Application of Paeonia lactiflora extracts in the preparation of antiviral active ingredients for swine viruses

Abstract

This invention belongs to the field of biomedicine, specifically relating to the application of stilbene extract (SEP) in the preparation of antiviral active ingredients against porcine viruses. This invention extracts SEP from peony seeds or pod shells, which significantly inhibits the replication of porcine PRV virus in porcine kidney PK15 cells. Its effective concentration is 0.0078-5 μg / mL, the cellular half-lethal dose is 15.81 μg / mL, and the half-inhibitory dose against porcine PRV virus is 0.164 μg / mL; furthermore, 0.625 μg / mL SEP resulted in a cell viability of 84.7% and a virus inhibition rate of 90.4%. SEP exhibits better antiviral efficacy, a lower effective dose, and a higher safety dose against porcine viruses than resveratrol, paclitaxel, or a mixture of resveratrol and paclitaxel, showing promising application prospects in the development of feed, veterinary drugs, and pharmaceuticals for combating porcine viruses.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of stillbens extracted from peony (SEP) in the preparation of anti-swine virus active ingredients. Background Technology

[0002] Porcine pseudorabies is an acute infectious disease of pigs caused by the porcine pseudorabies virus (PRV). This virus can infect a variety of mammals and is prevalent globally. Pigs are considered the only reservoir host and primary source of infection for PRV. It can cause abortion and stillbirth in pregnant sows, infertility in boars, high mortality rates in newborn piglets, and respiratory distress and growth retardation in finishing pigs. Therefore, it has a significant impact on the pig industry and is one of the major infectious diseases threatening the global pig industry.

[0003] PRV belongs to the Herpesviridae family, Alphaherpesvirinae subfamily, and is a linear DNA double-stranded virus. The virus particle is round or oval, consisting of a core, an icosahedral nucleocapsid, and an envelope. The envelope surface has radially arranged spikes approximately 8–10 nanometers long. The PRV genome is approximately 150 kb in size, encoding 70–100 viral proteins, mainly including capsid proteins, envelope glycoproteins, and various enzymes. PRV has only one serotype, but virulence varies among different strains. More effective drugs need to be developed based on prevalent PRV variants circulating in China.

[0004] my country boasts abundant traditional Chinese medicine resources, and its veterinary medicine products are characterized by their naturalness, multifunctionality (e.g., antipathogenic, regulatory, anti-stress, antioxidant, immune-enhancing, and nutritional effects), low toxicity and side effects, no drug residues, low likelihood of inducing drug resistance, no pollution to the ecological environment, safety, and ease of use. Currently, the research and development of veterinary medicine products and their application in veterinary clinical practice and livestock production have become a hot topic in the industry. The market size of veterinary medicine has rapidly increased from 3.684 billion yuan in 2018 to 6.126 billion yuan in 2021, with a compound annual growth rate of 18%, indicating strong market demand and broad application prospects.

[0005] In existing research on PRV, Zhao et al. (2016) found that resveratrol, a stilbene compound extracted from grapes, can inhibit PRV proliferation by inhibiting the NF-κB signaling pathway. Yang et al. (2020) used curcumin in vivo experiments in mice and found that curcumin exhibits neuroprotective effects against PRV infection by upregulating the BDNF / TrkB pathway. Curcumin can also reduce PRV-induced nitric oxide synthase expression and inhibit mitochondrial apoptosis, thus mitigating neuronal oxidative damage and protecting neurons from the effects of apoptosis. Other studies have shown that flavonoids such as quercetin and epigallocatechin gallate also inhibit PRV infection by inhibiting PRV adsorption. In addition, polysaccharides and terpenoids also have inhibitory effects on PRV. For example, Isatis indigotica polysaccharide can inhibit early PRV proliferation and replication and can directly inactivate the PRV virus; the terpenoid compound gemimazone can also inhibit early viral infection but cannot inactivate the PRV virus. The above studies demonstrate that plant-derived natural compounds play an important role in antiviral research. These compounds exert different functions in the antiviral process, such as inhibiting viral adsorption, invasion, and replication, and regulating signaling pathways. This provides an important reference for research on the antiviral properties of natural products.

[0006] The antiviral effects of stilbene compounds have been reported, primarily focusing on resveratrol, a representative stilbene compound. Palamara et al. (2005) showed that resveratrol inhibits the nucleocytoplasmic transport of influenza virus ribonucleoproteins, thereby inhibiting the virus. Resveratrol can also inhibit human herpesvirus by suppressing transcription and translation, thus inhibiting viral lysis and replication (Leo et al., 2012; Yiu et al., 2010). It inhibits early replication of herpes simplex virus (Docherty et al., 1999) and suppresses the expression of tyrosine kinase-like enzymes in respiratory syncytial virus, thereby inhibiting viral replication (Xie et al., 2012). Resveratrol can inhibit viral DNA synthesis during HIV-1 reverse transcription (Clouser et al., 2012). It can also inhibit the synthesis of early proteins in varicella-zoster virus and enterovirus, thereby inhibiting viral proliferation (Docherty et al., 2006). Inhibits the replication of duck enteritis virus nucleic acid and the expression of capsid protein after infection (Xu et al., 2013).

[0007] Currently, there are no research reports on the antiviral function of peony-astragalus compounds against PRV. In-depth research on the antiviral function of SEP is of great value and significance for the development of veterinary drugs and pharmaceuticals. Summary of the Invention

[0008] In view of the shortcomings of the existing technology, the purpose of this invention is to provide the application of strychnine extract (SEP) in the preparation of anti-swine virus active ingredients, which inhibits the replication of swine virus and significantly reduces the death of virus-infected cells.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] This invention provides the application of peony stilbene compound extract (SEP) in the preparation of anti-swine virus active ingredients.

[0011] Furthermore, the application of stilbene compound extract (SEP) in the preparation of antiviral active ingredients, including pharmaceutical preparations, disinfectants and / or feed additives.

[0012] In this invention, the peony stilbene compound extract (SEP) exerts an antiviral effect by inhibiting the replication of porcine pseudorabies virus (PRV).

[0013] Furthermore, this invention provides the application of peony stilbene compound extract (SEP) in the preparation of drugs against swine rabies virus (PRV). Peony stilbene compound extract (SEP) is obtained by extraction, separation, and purification from peony seed shells or peony pod shells. SEP primarily reduces the cytotoxic effects of PRV by inhibiting PRV replication.

[0014] In some embodiments of the present invention, the porcine pseudorabies virus also includes a variant of the porcine pseudorabies virus.

[0015] Furthermore, the preparation method of SEP is as follows:

[0016] 1. Extraction of crude extract of peony stilbene compounds: Take the ground peony seed shell powder and add it to a 50% ethanol solution (50% refers to the volume percentage concentration, and ethanol solution refers to an aqueous ethanol solution, which will not be elaborated elsewhere) at a solid-liquid ratio of 1g:20mL-1g:35mL. Soak for 1-2 weeks, mix, and then sonicate at room temperature for 15-30 minutes to obtain a well-mixed extract. Filter and centrifuge the well-mixed extract, and take the supernatant to concentrate it under reduced pressure at 45-60℃ using a rotary evaporator to obtain a concentrated extract. Freeze-dry the concentrated extract under vacuum at -40℃ to obtain the crude extract of peony stilbene compounds. The centrifugation conditions are: 3000-5000r / min, centrifugation for 3-5 minutes.

[0017] 2. Pretreatment of macroporous resin:

[0018] The macroporous resin used for pretreatment and purification was loaded with ultrapure water and stirred. After elution with ethanol, it was washed with water, then soaked in hydrochloric acid and washed with water until neutral. Soaked in sodium hydroxide solution and washed with water until neutral. It was then eluted with anhydrous ethanol and washed with water until there was no alcohol odor, thus obtaining the pretreated macroporous resin for use.

[0019] Preferably, HPD-100 macroporous resin is used. The resin is stirred and loaded with ultrapure water, then eluted with ethanol, washed with water, soaked in 4.5% hydrochloric acid for 3-4 hours, washed with water until neutral, soaked in 4.5% sodium hydroxide solution for 3-4 hours, washed with water until neutral, then eluted with 95% ethanol, washed with water until no alcohol odor is detected, and then processed for use.

[0020] 3. Wet packing of macroporous resins:

[0021] A wet packing method was used. The resin was poured into distilled water, allowed to stand, and the foam was removed. The resin was then slowly poured into the glass chromatography column to allow the resin particles to settle evenly. Excess water was drained from the bottom, ensuring the water level was at least 3 cm above the resin surface. After packing, the column volume (BV) was calculated to be 350 mL.

[0022] 4. Purification of crude extracts of peony-astragalus compounds:

[0023] The crude extract of peony stilbene compounds was dissolved in 60% ethanol at a solid-liquid ratio of 1g:3-10mL to obtain a stilbene compound loading solution. This solution was then slowly added along the column wall from the top, avoiding loosening the surface resin and causing it to float. The eluent was collected simultaneously, one tube per 100mL. The absorbance of the eluent was measured at 306nm using a UV spectrophotometer. Loading was stopped when the absorbance of the eluent was 1 / 10 of that of the stilbene compound loading solution. The eluent was then eluted sequentially with 3 BV of water three times, 3 BV of 30% ethanol once, 6 BV of 60% ethanol once, and finally 3 BV of 90% ethanol once. The eluent was collected during elution. The concentrated extract obtained after rotary evaporation of all collected ethanol solutions was then freeze-dried under vacuum for two days to obtain a dark brown solid with a certain metallic luster, which is the peony stilbene compound extract (SEP).

[0024] In some embodiments of the present invention, the active ingredient includes pharmaceutical preparations, disinfectants, and / or feed additives. Specifically, this includes: the use of SEP in the preparation of drugs for treating swine pseudorabies virus infection; and / or the use of SEP in the preparation of disinfectants for treating swine pseudorabies virus infection; and / or the use of SEP in the preparation of feed additives for treating swine pseudorabies virus infection.

[0025] In some preferred embodiments of the present invention, the pharmaceutical preparation further includes pharmaceutically acceptable salts and / or excipients.

[0026] Furthermore, pharmaceutically acceptable carriers refer to conventional drug carriers in the pharmaceutical field, such as: diluents, excipients like water, fillers like starch and sucrose; binders like cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; humectants like glycerin; disintegrants like agar, calcium carbonate, and sodium bicarbonate; absorption enhancers like quaternary ammonium compounds; surfactants like hexadecyl alcohol; adsorbents like kaolin and soap clay; lubricants like talc, calcium stearate, magnesium stearate, and polyethylene glycol. Other excipients such as sweeteners and flavorings can also be added to the composition.

[0027] Furthermore, the pharmaceutical preparation may be formulated as needed into any pharmaceutically acceptable formulation, including tablets, capsules, granules, injections, pills, syrups, powders, or ointments.

[0028] In some embodiments of the present invention, the concentration of the SEP is 0.0078 μg / mL to 5 μg / mL.

[0029] In some embodiments of the present invention, the half-inhibitory dose of the SEP against porcine PRV virus is 0.164 μg / mL.

[0030] In some embodiments of the present invention, the cellular half-lethal dose of the SEP is 15.81 μg / mL.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] This invention discloses for the first time the application of SEP in the preparation of active ingredients against porcine PRV virus. In vitro antiviral cell experiments confirmed that the alcohol-soluble SEP had a cell half-lethal dose of 15.81 μg / mL and a half-inhibitory dose of 0.164 μg / mL against porcine PRV virus. In cell culture medium containing 0.625 μg / mL SEP, the cell viability reached 84.7%, but the virus inhibition rate reached 90.4% compared to the control group. This invention compared the inhibitory effects of resveratrol and picaridin on the virus. Under the same experimental conditions, the half-inhibitory dose (WIC) of resveratrol was 5.305 μg / mL, and that of picaridin was 22.25 μg / mL. SEP showed better antiviral efficacy and lower dosage compared to resveratrol and picaridin alone. Furthermore, SEP showed better antiviral efficacy and lower dosage compared to a mixture of resveratrol and picaridin. By analyzing the characteristics of different stages of viral infection in cells after adding SEP, this invention determined that the main mechanism of action of SEP is to inhibit viral replication. Attached Figure Description

[0033] Figure 1This study investigates the cytotoxicity of SEP and its effect on PK15 cells infected with PRV-EGFP recombinant virus. Figures A and B show the cytotoxicity results of different concentrations of SEP and the control group on PK15 cells. Figure C shows the fluorescence of the viral fusion GFP protein in SEP-treated and control groups. Figures D and E show the fluorescence intensity of the viral fusion GFP protein at different SEP concentrations. Figure F shows the expression level analysis of the viral IE180 gene in PRV-EGFP-infected PK15 cells using 0.625 μg / mL SEP. Figure G shows the Western blot detection of the viral fusion gene protein GFP in PRV-EGFP-infected PK15 cells using 0.625 μg / mL SEP.

[0034] Figure 2 This experiment illustrates the effect of SEP on the infection of PK15 cells by PRV-EGFP. Figure A shows the expression level of the viral IE180 gene in PRV-EGFP-infected PK15 cells by SEP during the viral adsorption phase; Figure B shows the Western blot analysis of the viral fusion gene protein GFP in PRV-EGFP-infected PK15 cells by SEP during the viral adsorption phase; Figure C shows the expression level of the viral IE180 gene in PRV-EGFP-infected PK15 cells by SEP during the viral invasion phase; Figure D shows the Western blot analysis of the viral fusion gene protein GFP in PRV-EGFP-infected PK15 cells by SEP during the viral invasion phase; Figure E shows the expression level of the viral IE180 gene in PRV-EGFP-infected PK15 cells by SEP during the viral replication phase; and Figure F shows the Western blot analysis of the viral fusion gene protein GFP in PRV-EGFP-infected PK15 cells by SEP during the viral replication phase.

[0035] Figure 3 The graph shows the detection effect of resveratrol and paclitaxel on inhibiting PRV virus proliferation in cells; where A represents the fluorescence intensity of the viral fusion GFP protein at different resveratrol concentrations; and B represents the fluorescence intensity of the viral fusion GFP protein at different paclitaxel concentrations.

[0036] Figure 4 shows the detection results of resveratrol and paclitaxel in SEP, where 4A: paclitaxel standard curve, 4B: resveratrol standard curve, 4C: raw values ​​from the paclitaxel standard curve, 4D: raw values ​​from the resveratrol standard curve, 4E: liquid chromatogram of the blank sample, 4F: liquid chromatogram of the blank sample, 4G: liquid chromatogram of replicate 1 of the SEP sample, 4H: liquid chromatogram of replicate 1 of the SEP sample, 4I: liquid chromatogram of replicate 2 of the SEP sample, and 4J: liquid chromatogram of replicate 2 of the SEP sample.

[0037] Figure 5 Figure A shows the cytotoxicity test results of different concentrations of RPM on PK15 cells. Figure 5 B represents the fluorescence intensity of the viral fusion GFP protein in PRV-EGFP-infected PK15 cells at different concentrations of RPM. Detailed Implementation

[0038] The technical solution described in this invention uses a PRV-EGFP recombinant virus derived from a publicly available research project by Professor Wang Aibing of Hunan Agricultural University (Homologous recombination technology generated recombinant pseudorabies virus expressing EGFP facilitates to evaluate its susceptibility to different cells and screen antiviral compounds https: / / doi.org / 10.1016 / j.rvsc.2022.02.005). This strain, based on the PRV virus, uses the pCMV-EGFP-pSV40 gene to replace the gI and gE groups of the PRV virus, and its growth kinetics are close to those of the wild-type strain (Tan et al., 2022). Unless otherwise specified, other methods are conventional in the field; reagents or materials, unless otherwise specified, are all from commercial sources.

[0039] Example 1: Extraction, separation and purification of SEP

[0040] Extraction of crude extracts of peony-astragalus compounds:

[0041] Take 100g of ground peony seed shell powder, add 50% ethanol solution at a solid-liquid ratio of 1g:30mL and soak for one week, then sonicate at room temperature for 30min to obtain a well mixed extract; filter and centrifuge the well mixed extract at 5000r / min for 5min, take the supernatant and concentrate it under reduced pressure at 45℃ using a rotary evaporator to obtain a concentrated extract; freeze-dry the concentrated extract under vacuum at -40℃, and the freeze-dried powder is the crude extract of peony stilbene compounds.

[0042] Pretreatment of macroporous resins:

[0043] The HPD-100 macroporous resin used for pretreatment and purification is prepared by stirring the macroporous adsorption resin with ultrapure water, then eluting with ethanol, washing with water, soaking in 4.5% hydrochloric acid for 3-4 hours, washing with water until neutral, soaking in 4.5% sodium hydroxide for 3-4 hours, washing with water until neutral, eluting with 95% ethanol, washing with water until no alcohol odor is detected, and then processing for later use.

[0044] Wet packing of macroporous resins:

[0045] A wet packing method was used. The resin was poured into distilled water, allowed to stand, and the foam was removed. The resin was then slowly poured into the glass chromatography column to allow the resin particles to settle evenly. Excess water was drained from the bottom, ensuring the water level was at least 3 cm above the resin surface. After packing, the column volume (BV) was calculated to be 350 mL.

[0046] Purification of crude extracts of peony-astragalus compounds:

[0047] The crude extract of stilbene compounds from peony was dissolved in 60% ethanol at a solid-liquid ratio of 1g:3mL to obtain a stilbene compound loading solution. This solution was then slowly added along the column wall from the top, avoiding loosening the surface resin and causing it to float. The eluent was collected simultaneously, one tube per 100mL. The absorbance of the eluent was measured at 306nm using a UV spectrophotometer; loading was stopped when the absorbance of the eluent was 1 / 10 of the absorbance of the stilbene compound loading solution. The mixture was then eluted sequentially with 3 BV of water three times, 3 BV of 30% ethanol once, 6 BV of 60% ethanol once, and finally 3 BV of 90% ethanol once. The eluent was collected during elution, and all ethanol eluent was collected. The collected eluent was rotary evaporated, and the resulting concentrated extract was freeze-dried under vacuum for two days to obtain total stilbene compounds from peony seed shells (abbreviated as SEP, used in the following examples).

[0048] Example 2: SEP cytotoxicity detection

[0049] PK15 cells (8000 cells / well) were seeded in 96-well plates and cultured for 24 h. The original medium was replaced with 2% DMEM, and eight different concentrations of SEP were added to achieve final SEP concentrations of 0.078 μg / mL, 0.156 μg / mL, 0.3125 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL. Anhydrous ethanol was used as a control (SEP replaced with anhydrous ethanol), and a blank control (no SEP, no PRV-EGFP added) was also included. Four biological replicates were performed for each concentration. After 24 h of culture, the medium was aspirated, and the cells were washed twice with PBS. Then, 100 μL of SEP was added to each well. The reagent (Meilun Biotechnology, catalog number: PWL111) was used and allowed to stand for 5 minutes before chemiluminescence detection and data processing were performed.

[0050] Example 3: Detection of the effect of SEP on inhibiting viral proliferation in cells

[0051] PK15 cells (15,000 cells / well) were seeded in 96-well plates and cultured for 24 h. The original medium was replaced with 2% DMEM medium, and eight different concentrations of SEP were added to make the final concentrations of SEP in the medium 0.078 μg / mL, 0.156 μg / mL, 0.3125 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL, respectively. An ethanol control group (SEP replaced with anhydrous ethanol) and an untreated group (no SEP, no PRV-EGFP added) were set up as controls. Each concentration and the two controls were set up in triplicate. After 2 hours of incubation, each well in the experimental group was replaced with 0.1% multiplicity of infection (MOI) PRV-EGFP recombinant virus and SEP and 2% DMEM medium at the corresponding eight reaction concentrations, respectively. In the ethanol control group, each well was replaced with 0.1% MOI PRV-EGFP recombinant virus, anhydrous ethanol, and 2% DMEM medium, respectively. In the untreated group, no SEP or PRV-EGFP was added, and only 2% DMEM medium was used to replace the original medium. After 24 hours of incubation, the plates were scanned and photographed using a fluorescence microscope, and the images were processed in batches using ImageJ software.

[0052] Example 4: Western blot detection of viral fusion GFP protein

[0053] PK15 cells (4 × 10⁶ cells) were seeded in 6-well plates. 5 Cells were cultured at 100 cells / well until they adhered and reached a density of approximately 90%. The original culture medium was discarded, and the cells were pretreated with 2 mL of 0.625 μg / mL cell maintenance medium for 2 hours. Then, PRV-EGFP recombinant virus solution with an MOI of 0.1 was added to infect the cells for 1 hour. The virus solution was discarded, and the cells were washed three times with PBS. After incubation for 24 hours, 2 mL of cell maintenance medium containing 0.625 μg / mL SEP was added, followed by protein extraction. Anhydrous ethanol was replaced with SEP as an ethanol control group, and an untreated group (no SEP, no PRV-EGFP) was set up. Western blot experiments were performed according to the following steps:

[0054] (1) Cell lysis

[0055] a. Add 300 μL of 2×SDS Loading Buffer to each well of a 6-well plate. After lysing for 2-3 min, use a pipette tip to remove the cells from the culture plate and transfer them to 1.5 mL EP tubes. This process should be performed in a fume hood.

[0056] b. Place the EP tube in a heating and constant temperature mixer and heat at 95°C for 10 minutes;

[0057] c. Place the heated sample in an ultrasonic homogenizer and ultrasonically homogenize for 10 minutes. Set the ultrasonic program to 30 seconds on, 30 seconds off, 40% power, and 10 minutes.

[0058] d. Store the processed protein samples in a -20°C freezer.

[0059] (2) SDS-PAGE gel electrophoresis

[0060] a. Preparation of separating and stacking gels: Fix the glass plate on the support and prepare a separating gel of appropriate concentration (10%) according to the size of the protein. The specific components are shown in Table 1.

[0061] Table 1 Separating Gel Formulation Table

[0062]

[0063] After preparing the separating gel according to the above formula and sample addition order, use a pipette to mix the separating gel evenly and add it at a uniform speed between the two glass plates. When the separating gel is about 1 / 3 of the way from the top of the glass plate, stop adding the sample. Then add isopropyl alcohol to the middle of the glass plates and flatten the separating gel. After the separating gel solidifies, gently tilt the glass plates and use a pipette to remove the isopropyl alcohol. Use filter paper to absorb any remaining isopropyl alcohol. Then prepare a 5% stacking gel according to the formula in Table 2:

[0064] Table 2. Formulation of Concentrated Gel

[0065]

[0066]

[0067] After preparing the stacking gel according to the above formula and sample loading order, add the stacking gel between the glass plates, insert the matching gel casting comb, and remove the comb after the gel solidifies. Then you can perform SDS-PAGE gel electrophoresis.

[0068] b. After assembling the mold, place the prepared gel into the electrophoresis tank, fill the electrophoresis tank with 1×Running Buffer, and then add a certain volume of sample into the well;

[0069] c. Cover the electrophoresis tank with the lid, then connect it to the electrophoresis tank power supply and set the program to run at 80V for 30 minutes and at 120V for 90 minutes.

[0070] (3) Transfer and sealing

[0071] a. After the transfer solution is prepared, it should be pre-cooled at 4°C;

[0072] b. Immerse the NC membrane, filter paper, and sponge in the transfer solution and pre-cool at 4°C;

[0073] c. After electrophoresis, remove the gel block and cut off the top layer of gel and any excess gel at the bottom and sides (avoid cutting the target protein);

[0074] d. Arrange the transfer apparatus in the following order: blackboard at the bottom, sponge, filter paper, glue block, NC membrane (nitrocellulose membrane), and whiteboard at the top. Carefully remove any air bubbles between the NC membrane and the glue block. Clamp the apparatus and install it onto the wet transfer apparatus. Fill the center of the transfer apparatus with transfer solution. The entire transfer apparatus should be immersed in an ice-water mixture to prevent the temperature from getting too high.

[0075] e. Connect the power supply and run at a constant voltage of 100V for 80 minutes to transfer the film;

[0076] f. Remove the transferred membrane, stain it with Ponceau S, and then wash off the Ponceau S dye with PBS-T or deionized water before blocking.

[0077] g. Soak the NC membrane in 5% skim milk at room temperature for 2 hours.

[0078] (4) Primary and secondary antibody incubation and exposure imaging

[0079] a. Wash the sealed NC membrane three times with PBS-T, 5 min each time;

[0080] b. Prepare the primary antibody (Recombinant Anti-betaActin antibody, Seville) using 1% BSA (bovine serum albumin) according to the instructions. Soak the NC membrane in the primary antibody and incubate at room temperature for 2 hours or overnight at 4°C.

[0081] c. Recover the primary antibody and wash the NC membrane three times with PBS-T, 5 min each time;

[0082] d. Prepare secondary antibody (HRP-labeled goat anti-rabbit IgG, Seville) with 1% BSA according to the instructions. Immerse the NC membrane in the secondary antibody and incubate at room temperature for 50 min–1 h. Recover the secondary antibody and wash the NC membrane three times.

[0083] e. Prepare the developing solution according to the ratio of solution A to solution B of 1:1, and expose it using a chemiluminescence imaging system.

[0084] Experimental results of Examples 1-4: The results of the relative activity assay of SEP on PK cells showed that the relative cell activity gradually decreased with increasing SEP concentration in the culture medium (0.078 μg / mL - 10 μg / mL). The calculated median lethal dose (LD50) of SEP on PK cells was 15.81 μg / mL. Figure 1 A, Figure 1 B); The fluorescence intensity of recombinant viral EGFP in PK cells was detected using the same concentration of SEP. The results showed that the fluorescence intensity of recombinant viral EGFP gradually decreased with increasing SEP concentration. Figure 1 D、 Figure 1 E) The half-maximal inhibitory concentration (HIC) of SEP against porcine PRV virus was calculated to be 0.164 μg / mL. Based on the above experimental results, the inhibitory effect of SEP on the virus was analyzed at a relatively safe SEP concentration (cell viability reached 84.7% in cell culture medium containing 0.625 μg / mL SEP). Figure 1 C Figure 1 E), the results showed that the inhibition effect of this concentration of SEP reached 90.4% compared with the control group. (The viral fluorescence virulence of the ethanol control group was 100%, the fluorescence intensity of the experimental group was 9.6%, and the inhibition rate reached 90.4%. The fluorescence intensity of the untreated group was 0%).

[0085] Example 5: qRT-PCR detection of the PRV virus IE180 gene

[0086] PK15 cells (4 × 10⁶ cells) were seeded in 6-well plates. 5 Cells were cultured at 100 cells / well until they adhered and reached a density of approximately 90%. The original culture medium was removed, and the cells were pretreated with 2 mL of 0.625 μg / mL cell maintenance medium for 2 hours. Then, PRV-EGFP recombinant virus solution with an MOI of 0.1 was added to infect the cells for 1 hour. The virus solution was discarded, and the cells were washed three times with PBS. After incubation with 2 mL of 0.625 μg / mL cell maintenance medium for 24 hours, protein was extracted. SEP was replaced with anhydrous ethanol as an ethanol control group, and an untreated group (no SEP, no PRV-EGFP) was also set up. The qRT-PCR experiment was performed according to the following steps:

[0087] (1) RNA extraction

[0088] a. Add 1 mL of TransZol Up (6-well plate) to each well and lyse cells at room temperature for 2-3 min;

[0089] b. Transfer the cell lysate to a 1.5 mL enzyme-free EP tube, add 200 μL of chloroform, vortex to mix for 30 s, and let stand at room temperature for 2-3 min;

[0090] c. Centrifuge at 12000g at 4℃ for 10min. At this time, the liquid in the EP tube will separate into three layers. Carefully aspirate the upper aqueous phase into another new EP tube.

[0091] d. Add 500 μL of isopropanol, gently invert to mix, and precipitate at room temperature for 10-30 min;

[0092] f. Centrifuge the EP tube at 12000g for 10 min at 4℃. A white feather-like precipitate will be visible at the bottom of the tube. Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) to each tube, mix by inverting, and centrifuge at 12000g for 10 min at 4℃. Repeat this step.

[0093] g. Centrifuge the EP tube at 12000g for 2 min at 4℃, remove the residual liquid in the tube with a pipette tip, invert the EP tube and dry it at room temperature for 5 min;

[0094] h. Add 50 μL LEPC water to each tube to dissolve the RNA precipitate. This step must be performed on ice.

[0095] (2) cDNA synthesis

[0096] a. Determination of RNA concentration: The RNA concentration of the extracted sample was detected using a spectrophotometer;

[0097] b. Calculate the sample volume based on the RNA concentration;

[0098] c. Prepare enzyme-free PCR tubes. According to the instructions of the reverse transcription kit (TransGold, catalog number: AU341-02), add 4 μL of 5× enzyme solution to each PCR tube. Uni All-in-One Super Mix for qPCR, 1 μL g DNA remover, sample (RNA sample), then add RNase-free water to bring the reaction volume to 20 μL;

[0099] d. After shaking and centrifuging the PCR tube, place it in a PCR instrument, set the PCR reaction program to 50℃ for 30 min and 85℃ for 5 s, and finally obtain the required cDNA. Store at 4℃.

[0100] e. RNA should be placed on ice during the experiment, and the extracted RNA sample should be stored at -80℃ after the experiment.

[0101] (3) Real-time PCR

[0102] Quantitative real-time PCR was performed according to the instructions of the 2×Q3 SYBR qPCR Master mix (Universal) kit (ToloBio, catalog number: 22204). The primers for the viral internal control gene were GAPDH-F / R, and the primers for the IE180 viral gene were IE180-F / R. The primer sequences are shown in Table 3. The PCR reaction system consisted of 5 μL qPCR Mix, 0.4 μL each of forward and reverse primers, 4.1 μL ddH2O, and 0.1 μL cDNA. The PCR program was: 95℃ pre-denaturation for 5 min; 95℃ for 10 s, 60℃ for 35 s, 40 cycles; through 2... -ΔΔCt The relative expression levels of the target gene were calculated, and the experimental results were analyzed for differential expression using GraphPad Prism 8.0.2.

[0103] Table 3 Primer list for quantitative real-time PCR

[0104]

[0105]

[0106] Example 6: Study on virus adsorption to cells

[0107] 1. Seed PK-15 cells (3 × 10⁶ cells / well) into 6-well plates. 5 (1 cell / well), and wait for it to adhere to the wall and grow to a density of about 90%.

[0108] 2. Remove the supernatant and divide the cells into three groups: 2 mL of cell maintenance medium containing 0.625 μg / mL LSP and 0.1 MOI of PRV-EGFP fusion virus was added to the experimental group cells; 2 mL of cell maintenance medium containing the same concentration of ethanol solvent and PRV-EGFP as the experimental group was added to the ethanol control group; and 2 mL of cell maintenance medium was added to the untreated group. The cells were then placed in a 4°C refrigerator for 1 hour to allow the virus to adsorb.

[0109] 3. Discard the supernatant, wash three times with PBS, add cell maintenance medium, and incubate at 37°C for 24 hours;

[0110] 4. The detection experiment of viral fusion GFP protein was carried out according to the Western blot method in Example 4, and the expression level of PRV viral gene was detected according to the RNA extraction and fluorescence quantification experimental methods in Example 5.

[0111] Example 7: Study on PRV virus invasion of cells

[0112] 1. Seed PK-15 cells (3 × 10⁶ cells / well) into 6-well plates. 5 (1 cell / well), and wait for it to adhere to the wall and grow to a density of about 90%;

[0113] 2. Remove the supernatant and divide the cells into three groups. Add 0.1 MOI of PRV-EGFP fusion virus to the cell wells of the experimental group and the ethanol control group. No treatment is performed on the untreated group. Place the cells in a 4°C refrigerator for virus adsorption for 1 hour.

[0114] 3. After removing the supernatant, all groups were washed three times with PBS. The experimental group was added with 2 mL of cell maintenance medium containing 0.625 μg / mL SEP, the ethanol control group was added with 2 mL of cell maintenance medium containing the same concentration of ethanol solvent and PRV-EGFP as the experimental group, and the untreated group was added with 2 mL of cell maintenance medium. The cells were incubated at 37°C for 2 h until the virus entered the cells. The supernatant was then discarded, the cells were washed three times with PBS, and cell maintenance medium was added and incubated for 24 h.

[0115] 4. The detection experiment of viral fusion GFP protein was carried out according to the Western blot method in Example 4, and the expression level of PRV viral gene was detected according to the RNA extraction and fluorescence quantification experimental methods in Example 5.

[0116] Example 8: Study on viral replication in cells

[0117] PK-15 cells (3 × 10⁶ cells / well) were seeded in 1.6-well plates. 5 (1 cell / well), and wait for it to adhere to the wall and grow to a density of about 90%;

[0118] 2. Remove the supernatant and divide the cells into three groups. Add 0.1 MOI of PRV-EGFP to the cell wells of the experimental group and the ethanol control group. No treatment is given to the blank group. Incubate at 37°C for 2 hours for virus invasion into the cells.

[0119] 3. Discard the supernatant, wash 3 times with PBS, add 2 mL of cell maintenance medium containing 0.625 μg / mL LSEP to the experimental group, add 2 mL of cell maintenance medium containing the same concentration of ethanol solvent as the experimental group to the ethanol control group, and add 2 mL of cell maintenance medium to the untreated group. Incubate at 37°C for 24 h.

[0120] 4. The detection experiment of viral fusion GFP protein was carried out according to the Western blot method in Example 4, and the expression level of PRV viral gene was detected according to the RNA extraction and fluorescence quantification experimental methods in Example 5.

[0121] Experimental results of Examples 5-8: The expression of the viral gene IE180 was detected by quantitative real-time fluorescence method. The results showed that in cell culture medium containing 0.625 μg / mL SEP, the relative expression level of IE180 was only 24% of that in the ethanol control group, and no expression of this gene was detected in the untreated group. Figure 1F). Western blot analysis was used to verify the content of the fusion virus tag protein GFP. This experiment further confirmed that in cell culture medium containing 0.625 μg / mL SEP, the GFP protein band was significantly weaker than that in the ethanol control group, while no band was detected in the untreated group. Figure 1 G).

[0122] Experiments on the effect of SEP on PRV virus adsorption in PK cells confirmed that the addition of SEP had no significant inhibitory effect on the relative expression level of IE180, and the expression of this gene was not detected in the untreated group. Figure 2 A), which was also confirmed in subsequent Western blot analysis, showed that the GFP protein content was not significantly different from that in the ethanol control group, while no band was detected in the untreated group. Figure 2 B). This indicates that SEP has no significant effect on the process by which PRV virus adsorbs onto PK cells.

[0123] Experiments on the effect of SEP on PRV virus invasion of PK cells confirmed that the addition of SEP had no significant inhibitory effect on the relative expression level of IE180, and the expression of this gene was not detected in the untreated group. Figure 2 C), Western blot analysis also showed that the content of GFP protein was not significantly different from that of the ethanol control group, and no band was detected in the untreated group. Figure 2 D). This indicates that SEP has no significant effect on the process of PRV virus invading PK cells.

[0124] Experiments demonstrating the effect of SEP on PRV virus replication in PK cells confirmed that, during the viral replication phase, the added SEP significantly inhibited the relative expression level of IE180, reducing it to only 5.5% of the relative expression level of IE180 in the ethanol-treated group. No expression of this gene was detected in the untreated group. Figure 2 E), Western blot analysis also showed that the GFP protein content in the SEP-treated group was significantly lower than that in the ethanol control group, and no band was detected in the untreated group. Figure 2 F). This indicates that SEP has a significant inhibitory effect on PRV virus replication in PK cells.

[0125] Example 9: Detection of the inhibitory effect of resveratrol (Res) on viral proliferation in cells

[0126] PK15 cells (15,000 cells / well) were seeded in 96-well plates and cultured for 24 h. The original medium was replaced with 2% DMEM, and eight different concentrations of Res were added to achieve final Res concentrations of 0.78 μg / mL, 1.56 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. An ethanol control group (using anhydrous ethanol) and a no-treatment group were also included. Each concentration and each of the two controls was performed in triplicate. After 2 h of culture, each well was treated with 0.1 MO IPRV-EGFP recombinant virus and either the corresponding eight Res concentrations, ethanol control, or no treatment (blank group). The original medium was replaced with 2% DMEM. After 24 h of culture, the plates were scanned using a fluorescence microscope, and the images were processed in batches using ImageJ software.

[0127] Example 10: Detection of the inhibitory effect of paclitaxel on viral proliferation in cells

[0128] PK15 cells (15,000 cells / well) were seeded in 96-well plates and cultured for 24 h. The original medium was replaced with 2% DMEM, and eight different concentrations of paclitaxel were added to achieve final SEP concentrations of 0.78 μg / mL, 1.56 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. An ethanol control group (using anhydrous ethanol) and a no-treatment group were also included. Each concentration and each control group had three biological replicates. After 2 h of culture, each well was treated with 0.1 MOI of PVRV-EGFP recombinant virus, and either the corresponding eight concentrations of paclitaxel or ethanol (control group) or no treatment (blank group). The original medium was replaced with 2% DMEM. After 24 h of culture, the plates were scanned using a fluorescence microscope, and the images were processed in batches using ImageJ software.

[0129] Experimental results of Examples 9 and 10: The experiments on the inhibitory effects of resveratrol and picaridin on PRV virus showed that, with the increase of resveratrol and picaridin concentrations, the relative fluorescence value of the recombinant virus EGFP gradually decreased. Figure 3 The calculated half-maximal inhibitory concentration (IC50) of resveratrol against PRV virus is 5.305 μg / L. Figure 3 A) The half-maximal inhibitory concentration (WMC) of paclitaxel against PRV virus is 22.25 μg / L. Figure 3 B).

[0130] Example 11: Quantitative determination of resveratrol and paclitaxel in SEP using liquid chromatography (LC-20AT, Shimadzu).

[0131] Preparation of standard curve:

[0132] Accurately weigh resveratrol (CAS No.: 501-36-0) and piperidine standard (CAS No.: 10083-24-6) and dissolve them completely. Prepare a 100 μg / mL mixed standard solution with methanol, and then prepare a series of standard solutions with final concentrations of 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL and 50 μg / mL with 75% ethanol.

[0133] SEP sample preparation: (1) Take about 10 mg of SEP sample and add 1 mL of 75% ethanol solution. (2) Shake for 30 s, sonicate for 10 min, filter through a 0.22 μm organic phase membrane, and perform instrument detection; set up 2 replicates.

[0134] Chromatographic detection conditions: Column: Athena-C18 (4.6mm×250mm, 5μm); Column temperature: 30℃; Mobile phase: Phase A: 0.2% phosphoric acid water; Phase B: acetonitrile; Flow rate: 1.0mL / min; Injection volume: 10μL; Cell temperature: 30℃; Detection wavelength: 320nm.

[0135] The elution process of the mobile phase is as follows: mobile phase A: mobile phase B = 72:28, column chromatography for 18 min; mobile phase A: mobile phase B = 10:90, column chromatography for 6 min; mobile phase A: mobile phase B = 72:28, column chromatography for 6 min; detection is completed, and a chromatogram is obtained.

[0136] Results of Example 11: Results obtained from the preparation of standard curves for resveratrol and piperidine. The R-value of the standard curves... 2 The values ​​are 0.9999322 and 0.9999510 respectively. The standard curve preparation results are excellent and can be used for subsequent detection of the corresponding products. Figure 4A (B, C, D). Liquid chromatography analysis of the SEP sample revealed the following results: the average of two replicates showed that the content of paclitaxel in the SEP was 32.705 μg / g; the content of resveratrol in the SEP was 678.56 μg / g. Figure 4G H, I, J).

[0137] Example 12: Effect of resveratrol + paclitaxel mixture (RPM) on PRV

[0138] PK15 cells (15,000 cells / well) were seeded in 96-well plates and cultured for 24 h. The original medium was replaced with 2% DMEM medium, and eight different concentrations of a mixture of resveratrol and paclitaxel (20.75:1, RPM) were added to achieve final RPM concentrations of 0.78 μg / mL, 1.56 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. An ethanol control group (using anhydrous ethanol) and an untreated group were set up as controls. Each concentration and the two controls were configured in triplicate. After 2 hours of culture, each well was treated with 0.1 MOI RV-EGFP recombinant virus and 8 corresponding concentrations of RPM or ethanol as controls or no treatment (blank group). At the same time, 2% DMEM medium was used to replace the original medium. After 24 hours of culture, the plate was scanned and photographed using a fluorescence microscope, and the images were processed in batches using ImageJ software.

[0139] Example 12 Experimental Results: The CC50 of RPM was 44.05 μg / mL ( Figure 5 A), the EC50 of RPM is 13.06 μg / mL ( Figure 5 B), its half-inhibitory concentration (WIC) for inhibiting the virus is between that of resveratrol and picaridin, indicating that the inhibitory effect of the mixture of resveratrol and picaridin in SEP is not even as strong as that of resveratrol alone. The half-inhibitory concentration of PRV virus in SEP is 0.165 μg / mL, indicating that the antiviral effect of SEP mainly comes from other components besides resveratrol and picaridin.

Claims

1. The application of peony-astragalus compound extracts in the preparation of antiviral active ingredients for swine viruses, characterized in that, The active ingredients include pharmaceutical preparations, disinfectants, and / or feed additives; The preparation method of the peony stilbene compound extract includes the following steps: 1) Extraction of crude extract of peony stilbene compounds: Take the ground peony seed shell powder, soak it in 50% ethanol solution, sonicate and mix it, filter and centrifuge, take the supernatant and concentrate it under reduced pressure to obtain concentrated extract, and freeze dry to obtain crude extract of peony stilbene compounds. 2) Pretreatment of macroporous resins: The macroporous resin used for pretreatment and purification was stirred with ultrapure water and loaded onto the sample. After elution with ethanol, it was washed with water, then soaked in hydrochloric acid and washed with water until neutral. Soaked in sodium hydroxide solution and washed with water until neutral. After elution with 95% ethanol and washing with water until there was no alcohol odor, the pretreated macroporous resin was obtained for use. 3) Wet packing of macroporous resins: Using the wet packing method, pour the pretreated macroporous resin from step 2) into distilled water, let it stand, remove the foam, and slowly pour it into the glass chromatography column to allow the resin particles to settle evenly. Remove excess water from the bottom and keep the water level at least 3 cm above the resin surface to complete the packing. 4) Purification of crude extracts of peony-astragalus compounds: The crude extract of stilbene compounds obtained in step 1) was dissolved in 60% ethanol at a solid-liquid ratio of 1g:3-10mL to obtain a stilbene compound loading solution. The stilbene compound loading solution was then slowly added from the top of the glass chromatography column along the column wall. The eluent was collected at the same time, and the absorbance of the eluent was detected at 306nm. The loading was stopped when the absorbance of the eluent was 1 / 10 of the absorbance of the stilbene compound loading solution. The mixture is eluted at least 6 times, and the eluent is collected during elution. The collected eluent is then rotary evaporated to obtain a concentrated extract, which is then freeze-dried under vacuum to obtain an extract of peony-astragalus compounds. The porcine virus includes porcine pseudorabies virus and / or mutated porcine pseudorabies virus.

2. The application according to claim 1, characterized in that, The concentration of the peony stilbene compound extract is 0.078 μg / mL to 10 μg / mL.

3. The application according to claim 1, characterized in that, The half-inhibitory dose of the peony-astragalus compound extract against porcine PRV virus is 0.164 μg / mL.

4. The application according to claim 1, characterized in that, The cellular half-lethal dose of the extract of peony stilbene compounds was 15.81 μg / mL.

5. The application according to claim 1, characterized in that, In step 1), take the ground peony seed shell powder, add it to 50% ethanol solution at a solid-liquid ratio of 1g:20mL-1g:35mL and soak for 1-2 weeks. After mixing, sonicate at room temperature for 15-30 minutes to obtain a well-mixed extract. Filter and centrifuge the well-mixed extract, take the supernatant and concentrate it under reduced pressure at 45-60℃ using a rotary evaporator to obtain a concentrated extract. Freeze-dry the concentrated extract under vacuum at -40℃ to obtain a crude extract of peony stilbene compounds. The centrifugation conditions are: 3000-5000r / min, centrifuge for 3-5min.

6. The application according to claim 1, characterized in that, In step 4), the elution process specifically involves eluting three times with 3BV of water, once with 3BV of 30% ethanol, once with 6BV of 60% ethanol, and once with 3BV of 90% ethanol.

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