Application of biochanin A in the preparation of drugs against porcine epidemic diarrhea virus infection

Abstract

The present invention discloses the use of biochanin A in the preparation of a drug for preventing porcine epidemic diarrhea virus infection. Pharmacological tests have shown that biochanin A can effectively inhibit the proliferation of porcine epidemic diarrhea virus in cells and piglets. It can be used as a pharmaceutical ingredient and added to adjuvants to formulate a preparation for preventing and treating diseases caused by porcine epidemic diarrhea virus infection.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to the application of biochanin A in drugs for resisting porcine epidemic diarrhea virus infection. Background Art

[0002] Porcine epidemic diarrhea (PED) is an infectious disease of piglets caused by infection with the porcine epidemic diarrhea virus (PEDV), resulting in acute diarrhea and vomiting, dehydration, and high mortality. PEDV is an encapsulated, single-stranded, positive-sense RNA virus with an approximately 28 kb RNA genome. The genome consists of a 5' end, a 5' cap, a 5' untranslated region (UTR), seven open reading frames (ORFs), a 3' UTR, and a polyadenylation (polyA) tail at the 3' end. The seven ORFs encode four structural proteins: S (spike), M (membrane), E (envelope), and N (nucleocapsid), as well as pp1a, pp1ab, and ORF3 proteins. PEDV primarily infects the small intestinal epithelial cells of pigs and can cause severe mucosal atrophy and malabsorption. The virus can infect pigs of all ages, but lactating piglets are most susceptible. Due to the immature development of their organs and tissues, the mortality rate in one-week-old piglets can reach 100%. PEDV is primarily transmitted through fecal-oral transmission. In newborn pigs, PEDV is excreted in the feces, causing acute viremia and severe atrophic enteritis in the jejunum and ileum.

[0003] Currently, the prevention and control of PED mainly relies on biosafety prevention and control measures and vaccination. However, due to the wide variety of PEDV genotypes and extremely rapid virus mutations, especially the high variability of the S protein, the main target of neutralizing antibodies, new mutant strains frequently appear, making the vaccine have a serious lag. Therefore, there is an urgent need to develop effective broad-spectrum anti-PEDV drugs for the treatment of PED, so as to make up for the losses caused when the vaccine cannot provide protection.

[0004] Biochanin A (BCA) is an isoflavone compound found in red clover, cabbage, alfalfa, and many other herbs. It can also be extracted from various natural plants, including rosewood leaves, dalbergia odorifera leaves, and chickpeas. Recent research on biochanin A has focused on its antioxidant and anti-inflammatory properties. Some studies have also shown that biochanin A can improve insulin sensitivity and control hyperglycemia in patients with type 2 diabetes. However, there are no reports on the anti-poisonous diarrhea virus (PEDV) activity of biochanin A and its application in preventing and treating PEDV infection. Summary of the Invention

[0005] The present invention aims to solve at least one of the problems in the related art to a certain extent. To this end, the present invention aims to provide a use of biochanin A in the preparation of a drug for resisting porcine epidemic diarrhea virus infection.

[0006] Biochanin A is obtained by the following method: (1) crushing chickpea seeds to 50-100 mesh, mixing with acid water of pH 4-6, adding biological enzymes and keeping them at 35-40°C for enzymolysis to obtain an enzymolysis material. (2) adding ethanol to the enzymolysis material for hot reflux extraction, extracting 2-3 times, recovering ethanol from the extract to obtain a crude extract. (3) putting the crude extract into a supercritical CO2 extraction reactor, injecting an entrainer, passing CO2, and extracting at an extraction pressure of 20-55MPa and a temperature of 30-55°C. After 1-2 hours of extraction, collecting the extract, and evaporating the solvent to obtain a crude extract. (4) The crude extract obtained by extraction was dissolved in anhydrous ethanol, and then mixed with 100-200 mesh polyamide. The ethanol was evaporated to obtain a sample, which was loaded onto a pretreated polyamide column for chromatography, and gradient eluted with petroleum ether-ethyl acetate (10:1, 9:1, 4:1). TLC detection was performed, and the biochanin A fraction was collected, concentrated and crystallized, and then recrystallized with acetone to obtain a white crystalline powder, namely, biochanin A.

[0007] The drug in this application also contains pharmaceutically acceptable excipients; the dosage form of the drug is tablets, capsules, granules, powders, syrups, oral solutions or injections.

[0008] In the present application, biochanin A has antiviral activity against porcine epidemic diarrhea virus.

[0009] In the present application, biochanin A can inhibit the synthesis of N protein in porcine epidemic diarrhea virus, and the concentration of biochanin A used to reduce the expression amount of N protein in porcine epidemic diarrhea virus is 7.5-30 μM.

[0010] In the present application, biochanin A can inhibit the transcription of RNA in porcine epidemic diarrhea virus.

[0011] In the present application, the concentration of biochanin A that inhibits RNA transcription in porcine epidemic diarrhea virus is 7.5-30 μM.

[0012] In the present application, biochanin A can inhibit the replication of progeny viruses of porcine epidemic diarrhea virus.

[0013] In the present application, the concentration of biochanin A that inhibits the replication of progeny viruses in porcine epidemic diarrhea virus is 7.5-30 μM.

[0014] In the present application, biochanin A has an inhibitory effect on the replication of porcine epidemic diarrhea virus in piglets.

[0015] In the present application, porcine epidemic diarrhea virus (PEDV) infection can cause porcine epidemic diarrhea (PED) disease. In the present application, biochanin A is used to prepare a drug for resisting porcine epidemic diarrhea virus infection, but is not limited to the use of an effective amount of the compound of the present invention for preventing or treating diseases caused by porcine epidemic diarrhea virus, alleviating disease symptoms caused by porcine epidemic diarrhea virus, or delaying the development of diseases caused by porcine epidemic diarrhea virus.

[0016] The medicine described in this application also contains pharmaceutically acceptable excipients, which may be carriers, excipients, diluents, vehicles, etc.

[0017] The present application has the following beneficial effects: The present application has discovered a new use of biochanin A in inhibiting porcine epidemic diarrhea virus. On Vero cells, the half effective concentration (EC 50 ) value was 6.9 μM. At the same time, biochanin A can effectively reduce the viral titer of PEDV in Vero cells. At the highest dose of 30 μM, the virus inhibition rate reached more than 90% after 48 hours of administration. In addition, an 8 mg / kg dose of biochanin A was gavage-fed to newborn piglets artificially infected with PEDV, which significantly inhibited the replication of PEDV in the piglet digestive tract. This fully demonstrates that the biochanin A of the present application has a significant inhibitory effect on porcine epidemic diarrhea virus and has application value in the clinical treatment of PEDV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0020] In the attached figure:

[0021] Figure 1 The cytotoxic effect of biochanin A on Vero cells (CC 50 ) and its anti-PEDV activity on cells (EC 50 ) statistical chart;

[0022] Figure 2 This is an immunofluorescence image of the immunofluorescence analysis of the effect of different concentrations of biochanin A on reducing the expression of PEDV virus N protein in cells in Example 2;

[0023] Figure 3 This is a Western Blot analysis of the inhibitory effect of different concentrations of biochanin A on the synthesis of PEDV virus N protein in cells in Example 3;

[0024] Figure 4 This is a statistical graph of the relative mRNA expression corresponding to the inhibitory effect of different concentrations of biochanin A on PEDV viral RNA proliferation in cells analyzed by qRT-PCR in Example 4;

[0025] Figure 5 This is a statistical graph of viral titers corresponding to the inhibitory effects of different concentrations of biochanin A on PEDV progeny viruses in cells analyzed by the endpoint dilution method in Example 5.

[0026] Figure 6 This is a statistical graph of viral mRNA corresponding to the qRT-PCR analysis of the inhibitory effect of biochanin A on PEDV replication in piglets in Example 6. DETAILED DESCRIPTION

[0027] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention, without departing from the spirit and substance of the present invention, fall within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, and instruments used in the examples herein are commercially available; unless otherwise specified, all technical means in the examples herein are conventional means well known to those skilled in the art.

[0028] Example 1: Evaluation of the cytotoxic effect of biochanin A on Vero cells (CC 50 ) and its anti-PEDV activity on cells (EC 50 ).

[0029] The density is 1.5×10 5Vero cells were seeded at a concentration of 100 μL / well in a 96-well plate. After confluence and monolayer formation, the cells were washed twice with PBS and diluted two-fold in DMEM containing 2% FBS. Eight concentrations of biochanin A (BCA) were added to the treatment group, ranging from 1.87 to 240 μM, along with a solvent control group containing 4‰ DMSO and a blank control group (100 μL / well). After 48 hours of incubation at 37°C and 5% CO₂, the supernatant was discarded and 100 μL / well of 0.5 mg / mL MTT solution was added and incubated at 37°C in the dark. After 4 hours of incubation, the supernatant was discarded and 150 μL of DMSO was added to each well. The cells were shaken at low speed for 10 minutes to dissolve the formazan crystals. OD values ​​were measured at 570 nm using a full-wavelength microplate reader, and cell viability was calculated. GraphPad Prism 9.0 software was used to calculate the median cytotoxic concentration (CC) by nonlinear regression. 50 ).

[0030] The calculation formula is as follows:

[0031]

[0032] As above, after the Vero cells have grown to a monolayer, they are washed twice with PBS, and the compound is diluted twice with DMEM maintenance solution containing 2% FBS. A total of 8 BCA drug groups with a concentration gradient of 3.75-480 μM, a solvent control group containing 8‰ DMSO, and a blank control group are set, with 100 μL per well. The culture is terminated after 48 hours of culture in a constant temperature incubator at 37°C and 5% CO2. After observing the cell morphology, the cells are fixed with 4% paraformaldehyde for IFA detection, and observed and photographed using a fluorescent inverted microscope. The fluorescence intensity (blue fluorescence and red fluorescence) of each well is quantified using ImageJ software, and the DMSO-treated control group is set as 100%. The other groups are compared with the DMSO-treated group, and the half-effective concentration (EC50) is determined by quantifying the cell protection rate of the drug-treated group. 50 ) values ​​and calculated by nonlinear regression function using GraphPad Prism 9.0 software.

[0033] The calculation formula is as follows:

[0034]

[0035] The test results are as follows Figure 1 As shown, the drug biochanin A of the present invention has antiviral activity against PEDV virus, and its EC 50 The value is 6.9μM. After biochanin A acts on Vero cells for 48 hours, its cytotoxicity index CC 50 The value was 270.6 μM, indicating that biochanin A was low toxic to Vero cells.

[0036] Example 2: Immunofluorescence analysis of the inhibitory effect of different concentrations of biochanin A on the synthesis of PEDV virus N protein in cells.

[0037] Vero cells were plated in 96-well plates and washed twice with PBS until the monolayer was confluent. The virus was diluted to 100 TCID in DMEM containing 2% FBS. 50 A blank control group was set up, and cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. The cells were then washed twice with PBS to remove unbound viral particles. A blank control group, a solvent control group, and groups containing different concentrations of BCA were set up, with 100 μL per well. After 48 hours of culture, the supernatant was discarded, the culture was terminated, and the cells were fixed with 4% paraformaldehyde at room temperature, with 150 μL per well. After 15 minutes, the wells were gently washed three times with PBS, and 50 μL of 0.3% Triton-X100 solution was added to each well and incubated at room temperature. After 10 minutes, the wells were gently washed three times with PBS, and 100 μL of 2% BSA solution was added to each well and incubated at 37°C. After 1 hour, the wells were gently washed three times with PBS, and 50 μL of anti-PEDV-antibody (1:1000 dilution) was added to each well and incubated overnight at 4°C. After three 5-minute washes with PBS, 50 μL of AlexaFluor 568-labeled goat anti-mouse IgG (H+L) (ab175473) was added to each well and incubated at 37°C in the dark. After 1 hour, the wells were washed three times with PBS for 5 minutes each, and 100 μL of DAPI (300 nM) solution was added to each well in the dark and incubated at room temperature. After 5 minutes, the wells were washed three times with PBS for 5 minutes each, and the wells were observed and photographed using an inverted fluorescence microscope.

[0038] The test results are as follows Figure 2 As shown, the drug biochanin A of the present invention significantly reduced the expression level of PEDV N protein in Vero cells within the concentration range of 7.5-30 μM, and showed a good dose-effect relationship.

[0039] Example 3: Western Blot analysis of the inhibitory effect of different concentrations of biochanin A on the synthesis of PEDV virus N protein in cells.

[0040] Vero cells were seeded in a 6-well plate with 2 mL per well. After the cells grew to a monolayer, the following test was performed. The steps of infecting Vero cells with PEDV and adding drugs were the same as in Example 1. After incubation in the incubator for 48 hours, the culture was terminated, the supernatant was discarded, and the cells were washed twice with PBS. The cell culture plate was placed on ice, 120 μL / well of RIPA lysis buffer was added, and the liquid was aspirated into a centrifuge tube after repeated pipetting. Centrifugation was performed at 13,000 rpm for 20 minutes, and the supernatant was aspirated into another clean tube for later use. After determining the protein concentration of each sample by the BCA method, the bands of PEDV N protein and internal reference protein GAPDH were detected by Western Blot.

[0041] The test results are as follows Figure 3 As shown, the drug biochanin A of the present invention has a significant inhibitory effect on the synthesis of PEDV N protein in Vero cells within the concentration range of 7.5-30 μM, and shows a good dose-effect relationship.

[0042] Example 4: qRT-PCR analysis of the inhibitory effect of different concentrations of biochanin A on the proliferation of PEDV viral RNA in cells.

[0043] The steps of infecting Vero cells with PEDV and adding drugs were the same as in Example 1. After incubation in the incubator for 48 hours, the culture was terminated. After observing the cell morphology, the cell plate was repeatedly frozen and thawed three times at -80°C and 4°C to fully lyse the cells, causing all the virus in the cells to be released into the cell supernatant. The supernatant of each well was then collected. The collected cell supernatant was subjected to total RNA extraction using the recommended operating method of the Total RNA Rapid Extraction Kit (Shanghai Feijie Biotechnology Co., Ltd.). After RNA extraction, reverse transcription was immediately performed. Real-time PCR was used to detect the copy number of the PEDV N gene using cDNA as a template and β-Actin as an internal reference gene. The changes in N mRNA were evaluated using the virus control group as a reference.

[0044] PEDV N gene upstream and downstream primer sequences:

[0045] PEDV-NF: 5'-CGCAAAGACTGAACCCACTAATTT-3'

[0046] PEDV-NR: 5'-TTGCCTCTGTTGTTACTTGGAGAT-3'

[0047] β-Actin gene upstream and downstream primer sequences:

[0048] β-Actin-F: 5'-GGACTTCGAGCAGGAGATGG-3'

[0049] β-Actin-R: 5'-AGGAAGGAGGGCTGGAAGAG-3'

[0050] The test results are as follows Figure 4 As shown, the Mock group in the horizontal axis represents the blank control group, the DMSO group represents the virus-infected control group, and the BCA group represents the biochanin A-treated group; the vertical axis represents the relative expression of NmRNA in PEDV. In the figure, compared with the virus-infected control group, *, P<0.05; **, P<0.01; ***, P<0.001, indicate significant differences. The experimental results are shown in Figure 4 As shown, the drug biochanin A of the present invention has a significant inhibitory effect on PEDV RNA transcription in Vero cells within a concentration range of 7.5-30 μM, and exhibits a good dose-effect relationship. At the highest dose of 30 μM, the viral inhibition rate reached 100% after 48 hours of administration.

[0051] Example 5: The endpoint dilution method was used to analyze the inhibitory effect of different concentrations of biochanin A on PEDV progeny viruses in cells.

[0052] Vero cells were plated in 24-well plates and washed twice with PBS until the monolayer was fully grown. A blank control group, a solvent control group, and groups containing different concentrations of BCA drugs were set up. Except for the blank control group, which was added with a maintenance solution containing 2% FBS, 100 TCID diluted in the maintenance solution was added to the other treatment wells. 50 100 μL of PEDV virus solution was added to each well and incubated in a 37°C, 5% CO2 incubator. After 2 hours, the cells were washed twice with PBS to remove unbound virions. The drug-treated groups were treated with the corresponding compound diluted twofold in DMEM with 2% FBS. The blank and solvent controls were treated with fresh DMEM, with 500 μL added to each well. The cells were then incubated in the incubator. After 48 hours, the culture was terminated and the cell plates were frozen and thawed three times at -80°C and 4°C to fully release the virus into the supernatant. 120 μL of the freeze-thaw solution was collected per tube, and 3-4 replicates were collected for subsequent experiments.

[0053] The density is 1.5×10 5 Vero cells were seeded into a 96-well plate at 100 μL per well. After confluent monolayers were grown, the cells were washed twice with PBS. The sample solution was diluted 10-fold in DMEM and added to the plate at 100 μL per well, with replicates of 4 wells. After 48 hours of incubation, the cells were observed under an inverted microscope and the cytopathic effect was recorded. Wells showing CPE were designated as positive.

[0054] The test results are as follows Figure 5As shown in the figure, the Mock group in the horizontal axis represents the blank control group, the DMSO group represents the virus-infected control group, and the BCA group represents the biochanin A-treated group; the vertical axis represents the virus titer. In the figure, compared with the virus-infected control group, *, P < 0.05; **, P < 0.01; ***, P < 0.001, indicate significant differences. The experimental results are shown in the figure. Figure 5 As shown, the drug biochanin A of the present invention has a significant inhibitory effect on the replication of PEDV progeny viruses in Vero cells within the concentration range of 7.5-30 μM, and shows a good dose-effect relationship.

[0055] Example 6: qRT-PCR analysis of the inhibitory effect of biochanin A on PEDV replication in piglets

[0056] Nine three-day-old newborn piglets were randomly divided into three groups: a PEDV-challenged group, a chickpea sprout A-treated group, and a negative control group. Each piglet was housed in a separate cage and the experiment was conducted after 1 day of adaptation. The PEDV-challenged group and the chickpea sprout A-treated group were orally challenged with PEDV, while the negative control group was orally administered with PBS. When the piglets developed diarrhea, they were treated with 8.0 mg / kg of chickpea sprout A, administered orally twice daily for 4 consecutive days. After the challenge, fecal samples of the piglets in the experimental group were collected at fixed times every day and stored in a -80°C refrigerator. 0.1 g of fecal sample was weighed, 1 mL of PBS was added, mixed evenly, and centrifuged (12000 rpm, 10 min, 4°C). The fecal supernatant was collected and the viral mRNA level was detected using the qRT-PCR method.

[0057] The test results are as follows Figure 6 As shown, the vertical axis represents the logarithmic value of the initial copy number of RNA in PEDV, that is, PEDV RNA (log 10 The vertical axis represents the number of days after infection. The PBS group represents the negative control group, the DMSO group represents the virus-infected control group, and the BCA group represents the biochanin A-treated group. In the figure, *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with the virus-infected control group, indicate significant differences. Figure 6 As shown in the results, oral administration of biochanin A significantly reduced the PEDV viral load in the feces of infected piglets compared with the DMSO group, suggesting that biochanin A has an inhibitory effect on the replication of PEDV in piglets.

[0058] It is understandable that the above embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can be made, all of which fall within the scope of protection of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should fall within the scope of coverage of the claims of the present invention.

Claims

1. Use of biochanin A in the preparation of a drug for resisting porcine epidemic diarrhea virus infection; the concentration of the biochanin A is 7.5-30 μM.

2. The use of biochanin A according to claim 1 in the preparation of a drug for resisting porcine epidemic diarrhea virus infection, characterized in that: The medicine further contains pharmaceutically acceptable excipients; the dosage form of the medicine is tablets, capsules, granules, powders, syrups, oral solutions or injections.

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