Application of paeonia suffruticosa stilbene compound extract in preparation of anti-porcine virus active component

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of a peony stilbene compound extract (SEP) in preparation of an anti-porcine virus active component. According to the invention, SEP is extracted from peony seeds or pod shells, and can significantly inhibit the replication of porcine PRV virus in porcine kidney PK15 cells. The effective concentration is 0.0078-5 [mu] g / mL, the cell median lethal dose is 15.81 [mu] g / mL, and the anti-porcine PRV virus semi-inhibitor dose is 0.164 [mu] g / mL; and the cell survival rate reaches 84.7% and the virus inhibition rate reaches 90.4% due to 0.625 [mu] g / mL of SEP. Compared with resveratrol or piceatannol and a resveratrol and piceatannol mixture, the SEP has a better antiviral effect, a lower onset dose and a larger safe dose on porcine viruses, and has a very good application prospect in the fields of porcine virus-resistant feed development, veterinary drug, medicine development and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of Stilbene compounds extracted from peony (SEP) in the preparation of active ingredients against porcine viruses. Background Art

[0002] Porcine pseudorabies is an acute infectious disease of pigs caused by Porcine pseudorabies virus (PRV). This virus can infect a variety of mammals and is prevalent worldwide. Pigs are considered the only reservoir host and the main source of infection of PRV, which can cause abortion and stillbirth of pregnant sows, infertility of boars, massive death of newborn piglets, dyspnea and growth stagnation of fattening pigs, etc. Therefore, it has a great impact on the pig industry and is also one of the major infectious diseases endangering the global pig industry.

[0003] PRV belongs to the Herpesviridae family, the Alphaherpesvirinae subfamily, and is a linear double-stranded DNA virus. The virus particles are round or oval, composed of a core, an icosahedral capsid, and an envelope. There are radially arranged spikes about 8-10 nanometers long on the surface of the envelope. The PRV genome is about 150 kb in size and encodes 70-100 viral proteins, mainly including capsid proteins, envelope glycoproteins, and various enzymes. PRV has only one serotype, but there are differences in virulence among different strains. It is necessary to develop more effective drugs based on domestic PRV epidemic variants.

[0004] China is rich in traditional Chinese medicine resources. Traditional Chinese veterinary medicine products have the characteristics of naturalness, multi-functionality (such as anti-pathogen effects, regulatory effects, anti-stress effects, antioxidant effects, immune enhancement and nutritional effects, etc.), low toxicity and side effects, no drug residues, not easy to produce drug resistance, no pollution to the ecological environment, safe medication, and convenient use. At present, the research and development of traditional Chinese veterinary medicine products and their application in veterinary clinical practice and livestock production have become a hot topic in the industry. The market scale of traditional Chinese 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%. The market demand is large and the application prospect is broad.

[0005] During the existing anti-PRV research, Zhao et al. (2016) found that the stilbene compound resveratrol extracted from grapes can inhibit the proliferation of PRV by inhibiting the NF-κB signaling pathway. Yang et al. (2020) found in in vivo experiments on mice using curcumin that curcumin showed neuroprotective effects against PRV infection by upregulating the BDNF / TrkB pathway. At the same time, curcumin can also reduce the expression of PRV-induced nitric oxide synthase and inhibit the mitochondrial apoptosis pathway, reducing oxidative damage to neurons and protecting neurons from apoptosis. Another study showed that flavonoids such as quercetin and epigallocatechin gallate also inhibit virus infection by inhibiting the adsorption of PRV virus. In addition, polysaccharides and terpenoids also have the effect of inhibiting PRV virus. For example, isatis root polysaccharide can inhibit the early proliferation and replication of PRV and can directly inactivate PRV virus; the terpenoid germacrone can also inhibit the early infection of the virus but cannot inactivate PRV virus. The above studies show that plant natural compounds play an important role in antiviral research, and these compounds play different roles in the process of antiviral, such as inhibiting virus adsorption, invasion, replication, and regulating signaling pathways. This provides an important reference for the research on antiviral natural products.

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

[0007] At present, there is no research report on the anti-PRV of peony stilbene compounds. In-depth study of the antiviral function of SEP has important value for the development of veterinary drugs and medicines. Summary of the Invention

[0008] Aiming at the deficiencies existing in the prior art, the purpose of the present invention is to provide the application of paeonia qi compound extract (SEP) in the preparation of anti-swine virus active ingredients, inhibit the replication of swine virus, and significantly reduce the death of virus-infected cells.

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

[0010] The present invention provides the application of paeonia qi compound extract (SEP) in the preparation of anti-swine virus active ingredients.

[0011] Furthermore, the application of paeonia qi compound extract (SEP) in the preparation of anti-swine virus active ingredients, the active ingredients include pharmaceutical preparations, disinfectants and / or feed additives.

[0012] In the present invention, paeonia qi compound extract (SEP) plays an antiviral role by inhibiting the replication of porcine pseudorabies virus (PRV).

[0013] Furthermore, the present invention provides the application of paeonia qi compound extract (SEP) in the preparation of drugs against porcine rabies virus (PRV). The paeonia qi compound extract (SEP) is extracted, separated and purified from peony seed husks or peony fruit pod husks. SEP mainly reduces the cytotoxic effect of PRV by inhibiting the replication of PRV.

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

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

[0016] 1. Extraction of crude paeonia qi compound extract: Take the ground peony seed husk powder, add 50% ethanol solution (50% refers to the volume percentage concentration, and the ethanol solution refers to the ethanol aqueous solution, not elaborated elsewhere) according to the solid-liquid ratio of 1g:20mL - 1g:35mL, soak for 1 - 2 weeks, mix and ultrasonicate at room temperature for 15 - 30 min 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 °C using a rotary evaporator to obtain a concentrated extract; vacuum freeze-dry the concentrated extract at -40 °C to obtain the crude paeonia qi compound extract; the centrifugation conditions are: 3000 - 5000 r / min, centrifuge for 3 - 5 min.

[0017] 2. Pretreatment of macroporous resin:

[0018] Pretreat the macroporous resin used for purification in advance. Stir the macroporous resin with ultrapure water for loading, elute with ethanol and wash with water until clean, then soak with hydrochloric acid and wash to neutral, then soak with sodium hydroxide solution and wash to neutral, elute with anhydrous ethanol and wash until there is no alcohol smell to obtain the pretreated macroporous resin for standby;

[0019] Preferably, HPD-100 macroporous resin is used. The resin is stirred and loaded with ultrapure water, then eluted with ethanol, washed with water until clean, soaked in 4.5% hydrochloric acid for 3 - 4 h, washed with water until neutral, soaked in 4.5% sodium hydroxide solution for 3 - 4 h, washed with water until neutral, and then eluted with 95% ethanol, washed with water until there is no alcohol smell, and then it is ready for use after treatment.

[0020] 3. Wet packing of macroporous resin:

[0021] Adopt wet packing. Pour the resin into distilled water, let it stand and pour off the floating foam, then slowly pour it into a glass chromatography column to make the resin particles settle evenly, and drain the excess water from the bottom, keeping the water surface at least 3 cm above the resin surface. After the packing is completed, calculate the column volume (BV) of this resin column to be 350 mL.

[0022] 4. Purification of crude extract of paeonolide compounds:

[0023] Add the above-mentioned crude extract of paeonolide compounds to 60% ethanol to dissolve according to the solid-liquid ratio of 1 g: 3 - 10 mL to obtain the paeonolide compound loading solution, and then slowly add the paeonolide compound loading solution along the tube wall from the upper part of the column, avoiding loosening the surface resin and making it float. At the same time, collect the effluent, collect 1 tube for every 100 mL. When using an ultraviolet spectrophotometer, detect the absorbance value of the effluent at 306 nm; when the absorbance value of the effluent is 1 / 10 of the absorbance value of the paeonolide compound loading solution, stop loading; then elute 3 times with 3 BV of water, elute 1 time with 3 BV of 30% ethanol, then elute 1 time with 6 BV of 60% ethanol, and finally elute 1 time with 3 BV of 90% ethanol. Collect the eluate while eluting, and vacuum freeze-dry the concentrated extract obtained by rotary evaporation of all the collected ethanol solution eluate for two days to obtain a dark brown solid with a certain metallic luster, which is the extract of paeonolide compounds (SEP);

[0024] In some embodiments of the present invention, the active ingredient includes a pharmaceutical preparation, a disinfectant and / or a feed additive. That is, it includes: the application of SEP in the preparation of a drug for preventing porcine pseudorabies virus infection; and / or the application of SEP in the preparation of a disinfectant for preventing porcine pseudorabies virus infection; and / or the application of SEP in the preparation of a feed additive for preventing porcine pseudorabies virus infection.

[0025] In some preferred embodiments of the present invention, the pharmaceutical preparation further includes a pharmaceutically acceptable salt and / or an excipient.

[0026] Further, a pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as diluents, excipients like water, fillers like starch, sucrose, etc.; binders like cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; wetting agents like glycerol; disintegrants like agar, calcium carbonate, and sodium bicarbonate; absorption promoters like quaternary ammonium compounds; surfactants like cetyl alcohol; adsorption carriers like kaolin and bentonite; lubricants like talc, calcium stearate, magnesium stearate, and polyethylene glycol, etc. Additionally, other adjuvants such as sweeteners, flavoring agents, etc. can be added to the composition.

[0027] Further, the pharmaceutical preparation is made into any pharmaceutically acceptable preparation as needed, 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 - 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 half-lethal dose of the SEP to cells is: 15.81 μg / mL.

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

[0032] The present invention discloses for the first time the application of SEP in the preparation of an active ingredient against porcine PRV virus. It is confirmed by in vitro antiviral cell experiments that the half-lethal dose of alcohol-soluble SEP to cells is: 15.81 μg / mL, and the half-inhibitory dose against porcine PRV virus is: 0.164 μg / mL. In the cell culture medium containing 0.625 μg / mL SEP, the cell survival rate reaches 84.7%, but the virus inhibition rate compared with the control group reaches 90.4%. The present invention also compares the inhibitory effects of resveratrol and piceatannol on this virus. Under the same experimental conditions, the half-inhibitory dose of resveratrol is 5.305 μg / mL, and the half-inhibitory dose of piceatannol is 22.25 μg / mL. SEP has a better antiviral effect and a lower dosage compared with resveratrol and piceatannol, and SEP has a better antiviral effect and a lower dosage compared with the mixture of resveratrol + piceatannol; by analyzing the characteristics of different stages of virus-infected cells after adding SEP, the present invention determines that the main mechanism of action of SEP is to inhibit virus replication. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1Detection of the cytotoxicity of SEP and its effect on PK15 cells infected with PRV-EGFP recombinant virus; among them, A and B are the detection result graphs of the cytotoxicity of different concentrations of SEP and the control group on PK15 cells; C is the fluorescence graph of the virus-fused GFP protein in the SEP treatment group and the control group; D and E are the fluorescence intensities of the virus-fused GFP protein at different SEP concentrations; F is the analysis graph of the expression level of the viral IE180 gene of PRV-EGFP-infected PK15 cells treated with 0.625 μg / mL SEP; G is the Western blot detection graph of the virus-fused gene protein GFP of PRV-EGFP-infected PK15 cells treated with 0.625 μg / mL SEP.

[0034] Figure 2 Experiment on the effect of SEP on the process of PRV-EGFP infecting PK15 cells; among them, A is the analysis graph of the expression level of the viral IE180 gene of PRV-EGFP-infected PK15 cells in the virus adsorption stage by SEP; B is the Western blot detection graph of the virus-fused gene protein GFP of PRV-EGFP-infected PK15 cells in the virus adsorption stage by SEP; C is the analysis graph of the expression level of the viral IE180 gene of PRV-EGFP-infected PK15 cells in the virus penetration stage by SEP; D is the Western blot detection graph of the virus-fused gene protein GFP of PRV-EGFP-infected PK15 cells in the virus penetration stage by SEP; E is the analysis graph of the expression level of the viral IE180 gene of PRV-EGFP-infected PK15 cells in the virus replication stage by SEP; F is the Western blot detection graph of the virus-fused gene protein GFP of PRV-EGFP-infected PK15 cells in the virus replication stage by SEP.

[0035] Figure 3 Detection graph of the inhibitory effect of resveratrol and piceatannol on the proliferation of PRV virus in cells; among them, A is the fluorescence intensity of the virus-fused GFP protein at different resveratrol concentrations; B is the fluorescence intensity of the virus-fused GFP protein at different piceatannol concentrations.

[0036] Figure 4 is the detection result graph of the contents of resveratrol and piceatannol in SEP. Among them, 4A: standard curve graph of piceatannol, 4B: standard curve graph of resveratrol, 4C: original numerical values for making the standard curve of piceatannol, 4D: original numerical values for making the standard curve of resveratrol, 4E: liquid chromatography graph of the blank sample, 4F: liquid phase numerical values of the blank sample, 4G: liquid chromatography graph of replicate 1 of the SEP sample, 4H: liquid phase numerical values of replicate 1 of the SEP sample, 4I: liquid chromatography graph of replicate 2 of the SEP sample, 4J: liquid phase numerical values of replicate 2 of the SEP sample.

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

[0038] The technical solution of the present invention, wherein the PRV-EGFP recombinant virus is derived from the publicly available project of Professor Aibing Wang 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). On the basis of the PRV virus, this strain uses the pCMV-EGFP-pSV40 gene to replace the gI and gE genes of the PRV virus, and its growth kinetics is close to that of the wild-type strain (Tan et al., 2022). Other than as specifically described, all are conventional protocols in the art; the reagents or materials, unless otherwise specifically stated, are all from commercial sources.

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

[0040] Extraction of crude extract of paeonia qi compounds:

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

[0042] Pretreatment of macroporous resin:

[0043] Pre-treat the HPD-100 macroporous resin used for purification. Stir and load the macroporous adsorption resin with ultrapure water, then elute with ethanol, wash with water until clean, soak in 4.5% hydrochloric acid for 3 - 4 h, wash with water until neutral, soak in 4.5% sodium hydroxide for 3 - 4 h, wash with water until neutral, then elute with 95% ethanol, and wash with water until there is no alcohol smell. Set aside after proper treatment.

[0044] Wet packing of the macroporous resin column:

[0045] Adopt wet packing. Pour the resin into distilled water, let it stand and pour off the floating foam, slowly pour it into a glass chromatography column to make the resin particles settle evenly, drain the excess water from the bottom, and keep the water surface at least 3 cm above the resin surface. After the column packing is completed, calculate the column volume (BV) of this resin column to be 350 mL.

[0046] Purification of the crude extract of paeonia qi compounds:

[0047] Add 60% ethanol to dissolve the above-mentioned crude extract of paeonia qi compounds according to the solid-liquid ratio of 1 g:3 mL to obtain the loading solution of qi compounds, and then slowly add the loading solution of qi compounds along the tube wall from the upper part of the column, avoiding loosening the surface resin and causing it to float. At the same time, collect the effluent, collect 1 tube for every 100 mL. When using a UV spectrophotometer, detect the absorbance value of the effluent at 306 nm; when the absorbance value of the effluent is 1 / 10 of the absorbance value of the loading solution of qi compounds, stop loading. Then elute with 3 BV of water 3 times, elute with 3 BV of 30% ethanol 1 time, then elute with 6 BV of 60% ethanol 1 time, and finally elute with 3 BV of 90% ethanol 1 time. Collect the eluate while eluting, collect all the ethanol eluates, rotary evaporate the collected eluate, and then conduct vacuum freeze-drying for two days to obtain the total qi compounds from peony seed husks (abbreviation: SEP, used in the following examples).

[0048] Example 2: SEP cytotoxicity detection

[0049] Inoculate PK15 cells (8000 cells / well) in a 96-well plate and culture for 24 h. Replace the original medium with 2% DMEM medium and add 8 different concentrations of SEP so that the final concentrations of SEP in the medium are 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, 10 μg / mL respectively, and set up a solvent absolute ethanol control (replace SEP with absolute ethanol) and an untreated blank control (do not add SEP, do not add PRV-EGFP). Set 4 biological replicates for each concentration. After culturing for 24 h, aspirate the medium, wash 2 times with PBS, and then add 100 μL to each well Reagent (Meilun Biotechnology, catalog number: PWL111), let it stand for 5 min, and then use chemiluminescence method to detect and process the data.

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

[0051] Inoculate PK15 cells (15,000 cells / well) in a 96-well plate, culture for 24 h, replace the original medium with 2% DMEM medium, and add 8 different concentrations of SEP to make the final concentrations of SEP in the medium be 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, 10 μg / mL respectively. And set up an ethanol control group with anhydrous ethanol as the solvent (replace SEP with anhydrous ethanol) and an untreated group as the control (without adding SEP and not adding PRV-EGFP). Each concentration and the two controls are set with 3 biological replicates. After culturing for 2 h, in each well of the experimental group, use 0.1 multiplicity of infection (MOI) PRV-EGFP recombinant virus and replace the original medium with the corresponding 8 reaction concentrations of SEP and 2% DMEM medium respectively. In each well of the ethanol control group, use 0.1 MOI PRV-EGFP recombinant virus, anhydrous ethanol and 2% DMEM medium to replace the original medium. The untreated group does not add SEP, does not add PRV-EGFP, and only uses 2% DMEM medium to replace the original medium respectively. After culturing for 24 h, use a fluorescence microscope to scan the plate and take pictures, and use ImageJ software to batch process the pictures.

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

[0053] Inoculate PK15 cells (4×10 5 cells / well) in a 6-well plate, and wait for them to adhere and grow to a density of about 90%; discard the original culture medium, add 2 mL of cell maintenance solution with a concentration of 0.625 μg / mL and pretreat for 2 h, then add PRV-EGFP recombinant virus solution with MOI = 0.1 to infect the cells for 1 h. Discard the virus solution, wash 3 times with PBS, then add 2 mL of cell maintenance solution containing 0.625 μg / mL SEP and incubate for 24 h, and then extract the protein. Replace SEP with anhydrous ethanol as the ethanol control group and set up an untreated group (without adding SEP and not adding PRV-EGFP). Carry out the Western blot experiment according to the following steps:

[0054] (1) Lyse the cells

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

[0056] b. Place the EP tube in a heating and constant mixing instrument and heat at 95 °C for 10 min.

[0057] c. Place the heated sample in an ultrasonic crusher and ultrasonically crush it for 10 min. Set the ultrasonic program to turn on for 30 s, turn off for 30 s, 40% power, and a time of 10 min.

[0058] d. Store the processed protein sample in a -20 °C refrigerator.

[0059] (2) SDS-PAGE Gel Electrophoresis

[0060] a. Preparation of separating gel and stacking gel: Fix the glass plates on the bracket. According to the protein size, prepare a separating gel with an appropriate concentration (10%). The specific components are shown in Table 1.

[0061] Table 1 Separating Gel Recipe

[0062]

[0063] After preparing the separating gel according to the above recipe and loading order, use a pipette tip to mix the separating gel evenly and add it to the space between the two glass plates at a uniform speed. Stop adding when the separating gel reaches 1 / 3 of the distance from the top of the glass plate. Then add isopropanol to the middle of the glass plates to flatten the separating gel. After the separating gel solidifies, gently tilt the glass plates and use a pipette tip to suck out the isopropanol, and then use filter paper to suck out the remaining isopropanol. Then prepare a 5% stacking gel according to the recipe in Table 2:

[0064] Table 2 Stacking Gel Recipe

[0065]

[0066]

[0067] After preparing the stacking gel according to the above recipe and loading order, add the stacking gel to the space between the glass plates, insert the matching gel comb, and pull out the comb after the gel solidifies to perform SDS-PAGE gel electrophoresis.

[0068] b. Assemble the mold, place the prepared gel in the electrophoresis tank, fill the electrophoresis tank with 1×Running Buffer (electrophoresis buffer), and then add a certain volume of sample to the wells.

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

[0070] (3) Transfer membrane and blocking

[0071] a. After preparing the transfer buffer, pre-cool it at 4°C.

[0072] b. Soak the NC membrane, filter paper, and sponge in the transfer buffer and pre-cool them at 4°C.

[0073] c. After electrophoresis, take out the gel block, cut off the upper gel and the extra gel at the bottom and both sides (avoid cutting the target protein).

[0074] d. Arrange the transfer apparatus in the order of blackboard at the bottom, sponge, filter paper, gel block, NC membrane (nitrocellulose membrane), and whiteboard at the top. Carefully remove the air bubbles between the NC membrane and the gel block, clamp the apparatus, install the transfer apparatus on the wet transfer device, pour the transfer buffer into the middle of the transfer apparatus, and soak the whole transfer apparatus in the ice-water mixture to avoid too high temperature.

[0075] e. Connect the power supply and transfer the membrane at a constant voltage of 100V for 80 min.

[0076] f. Take out the transferred membrane, soak it in Ponceau S for staining, and then wash the Ponceau S dye on the membrane with PBS-T or deionized water and start blocking.

[0077] g. Soak the NC membrane in 5% non-fat milk and block it at room temperature for 2 h.

[0078] (4) Incubation with primary antibody, secondary antibody and exposure imaging

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

[0080] b. Prepare the primary antibody (Recombinant Anti-betaActin antibody, Sevier) with 1% BSA according to the instruction manual, and soak the NC membrane in the primary antibody and incubate it at room temperature for 2 h or overnight at 4°C.

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

[0082] d. Prepare the secondary antibody (HRP-labeled goat anti-rabbit IgG, Sevier) with 1% BSA according to the instruction manual, soak the NC membrane in the secondary antibody and incubate it 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 volume ratio of A solution:B solution = 1:1, and expose and image with a chemiluminescence imaging system.

[0084] Experimental results of Examples 1-4: From the detection results of the relative activity of PK cells by SEP, it can be seen that as the concentration of SEP in the culture medium increases (0.078 μg / mL - 10 μg / mL), the relative cell activity shows a gradually decreasing trend. By calculation, the median lethal dose of SEP for PK cells is: 15.81 μg / mL( Figure 1 A、 Figure 1 B); The fluorescence intensity of recombinant virus EGFP in PK cells was detected using the same concentration of SEP. The results showed that as the concentration of SEP increased, the fluorescence intensity of recombinant virus EGFP gradually decreased( Figure 1 D、 Figure 1 E). By calculation, the half-inhibitory dose of SEP against porcine PRV virus is: 0.164 μg / mL. According to the above experimental results, at a relatively safe SEP concentration for cells (in the cell culture medium containing 0.625 μg / mL SEP, the cell survival rate reached 84.7%), the inhibitory effect of SEP on the virus was analyzed( Figure 1 C、 Figure 1 E). The results showed that the inhibitory effect of SEP at this concentration reached 90.4% compared with the control group. (The virus fluorescence strong poison 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 PRV virus IE180 gene

[0086] PK15 cells were seeded in a 6-well plate (4×10 5 cells / well), and waited for them to adhere and grow to a density of about 90%; the original culture medium was aspirated, 2 mL of cell maintenance medium containing 0.625 μg / mL was added for pretreatment for 2 h, and then PRV-EGFP recombinant virus solution with MOI = 0.1 was added to infect the cells for 1 h. The virus solution was discarded and the cells were washed 3 times with PBS, and then 2 mL of cell maintenance medium containing 0.625 μg / mL was added and incubated for 24 h, and then the protein was extracted. SEP was replaced with absolute ethanol as the ethanol control group and an untreated group was set (without adding SEP and without adding PRV-EGFP). The qRT-PCR experiment was carried out according to the following steps:

[0087] (1) RNA extraction

[0088] a. Add 1 mL of TransZol Up (for 6-well plate) to each well and lyse the 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, shake well for 30 s, and let stand at room temperature for 2 - 3 min;

[0090] c. Centrifuge at 12,000 g for 10 min at 4°C. At this time, the liquid in the EP tube is divided into three layers. Carefully aspirate the upper aqueous phase into another new EP tube.

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

[0092] f. Centrifuge the EP tube at 12,000 g for 10 min at 4°C. A white feathery precipitate can be seen at the bottom of the tube. Pour off the supernatant. Add 1 mL of 75% ethanol (prepared with DEPC water) to each tube, invert and mix well, centrifuge at 12,000 g for 10 min at 4°C, and repeat this step.

[0093] g. Centrifuge the EP tube at 12,000 g for 2 min at 4°C without sample, use a pipette tip to aspirate the residual liquid in the tube, invert the EP tube, and dry at room temperature for 5 min.

[0094] h. Add 50 μL of DEPC water to each tube to dissolve the RNA precipitate. This step needs to be carried out on ice.

[0095] (2) cDNA synthesis

[0096] a. Measurement of RNA concentration: Detect the RNA concentration of the extracted sample using a spectrophotometer.

[0097] b. Calculate the sample volume according to the RNA concentration.

[0098] c. Prepare a DNase - free PCR tube. According to the instructions of the reverse transcription kit (TransGen Biotech, catalog number: AU341 - 02), add 4 μL of 5× Uni All-in-One Super Mix for qPCR, 1 μL of gDNA remover, and the sample (RNA sample) into the PCR tube in sequence, and then supplement the reaction system to 20 μL with RNase-free Water.

[0099] d. After oscillating and centrifuging the PCR tube, place it in a PCR instrument, set the PCR reaction program as 50°C for 30 min, 85°C for 5 s, and finally obtain the required cDNA, and store it at 4°C.

[0100] e. During the experiment, RNA needs to be placed on ice. After the experiment, store the extracted RNA sample at -80°C.

[0101] (3) Fluorescent quantitative PCR

[0102] Fluorescence quantitative 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 reference gene GAPDH-F / R and the quantitative primers for the viral IE180 gene IE180-F / R are shown in Table 3; the reaction system for PCR: 5 μL of qPCR Mix, 0.4 μL of each upstream and downstream primer, 4.1 μL of ddH 2 O4, 0.1 μL of cDNA. PCR program: pre-denaturation at 95 °C for 5 min; 95 °C for 10 s, 60 °C for 35 s, 40 cycles; the relative expression level of the target gene was calculated by the 2 -ΔΔCt method, and the experimental results were analyzed for differences using GraphPad Prism 8.0.2.

[0103] Table 3 Fluorescence quantitative PCR primer table

[0104]

[0105]

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

[0107] 1. PK-15 cells (3×10 5 cells / well) were seeded in a 6-well plate and allowed to adhere and grow to a density of approximately 90%.

[0108] 2. The supernatant was removed and divided into three groups: 2 mL of cell maintenance medium containing 0.625 μg / mL SEP 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 as in the experimental group and PRV-EGFP was added to the ethanol control group, and 2 mL of cell maintenance medium was added to the untreated group. Then, it was placed in a 4 °C refrigerator for 1 h for virus adsorption;

[0109] 3. The supernatant was discarded, washed three times with PBS, and then cell maintenance medium was added and incubated at 37 °C for 24 h;

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

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

[0112] 1. PK-15 cells (3×10 5 cells / well) were seeded in a 6-well plate and allowed to adhere and grow to a density of approximately 90%;

[0113] 2. Remove the supernatant and divide it 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. Do not treat the untreated group. Place it in a refrigerator at 4°C for 1 h for virus adsorption.

[0114] 3. Remove the supernatant. After washing all groups 3 times with PBS, add 2 mL of cell maintenance medium containing 0.625 μg / mL SEP to the experimental group, add 2 mL of cell maintenance medium containing the same concentration of ethanol solvent and PRV-EGFP as in the experimental group to the ethanol control group, and add 2 mL of cell maintenance medium to the untreated group. Place it in an incubator at 37°C for 2 h. After the virus enters the cells, discard the supernatant, wash 3 times with PBS, and add cell maintenance medium to incubate for 24 h.

[0115] 4. Conduct the detection experiment of the virus-fused GFP protein according to the Western blot method in Example 4. At the same time, conduct the detection experiment of the expression level of the PRV virus gene according to the experimental methods of RNA extraction and fluorescence quantification in Example 5.

[0116] Example 8: Study on the replication of virus in cells

[0117] 1. Seed PK-15 cells (3×10 5 cells / well) in a 6-well plate and wait for them to adhere and grow to a density of about 90%.

[0118] 2. Remove the supernatant and divide it into three groups. Add 0.1 MOI of PRV-EGFP to the cell wells of the experimental group and the ethanol control group. Do not treat the blank group. Place it in an incubator at 37°C for 2 h 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 SEP to the experimental group, add 2 mL of cell maintenance medium containing the same concentration of ethanol solvent as in the experimental group to the ethanol control group, and add 2 mL of cell maintenance medium to the untreated group. Place it in an incubator at 37°C and incubate for 24 h.

[0120] 4. Conduct the detection experiment of the virus-fused GFP protein according to the Western blot method in Example 4. At the same time, conduct the detection experiment of the expression level of the PRV virus gene according to the experimental methods of RNA extraction and fluorescence quantification in Example 5.

[0121] Experimental results of Examples 5-8: Detect the expression of the virus gene IE180 by fluorescence quantification method. The results show that in the cell culture medium containing 0.625 μg / mL SEP, the relative expression level of IE180 is only 24% of the relative expression level of IE180 in the ethanol control group, and the expression of this gene is not detected in the untreated group ( Figure 1F). Meanwhile, the content of the fusion virus tag protein GFP was verified by Western blot experiment, and this experiment further confirmed that in the cell culture medium containing 0.625 μg / mL SEP, the band of GFP protein was significantly weaker than that of the ethanol control group, and no band was detected in the untreated group ( Figure 1 G).

[0122] The experiment on the effect of SEP on the adsorption of PRV virus to PK cells confirmed that adding SEP had no obvious 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 the subsequent Western blot detection experiment. The content of GFP protein had no obvious difference from that of the ethanol control group, and no band was detected in the untreated group ( Figure 2 B). It shows that SEP has no obvious effect on the process of PRV virus adsorbing to PK cells.

[0123] The experiment on the effect of SEP on the invasion of PRV virus into PK cells confirmed that adding SEP had no obvious 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), and the Western blot detection experiment also showed that the content of GFP protein had no obvious difference from that of the ethanol control group, and no band was detected in the untreated group ( Figure 2 D). It shows that SEP has no obvious effect on the process of PRV virus invading PK cells.

[0124] The experiment on the effect of SEP on the replication process of PRV virus in PK cells confirmed that during the virus replication stage, the added SEP had a significant inhibitory effect on the relative expression level of IE180, and its relative expression level was only 5.5% of that of IE180 in the ethanol-treated group, and the expression of this gene was not detected in the untreated group ( Figure 2 E), and the Western blot detection experiment also showed that the content of GFP protein in the SEP-treated group was significantly lower than that of the ethanol control group, and no band was detected in the untreated group ( Figure 2 F). It shows that SEP has a significant inhibitory effect on the replication of PRV virus in PK cells.

[0125] Example 9: Detection of the effect of resveratrol (Res) on inhibiting virus 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 medium, and 8 different concentrations of Res were added to make the final concentrations of Res in the medium 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, respectively. An ethanol control group with anhydrous ethanol as the solvent and an untreated group were set as controls. Three biological replicates were set for each concentration and the two controls. After culturing for 2 h, 0.1 MO IPRV-EGFP recombinant virus was used in each well, and the corresponding 8 reaction concentrations of Res or ethanol control or no treatment (blank group) were added respectively. At the same time, the original medium was replaced with 2% DMEM medium respectively. After culturing for 24 h, the plates were scanned and photographed using a fluorescence microscope, and the images were batch-processed using ImageJ software.

[0127] Example 10: Detection of the effect of piceatannol on virus 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 medium, and 8 different concentrations of piceatannol were added to make the final concentrations of SEP in the medium 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, respectively. An ethanol control group with anhydrous ethanol as the solvent and an untreated group were set as controls. Three biological replicates were set for each concentration and the two controls. After culturing for 2 h, 0.1 MO IPRV-EGFP recombinant virus was used in each well, and the corresponding 8 reaction concentrations of piceatannol or ethanol control or no treatment (blank group) were added respectively. At the same time, the original medium was replaced with 2% DMEM medium respectively. After culturing for 24 h, the plates were scanned and photographed using a fluorescence microscope, and the images were batch-processed using ImageJ software.

[0129] Experimental results of Examples 9 and 10: Experiments on the inhibitory effects of resveratrol and piceatannol on PRV virus. The results showed that as the concentrations of resveratrol and piceatannol increased, the relative fluorescence values of the virus recombinant virus EGFP gradually decreased ( Figure 3 ). Among them, after calculation, the half-inhibitory concentration of resveratrol on PRV virus was: 5.305 μg / L ( Figure 3 A), and the half-inhibitory concentration of piceatannol on PRV virus was: 22.25 μg / L ( Figure 3 B).

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

[0131] Preparation of standard curve:

[0132] Accurately weigh resveratrol (CAS No.: 501-36-0) and piceatannol reference standard (CAS No.: 10083-24-6), and dissolve them completely. Prepare a mixed standard solution of 100 μg / mL with methanol, and then prepare standard series concentration 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 solution.

[0133] Preparation of SEP sample for loading: (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 filter membrane, and perform on-machine detection; set 2 replicates.

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

[0135] The mobile phase elution process is as follows: Mobile phase A: Mobile phase B = 72:28 passes through the column for 18 min; Mobile phase A: Mobile phase B = 10:90 passes through the column for 6 min; Mobile phase A: Mobile phase B = 72:28 passes through the column for 6 min; Complete the detection to obtain the chromatogram.

[0136] Results of Example 11: Through the results of preparing the standard curves of resveratrol and piceatannol, the R 2 are respectively equal to 0.9999322 and 0.9999510. The results of preparing the standard curves are very good and can be used for the detection of subsequent corresponding products ( Figure 4A 、B、C、D). Detect the content of the SEP sample by liquid phase. From the results, the average results of the two replicates are: the content of piceatannol in SEP is: 32.705 μg / g; the content of resveratrol in SEP is: 678.56 μg / g ( Figure 4G 、H、I、J).

[0137] Example 12: Effect of resveratrol + piceatannol 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 a mixture of resveratrol and piceatannol (20.75:1, RPM) at 8 different concentrations was added to make the final concentrations of RPM in the medium 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, respectively. An ethanol control group with anhydrous ethanol as the solvent and an untreated group were set as controls. Three biological replicates were set for each concentration and the two controls. After 2 h of culture, 0.1 MOI PRV-EGFP recombinant virus was used in each well, and the corresponding 8 reaction concentrations of RPM or ethanol control or no treatment (blank group) were added respectively. At the same time, the original medium was replaced with 2% DMEM medium. After 24 h of culture, the plates were scanned and photographed using a fluorescence microscope, and the images were processed in batches using ImageJ software.

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

Claims

1. The use of peony stilbene compound extract in the preparation of anti-swine virus active ingredients, characterized in that: The active ingredients include pharmaceutical preparations, disinfectants and / or feed additives.

2. Application of Paeonia lactiflora compound extracts in the preparation of anti-swine virus drugs.

3. Application of peony stilbene compound extracts in the preparation of drugs for inhibiting porcine virus replication.

4. The use according to claim 1, 2 or 3, wherein the porcine virus comprises porcine pseudorabies virus and / or a mutated porcine pseudorabies virus.

5. The use according to claim 1, 2 or 3, characterized in that: The concentration of the peony stilbene compound extract is 0.078 μg / mL-10 μg / mL.

6. The use according to claim 1, 2 or 3, characterized in that: The half-inhibitory dose of the peony stilbene compound extract against porcine PRV virus is: 0.164 μg / mL.

7. The use according to claim 1, 2 or 3, characterized in that: The cell median lethal dose of the peony stilbene compound extract is 15.81 μg / mL.

8. The use according to any one of claims 1 to 7, characterized in that: The preparation method of the peony stilbene compound extract comprises 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, mix it by ultrasonic, filter it and centrifuge it, take the supernatant and concentrate it under reduced pressure to obtain a concentrated extract, and freeze-dry it to obtain a crude extract of peony stilbene compounds; 2) Pretreatment of macroporous resin: Pre-treat the macroporous resin used for purification, stir the macroporous resin with ultrapure water, wash with water after eluting with ethanol, soak with hydrochloric acid, wash with water until neutral, soak with sodium hydroxide solution, wash with water until neutral, elute with 95% ethanol, wash with water until there is no alcohol smell, and obtain the pre-treated macroporous resin for use; 3) Wet column packing of macroporous resin: The macroporous resin pretreated in step 2) is poured into distilled water, allowed to stand to remove the foam, and slowly poured into a glass chromatography column to allow the resin particles to settle evenly. Excess water is released from the bottom to keep the water surface at least 3 cm above the resin surface to complete the column packing; 4) Purification of crude extracts of peony stilbene compounds: The crude extract of the peony stilbene compound described in step 1) is added with 60% ethanol at a solid-liquid ratio of 1 g: 3-10 mL to obtain a stilbene compound loading solution, and then the stilbene compound loading solution is slowly added from the top of the column along the wall of the glass chromatography column; at the same time, the effluent is received and the absorbance of the effluent is detected at 306 nm; when the absorbance of the effluent is 1 / 10 of the absorbance of the stilbene compound loading solution, the loading is stopped; The mixture is then eluted at least 6 times, and the eluate is received during the elution. The concentrated extract obtained by rotary evaporation of the received eluate is vacuum freeze-dried to obtain a peony stilbene compound extract.

9. The use according to claim 8, characterized in that: The step 1) takes ground peony seed shell powder, adds 50% ethanol solution at a solid-liquid ratio of 1g:20mL-1g:35mL, and soaks for 1-2 weeks, and then mixes and ultrasonicates at room temperature for 15-30min to obtain a fully mixed extract; the fully mixed extract is filtered and centrifuged, and the supernatant is concentrated under reduced pressure at 45-60°C using a rotary evaporator to obtain a concentrated extract; the concentrated extract is vacuum freeze-dried at -40°C to obtain a crude extract of peony stilbene compounds; the centrifugation conditions are: 3000-5000r / min, centrifugation for 3-5min.

10. The use according to claim 8, characterized in that: The elution in step 4) is specifically eluted with 3BV of water for 3 times, 3BV of 30% ethanol for 1 time, 6BV of 60% ethanol for 1 time, and 3BV of 90% ethanol for 1 time.

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