized in that: Atractylenolide at a concentration of 200 μM has no cytotoxicity after 24 h of treatment.

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