CN116650494B

CN116650494B - Alkaloid preparation and application thereof in preparation of preparation for resisting porcine epidemic diarrhea virus activity

Info

Publication number: CN116650494B
Application number: CN202310566604.1A
Authority: CN
Inventors: 郝智慧; 李凯远; 陈婷婷; 钟睦琪; 王雪
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2023-05-18
Filing date: 2023-05-18
Publication date: 2024-03-12
Anticipated expiration: 2043-05-18
Also published as: CN116650494A

Abstract

The invention relates to an alkaloid preparation and application thereof in preparing antiviral products. The alkaloid preparation comprises aloperine and/or evodiamine, and can obviously inhibit diarrhea caused by Porcine Epidemic Diarrhea Virus (PEDV). The invention provides a theoretical basis for searching new antiviral natural compounds and targets, and lays a foundation for developing new anti-coronavirus drugs.

Description

Alkaloid preparation and application thereof in preparation of preparation for resisting porcine epidemic diarrhea virus activity

Technical Field

The invention relates to the field of medicines, in particular to an alkaloid preparation and application thereof in preparing antiviral products.

Background

Coronavirus is one of the main viral pathogens causing porcine diarrhea, wherein diarrhea caused by Porcine Epidemic Diarrhea Virus (PEDV) is particularly serious, 100% of morbidity of piglets can be caused, and the suffering livestock mainly show symptoms such as anorexia, vomiting, severe diarrhea, dehydration and the like. PEDV was identified and isolated in our country as early as 1984. Although PEDV has now developed vaccines, their effect is not optimal and massive infection is still occurring.

At present, a method for using a lot of pig farms is to feed (backup) sows after treatment of disease materials containing PEDV by back feeding, so that the sows obtain immunity and transmit the immunity to piglets by milk containing sIgA at the same time, and a good effect is achieved.

The traditional Chinese medicine is a precious characteristic resource in China, has low price and obvious effect, and is easy to popularize. A total of 50 tens of thousands of plants are reported worldwide, of which about 5 tens of thousands are used in traditional medicine. The secondary substances in the natural products of the traditional Chinese medicine are generally low-toxicity small molecular substances, have high chemical novelty and biological value, and in recent years, students have carried out a great deal of researches on antiviral active ingredients and action mechanisms of the traditional Chinese medicine, and the researches on the antiviral active ingredients of the traditional Chinese medicine are mainly concentrated on alkaloids, flavones, polysaccharides, saponins, tannins, polyphenols and the like. Among them, alkaloids have broad-spectrum antiviral effects, but particularly on different viruses, antiviral mechanisms and action cycles thereof are rarely reported.

Disclosure of Invention

In view of the technical limitations, the invention provides an alkaloid preparation and application thereof in preparing antiviral products. The traditional Chinese medicine monomer has unique advantages in the aspect of treating animal virus infection diseases, due to the complexity of coronaviruses, the PEDV related vaccine cannot generate 100% of protection effect, and the biological control method cannot completely control the occurrence of diseases, so that the disease frequently occurs worldwide, the economic and social development is influenced, the sophocarpidine and dehydroevodiamine generate a certain inhibition effect on PEDV, and the effect is good. The invention provides a theoretical basis for searching new antiviral natural compounds and targets, and lays a foundation for developing new anti-coronavirus drugs.

The invention aims at realizing the following technical scheme:

the invention provides an alkaloid preparation, which comprises aloperine and/or evodiamine.

In some embodiments of the invention, the effective concentration of aloperine is 1 μg/mL to 25 μg/mL; further, the effective concentration of aloperine is 3 μg/mL to 12.5 μg/mL, preferably 12.5 μg/mL; also, for example, 1. Mu.g/mL, 3.13. Mu.g/mL, 6.25. Mu.g/mL, 7. Mu.g/mL, 8. Mu.g/mL, 9. Mu.g/mL, 10. Mu.g/mL, 12.5. Mu.g/mL, 15. Mu.g/mL, 18. Mu.g/mL, 25. Mu.g/mL, etc. may be used.

In some embodiments of the invention, the effective concentration of dehydroevodiamine is from 1 μg/mL to 10 μg/mL; further, the effective concentration of dehydroevodiamine is 1.5 μg/mL to 6.25 μg/mL, preferably 6.25 μg/mL; also, for example, 1. Mu.g/mL, 1.56. Mu.g/mL, 3.13. Mu.g/mL, 4. Mu.g/mL, 5. Mu.g/mL, 6.25. Mu.g/mL, 7. Mu.g/mL, 8. Mu.g/mL, 9. Mu.g/mL, 10. Mu.g/mL, etc. may be used.

If the preparation comprises aloperine and evodiamine at the same time, the effective concentration ratio of the aloperine and evodiamine is 1 (0.1-3) as follows: 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3. Alternatively, the ratio of the two may be defined by a mass ratio, and the ratio of the two may be the effective weight ratio of the concentration ratios.

In some embodiments of the invention, when the formulation includes both components, either dilution and then admixture of the drug or dilution and then admixture is required, provided that the concentration ranges are defined above. Further, if the preparation includes both aloperine and evodiamine, the aloperine and evodiamine may be dissolved in water and/or an organic solvent, respectively, and then diluted with a medium (such as DMEM medium), and then mixed at a concentration ratio of the two. In practice, the two medicines can be taken separately (solid medicines can be taken or dissolved medicines can be taken), or can be taken after mixing.

In some embodiments of the invention, the evodiamine is dehydroevodiamine; the formulation is a formulation having anti/inhibitory activity against viruses.

In some embodiments of the invention, the alkaloid interaction phase includes, but is not limited to, a replication phase of the viral lifecycle. The aloperine can act on the whole life cycle of the virus, and comprises four stages of virus adsorption, invasion, replication and assembly; whereas dehydroevodia rutaecarpa acts mainly in the viral replication phase.

In some embodiments of the invention, the virus comprises a diarrhea virus.

In some embodiments of the invention, the virus comprises an epidemic diarrhea virus.

In some embodiments of the invention, the virus comprises Porcine Epidemic Diarrhea Virus (PEDV).

The invention also provides application of the alkaloid preparation in preparing an antiviral product, wherein the alkaloid preparation comprises aloperine and/or evodiamine.

In some embodiments of the invention, the formulation is a single ingredient or a combination of two ingredients, the effective amounts of each ingredient being as described above.

In some embodiments of the invention, the virus is of the type defined in any one of the preceding claims.

In some embodiments of the invention, the product comprises a medicament.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) According to the technical scheme provided by the invention, the aloperine can effectively inhibit PEDV proliferation in vitro, has long action time and stable effect, and experiments prove that compared with a positive control group, almost no cytopathy appears in cells 36h of a aloperine test group with the concentration of 12.5 mu g/mL, and the expression of virus N protein can be inhibited at each stage of the virus life cycle.

(2) According to the technical scheme provided by the invention, the fact that the dehydroevodiamine can effectively inhibit PEDV proliferation in vitro is confirmed, and compared with a control drug ribavirin with the concentration of 100 mug/mL, the dehydroevodiamine with the concentration of 6.25 mug/mL has obvious advantages in reducing the relative expression quantity of virus N protein, and the effect is durable.

(3) The technical scheme provided by the invention defines the life cycle of the sophocarpidine and dehydroevodiamine for resisting PEDV, and supplements the defect of the prior art on the cognition of the action mechanism of alkaloids.

(4) The invention provides a theoretical basis for searching new antiviral natural compounds and targets, and lays a foundation for developing new anti-coronavirus drugs.

Drawings

FIG. 1 shows the effect of aloperine on Vero CCL81 and IPEC-J2 cell viability.

Figure 2 shows a time-of-action analysis of aloperine in inhibiting PEDV virus.

FIG. 3 shows the results of flow cytometry detection of aloperin-reduced PEDV infected cells.

FIG. 4 shows a relative expression level analysis of aloperin-reduced PEDV N protein.

FIG. 5 shows analysis of aloperine lowering mRNA transcription levels of PEDV N and ORF 3 genes.

FIG. 6 shows CC of aloperine on Vero cells ₅₀ And its IC for PEDV ₅₀ As a result.

Figure 7 shows a schematic of the design of a aloperine intervention assay at different stages during the PEDV single replication cycle.

Figure 8 shows the effect of aloperine on different phases of the PEDV replication cycle.

FIG. 9 shows the effect of dehydroevodiamine on Vero and IPEC-J2 cell viability.

Figure 10 shows a time-of-action analysis of dehydroevodiamine in inhibiting PEDV virus.

FIG. 11 shows that dehydroevodiamine reduces TCID in PEDV infected cells ₅₀ As a result.

FIG. 12 shows the results of flow cytometry detection of dehydroevodiamine reduced PEDV infected cells.

FIG. 13 shows the relative expression levels of dehydroevodiamine-inhibited PEDV N protein.

FIG. 14 shows analysis of dehydroevodiamine reduced PEDV N and ORF 3 gene mRNA transcription levels.

FIG. 15 shows CC of dehydroevodiamine on Vero cells ₅₀ And its IC for PEDV ₅₀ As a result.

Figure 16 shows the effect of dehydroevodiamine on various phases of the PEDV replication cycle.

Figure 17 shows the use of aloperine, dehydroevodiamine and ribavirin Lin Lian against PEDV.

Figure 18 shows the combination of aloperine and dehydroevodiamine against PEDV.

Detailed Description

The present invention will be described in further detail below in order to make the objects, technical solutions and advantages of the present invention more apparent. It is to be understood that the description is only intended to illustrate the invention and is not intended to limit the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. Reagents and instruments used herein are commercially available, and reference to characterization means is made to the relevant description of the prior art and will not be repeated herein.

The invention adopts alkaloid monomers with different concentrations to explore anti-PEDV. Firstly, performing a CCK-8 cytotoxicity test, determining the maximum safe concentration of aloperine and dehydroevodiamine on Vero cells, adopting the highest concentration to explore the optimal dosing mode, subsequently setting high, medium and low doses at the safe concentration to perform subsequent exploration, detecting the number of virus infected cells by adopting a Flow Cytometry (FCM), observing the expression level of virus N protein by using indirect Immunofluorescence (IFA), and fitting CC by cytotoxicity and IFA tests ₅₀ And IC ₅₀ And (3) determining the therapeutic index SI of the aloperine and the dehydroevodiamine by related data, detecting the expression level of the viral N protein by using Western blot, measuring the transcription level of viral N and ORF3 genes by using RT-qPCR, and determining the influence stage of the aloperine and the dehydroevodiamine in the PEDV life cycle according to the test.

The invention is described in further detail below with reference to a few examples.

Example 1 technical Effect test Material and method

1. Materials and methods

1.1 materials

1.1.1 reagents

(1) The reagents used in this example are shown in Table 1.

TABLE 1 Main reagents for the test

(2) Preparation of the Main reagent

Preparation of aloperine

Accurately weighing 0.020g of aloperine dry product, dissolving in 4mL of DMSO to prepare 5000 mug/mL stock solution, subpackaging, storing in a refrigerator at-80 ℃, and diluting the subsequent working solution to the corresponding concentration by using a DMEM culture medium.

Preparation of dehydroevodiamine

Accurately weighing 0.020g of dehydroevodiamine dry product, dissolving in 4mL of DMSO to prepare 5000 mug/mL of stock solution, subpackaging, storing in a refrigerator at-80 ℃, and diluting the subsequent working solution to the corresponding concentration by using a DMEM culture medium.

Preparation of ribavirin

Accurately weighing 0.100g of ribavirin dry product, dissolving in 10mL of enzyme-free sterile water, preparing 10000 mug/mL of liquid medicine, diluting into 1000 mug/mL of stock solution, subpackaging, storing in a refrigerator at-80 ℃, and diluting the subsequent working solution to the corresponding concentration by using a DMEM (medium).

Preparation of combined medicine

The two drug stock solutions for combined use are diluted to 2 times of the effective concentration respectively by using DMEM culture medium, and then 1:1 to the final working concentration, and adding the mixture to the cells after mixing.

Cell culture reagent

10% DMEM cell growth medium: 500mL of DMEM culture medium, 50mL of fetal bovine serum and 5mL of double antibody (10000 IU/mL), after being mixed uniformly, the mixture is placed in a refrigerator at 4 ℃ for preservation, and the mixture is placed in a water bath kettle at 37 ℃ for preheating before use so as to reduce the stimulation to cells.

1% DMEM cell maintenance medium: 500mL of DMEM culture medium, 5mL of fetal bovine serum and double antibody (10000 IU/mL) are added, and after being mixed evenly, the mixture is placed in a refrigerator at 4 ℃ for preservation;

cell cryopreservation solution: fetal bovine serum 9mL, dimethyl sulfoxide (DMSO) 1mL, and mixing upside down (i.e., ready-to-use).

Western blot reagent

Western blot electrophoresis liquid: 200mL of 5 XTris-glycine electrophoresis buffer solution is added into 800mL of distilled water to prepare 1 XWestern blot electrophoresis solution.

Western blot transfer solution: 100mL of electrophoresis transfer buffer (transfer solution) is taken, 200mL of methanol and 700mL of distilled water are added, the mixture is inverted and mixed uniformly, and 1 XWestern blot transfer solution is prepared, and the transfer solution is placed on a refrigerator or ice for precooling at 4 ℃ before use.

Sealing liquid: weighing 5g of skimmed milk powder, dissolving in 100mL of TBST solution, mixing well by vortex, and preserving at 4 ℃.

1.1.2 instruments

The apparatus used in this example is shown in Table 2.

Table 2 main instrument for the test

1.1.3 primers

(1) Universal primers

The universal primer for PEDV reverse transcription is Oligo dT (18).

(2) The real-time fluorescent quantitative PCR primers used in this example are shown in Table 3.

TABLE 3 real-time fluorescent quantitative PCR primer sequences

1.1.4 cells and viruses

(1) And (3) cells: the African green monkey kidney cell line (Vero CCL 81) and porcine small intestine epithelial cells (IPEC-J2) used in the study were maintained by the present laboratory. The culture medium used for cell culture is DMEM, and fetal calf serum with final concentration of 10% and 1% diabody are added, 37 deg.C and 5% CO ₂ Culturing under the condition.

(2) Virus: porcine epidemic diarrhea virus (PEDV CV 777) was kept by the present laboratory.

1.2 method

1.2.1CCK-8 method for measuring cell viability

Cells in the logarithmic growth phase (cell density about 80% -90%) were digested and then grown at 1X 10 ⁴ The density of each/mL is inoculated in a 96-hole cell culture plate, the last row of non-inoculated cells is reserved, PBS solution is added, the cell culture plate is placed in a cell culture box for culturing for 12-16 hours, a culture medium is sucked, the cell culture plate is washed 3 times by PBS buffer solution, 100 mu L of diluted liquid medicine is added into each hole, 8-10 repetitions of each concentration are set, the cell culture plate is placed in the cell culture box for incubation, and the specific incubation time is determined according to the investigation content. After the incubation, the liquid medicine is discarded, after the washing is carried out for 3 times by using PBS buffer solution, 100 mu L of 10 percent CCK-8 reagent is added into each hole, the mixture is placed in a 37 ℃ incubator for incubation for 1 to 4 hours in dark, a microplate reader is used for measuring absorbance value (lambda=450 nm) at intervals of 1 hour, and the cell vitality and CC after the treatment of medicines with different concentrations are calculated according to the absorbance value ₅₀ Values. The cell viability was calculated as follows:

1.2.2PEDV proliferation and TCID ₅₀ Measurement

Virus proliferation: vero CCL81 cells at 37℃with 5% CO ₂ Culturing under the condition of 80-90% of T25 cell bottle, discarding original culture medium, washing with sterile PBS buffer solution for 3 times, adding 0.5-1mL virus solution, and gently shaking cellsAnd (3) placing the bottle in a cell culture box after the virus liquid is completely contacted with the bottom cells, taking out the bottle every half an hour, and gently shaking the bottle to increase the contact area and the contact times of the virus liquid and the cells. After 2h, the cell culture flask was removed, 5mL of cell maintenance medium was added thereto, and the mixture was placed at 37℃with 5% CO ₂ The cells are cultured in a cell culture box under the condition for 12-24 hours. Observing cell CPE every 2h after 12h, wherein the cells are wrinkled, become smaller and have unclear outlines, sealing the cell bottles by using sealing films when the CPE reaches about 90%, transferring to a refrigerator at-80 ℃ for freezing and thawing for 3 times, transferring liquid in the cell bottles to a 15mL sterile centrifuge tube, centrifuging at 4 ℃ and 10000rpm for 10min, taking supernatant, transferring and split charging the supernatant into a 1.8mL freezing and storing tube, and placing the tube in the refrigerator at-80 ℃ for standby.

TCID ₅₀ And (3) measuring: vero CCL81 cells with good growth state were cultured at 1×10 ⁵ The cells were plated in 96-well cell culture plates at a density of one/mL and washed 3 times with sterile PBS buffer until the cells grew to a uniform monolayer. The new virus is diluted by 10 times ratio with PEDV diluent, 8 gradients are added to the washed 96-well plate, 100 MuL/well is inoculated, 8 compound wells are arranged for each concentration, and the mixture is placed at 37 ℃ and 5% CO ₂ Is cultured in a cell culture incubator for 72 hours. After the culture is finished, staining is carried out according to an operation method (IFA) of 1.2.5 indirect immunofluorescence test, observation is carried out under a fluorescence microscope, the number of holes with green fluorescence is counted, and virus TCID is calculated according to a Reed-Muench two-way method ₅₀ 。

1.2.3 viral or intracellular RNA extraction

After cell culture, inoculating virus, adding medicine, treating for different time, taking out 6-hole cell plate treated by virus and medicine from cell incubator, discarding original culture medium, washing 2 times by using precooled PBS buffer solution, adding 1mL RNAiso Plus lysate into each hole, standing for 3-5min at room temperature after blowing for several times, transferring cell lysate into 1.5mL enzyme-free centrifuge tube, adding 200 μl chloroform into each tube, standing for 5min at room temperature, centrifuging for 15min at 12000 Xg at room temperature after shaking and mixing uniformly, carefully sucking supernatant after centrifugation into a new 1.5mL enzyme-free centrifuge tube, adding 0.5-1 times of isopropanol by volume of RNAiso Plus into each tube, standing for 10min at room temperature, centrifuging for 10min at 12000 Xg, discarding isopropanol, adding 75% absolute ethanol equivalent to RNAiso Plus at the bottom of the tube, washing the precipitate at 4 ℃, centrifuging for 5min at 7500 Xg, centrifuging to remove supernatant after centrifugation, placing the supernatant into a safe biological cabinet, drying at room temperature, and drying for a proper amount of Rno. after drying, and determining the concentration of Naee-Naee by using water.

1.2.4 preparation of cDNA real-time fluorescent quantitative PCR reaction

After RNA extraction, cDNA preparation was performed according to reverse transcription kit instructions, and the following were added to an enzyme-free centrifuge tube:

universal primer Oligo (dT) 18:1 μl;

RNA template: 1 μg;

RNase free water: make up to 12 μl;

mixing the above components, centrifuging, placing on a PCR amplification instrument, adjusting the temperature to 65deg.C, melting for 5min, and cooling on ice for 2min. The other reagents of the reverse transcription reaction system are shown in Table 4, and the components in the table are added into a centrifuge tube, and the total system is 20. Mu.L.

TABLE 4 reverse transcription reaction system

Blowing and mixing the components uniformly, performing instantaneous centrifugation, placing on a PCR amplification instrument, adjusting the temperature to 42 ℃, incubating for 1h,75 ℃ and 5min to thermally inactivate reverse transcriptase, wherein a reverse transcribed cDNA product is a template for PCR reaction, and placing in a refrigerator at 4 ℃ or-20 ℃ for preservation after completion.

The NCBI database (https:// www.ncbi.nlm.nih.gov /) or literature resources are consulted to search and design the primer sequence of the target gene, and synthesis is carried out, wherein the primer sequence is shown in Table 3. Preparing cDNA, preparing qPCR reaction system according to Table 5, setting qPCR reaction program according to Table 6, quantifying target gene, and after reaction, preparing cDNA according to 2 ^—△△CT The method calculates the relative content of the target gene.

TABLE 5qPCR reaction System

TABLE 6qPCR reaction procedure

1.2.5 Indirect immunofluorescence assay (IFA)

Plating after cell culture, washing 2 times by using sterile PBS buffer solution after cell adherence, then adding PEDV and liquid medicine with different concentrations simultaneously, incubating for 2 hours in a 37 ℃ incubator, washing 2 times by using sterile PBS buffer solution, replacing with liquid medicine with different concentrations, 500 mu L/hole, and incubating in the 37 ℃ incubator for different times. Taking out the 24-hole cell culture plate subjected to virus and drug treatment from an incubator, sucking and discarding old culture medium, cleaning 3 times by using PBS buffer solution, adding 500 mu L of tissue and cell fixing solution into each hole, fixing for 25min at room temperature, discarding the fixing solution after fixing, cleaning 3 times by using PBS buffer solution, shaking 5 min/time by using shaking table, adding 250 mu L of 0.3% Triton-X100 penetrating solution into each hole after cleaning, cleaning 3 times by using PBS buffer solution after penetrating at room temperature, shaking 5 min/time by using shaking table, adding 500 mu L of 3% BSA sealing solution into each hole after cleaning, sealing for 1h by using PBS buffer solution at 37 ℃, cleaning 3 times by using shaking table after sealing, and shaking 5 min/time by using shaking table. Then 150 mu L of diluted primary antibody is added into each hole, the mixture is placed in a 37 ℃ incubator for incubation for 1h or a 4 ℃ refrigerator for overnight incubation, PBST buffer solution is used for cleaning for 3 times after the primary antibody incubation is completed, a shaking table is used for shaking for 5 min/time, 150 mu L of diluted FITC-labeled fluorescent secondary antibody is added into each hole after cleaning, the mixture is incubated for 1h in a 37 ℃ incubator in a dark place, PBST buffer solution is used for cleaning for 3 times after the secondary antibody incubation is completed, the shaking table is used for shaking for 5 min/time, 100 mu L of DAPI solution (10 mu g/mL) is added into each hole, the mixture is incubated for 5min in a dark place, the liquid in the hole is removed after incubation, the PBST buffer solution is used for cleaning for 3 times, the shaking table is used for 5 min/time, 150 mu L of PBST buffer solution is added into each hole after the cleaning is completed, the mixture is placed under an inverted fluorescent microscope for observation, the excitation wavelength is placed at 488nm, the green fluorescent position is viral protein, the excitation wavelength is placed at 350-360nm, the blue fluorescent position is cell nucleus, and data analysis is performed by collecting pictures.

1.2.6 Western immunoblotting (Western blot) experiments

(1) Protein extraction, quantification and denaturation

After the cells are cultured to the density of the virus to be inoculated and treated for different time, the cell plates subjected to virus and drug treatment are taken out of a cell culture box, placed on ice for precooling, the original culture medium is sucked and removed, precooled PBS buffer solution is used for cleaning for 3 times, then 500 mu L of PBS buffer solution is added into each hole, a sterile cell scraper is used for carefully scraping the cells at the bottom of the cell plates, the cells are transferred into a 1.5mL centrifuge tube, centrifugation is carried out at 4 ℃ for 5min at 2000rpm, the supernatant is carefully sucked up after centrifugation, then a proper amount of Western and IP cell lysate (protease and phosphatase inhibitor are added in advance) is added into each tube, the uniformly mixed resuspended cells are blown and placed into the centrifuge tube for being subjected to pyrolysis for 30min, centrifugation is carried out at 4 ℃ for 12000rpm, the supernatant is transferred into the centrifuge tube for 1.5mL newly, a standard curve is manufactured by using a BCA measuring kit, the protein concentration of a sample is detected, a proper amount of lysate is added for adjusting the protein concentration of each sample, a proper amount of the protein is added according to a diluted sample system, a proper amount of 6X Protein Loading Buffer is added, a 100 ℃ metal bath is boiled for 10min for protein denaturation, and the protein denaturation is carried out after the denaturation, and the instant centrifugation is carried out at a refrigerator for low temperature of-20 ℃.

(2) SDS-PAGE protein gel preparation

And taking two dry and clean glue-making glass plates, putting the two dry and clean glue-making glass plates into a glue-making clamping plate according to the front-back sequence, clamping and adjusting the heights of the two glass plates to be level, and ensuring no liquid leakage. Adding the prepared separating gel into the gel plate, preparing the separating gel, namely, pressing the separating gel by using isopropanol, discarding the isopropanol after the separating gel is solidified, sucking the residual isopropanol clean by using filter paper, adding the prepared concentrated gel, inserting a comb to be gelled, disassembling a gel preparation clamp, taking out the prepared protein for later use, and preparing the concentrated gel, namely, the table 8.

TABLE 7 preparation method of SDS-PAGE separating gel (10 mL)

TABLE 8SDS-PAGE gel preparation method (5 mL)

(3) SDS-PAGE electrophoresis

And (3) placing the prepared protein gel in an electrophoresis tank for assembly, checking to ensure no leakage, adding 1X electrophoresis buffer solution into the electrophoresis tank, and pulling out the comb in the concentrated gel. Adding protein samples to be detected into the gel holes sequentially by using a micropipette, setting the positions of protein markers, starting electrophoresis, and setting the electrophoresis program to 80V for 30min;120V,60min; and stopping electrophoresis when the protein Marker electrophoresis reaches an ideal position for separating the target protein.

(4) PVDF film transfer printing

And (3) disassembling the protein gel plate after electrophoresis, determining the position of the target protein according to the molecular weight of the protein indicated by the protein Marker, and cutting off the required protein gel region. Cutting PVDF film according to the cut area, activating the cut PVDF film in methanol for 2min, arranging the PVDF film, the filter paper, the albumin glue, the PVDF film, the paper, the sponge and the transfer white board according to the transfer printing clamp blackboard, sponge, and the paper, removing bubbles in the lamination process, carefully clamping the whole transfer printing system, placing the whole transfer printing system into a transfer printing groove, pouring 1X transfer printing liquid, placing ice cubes around the transfer printing groove, adjusting the current to 250mA, and setting the transfer printing time according to the size of target protein.

(5) Closure

Disassembling the clamp after transfer printing, putting the transferred PVDF film into a closed box, pouring a proper amount of 5% skimmed milk, and incubating and closing at room temperature for 1-2h;

(6) Antibody incubation

Taking out the sealed PVDF membrane, washing 3 times with TBST buffer solution, shaking for 5min each time, removing residual skimmed milk, diluting the primary antibody according to a proper proportion by using primary antibody diluent according to a primary antibody instruction, and placing the diluted primary antibody and PVDF membrane in a refrigerator at 4 ℃ for overnight incubation or room temperature for 1-2h. After incubation, recovering the primary antibody, washing 3 times by using TBST buffer solution, shaking the table for 5min each time, washing away residual primary antibody, diluting the secondary antibody by using secondary antibody diluent according to the specification of the secondary antibody, incubating the diluted secondary antibody and PVDF membrane for 1-2h at room temperature, discarding the secondary antibody after incubation, washing 3 times by using TBST buffer solution, shaking the table for 10min each time, and washing away residual secondary antibody.

(7) Development analysis

Opening a protein molecular imager in advance, incubating ECL chemiluminescent liquid on a PVDF film, incubating for 2min in a dark place, putting into the imager for developing and exposing, storing images after analysis, and analyzing by using Image J.

1.2.7 Flow Cytometry (FCM)

Taking out the cell plates subjected to virus and drug treatment from a cell culture box, washing 3 times by using PBS, adding a proper amount of trypsin into each hole, placing the cell culture box to digest cells, adding 3 times of serum-containing culture medium with the volume of pancreatin after the cells are completely shed, stopping digestion, collecting the cells into a sterile 1.5mL centrifuge tube, centrifuging for 5min at 2000 Xg, discarding supernatant, adding cell rupture fixing solution, incubating for 7h at room temperature or in a refrigerator at 4 ℃, centrifuging for 2000 Xg at 4 ℃ for 5min, diluting rupture liquid washing solution in advance (10 times by using PBS buffer), discarding rupture liquid, washing by using washing solution, centrifuging for 5min at 4 ℃, repeating for 3 times, adding PEDV NP-FITC antibody (SD-1F) diluted by using rupture liquid washing solution, incubating 100 mu L each tube overnight at 4 ℃ in a refrigerator, discarding antibody after incubation, washing the cells by using rupture liquid washing solution at 2000 Xg at 4 ℃, centrifuging for 5min at 3 times, adding 300 mu L of rupture liquid in advance, detecting by using a machine, and adjusting the voltage of the whole cell culture box.

1.2.8 statistical analysis

Results data are expressed as Mean ± standard deviation (Mean ± SD), all data being the results of 3 or more trials. The drawing software was GraphPad Prism 8, differential analysis was performed using IBM SPSS Statistics software, and the comparison of the two sets of data used a T-test (two-tailed); comparison of the sets of data used one-way analysis of variance (ANOVA).

EXAMPLE 2 analysis of Sophocarpidine study results

1. Effect of aloperine on Vero CCL81 and IPEC-J2 cell viability

Vero CCL81 and IPEC-J2 cell lines are cell lines commonly used in PEDV research, so in the example, toxicity tests of aloperine are carried out on the two cell lines, and the two cell lines are respectively processed for 12, 24 and 36 hours, and the measured absorbance values are calculated according to a formula to obtain cell activity data after different processing times, so that the result is shown in figure 1, and when the aloperine concentration is 12.5 mug/mL on Vero cells, the cell activity of 12, 24 and 36 hours is above 90%, so the maximum safe concentration of aloperine on Vero cells is determined to be 12.5 mug/mL; on IPEC-J2 cells, the cell activities of 12, 24 and 36 hours are above 90% when the concentration of aloperine is 25 mug/m L, so that the maximum safe concentration of aloperine on IPEC-J2 cells is 25 mug/mL; in combination with the cytotoxicity data of aloperine on both cells, a final concentration of 12.5 μg/mL was chosen as the maximum concentration for the subsequent antiviral assay.

As shown in fig. 1, after Alo treatment of cells 12, 24, 36h at different concentrations, absorbance at 490nm was detected using CCK-8 method, vero CCL81 (a) and IPEC-J2 (B) cell viability maps were plotted and analyzed after calculation according to cell viability formula, NS indicated no statistical difference compared to the blank group (i.e. concentration of 0), P indicated a statistical difference, P indicated a significant difference; * P represents the difference is very significant.

2. Results of time of action of aloperine in inhibiting PEDV Virus

The maximum safe concentration of aloperine determined by cytotoxicity test was investigated at which different time of action of aloperine against PEDV was carried out, and the test procedure is shown in fig. 2 (a): 2 hours before virus inoculation (Pre-treatment), simultaneous incubation of drug and virus (Co-treatment), post-treatment and full-course administration (Whole-treatment); after 24h treatment, cells and viral proteins were extracted for Western blot analysis, and the results are shown in fig. 2 (B): compared with a positive Control group (Virus), the Whole-course administration almost completely inhibits the expression of Virus N protein, and the western blot of the Control group is not different; compared with the positive control group, the Pre-treatment group cannot inhibit the expression of PEDV N protein, the Co-treatment group inhibits the expression of PEDV N protein to a certain extent, the Post-treatment group has the strongest effect of inhibiting virus N protein, and according to the result, the drug adding time of the subsequent test adopts a Co-treatment+post-treatment mode. As shown in FIG. 2 (C), after PEDV (MOI=0.1) was inoculated into Vero cells, and the cells of the PEDV group showed obvious cytopathy and were wrinkled and shed compared with the Control group by using Co-treatment + Post-treatment for 36h with different concentrations (12.5, 6.25, 3.13. Mu.g/mL) of aloperine, the situation was significantly improved by the test group with 3.13. Mu.g/mL of aloperine added, and the cell state gradually returned to normal with increasing concentration, and almost no cytopathy was seen in the cells of the 12.5. Mu.g/mL aloperine test group.

As shown in fig. 2, alo was added at different times to inhibit PEDV effect assay design (a); after PEDV inoculation and administration according to the experimental design, collecting cells 24h to analyze PEDV N protein expression (B) and comparing the influence of different administration times on PEDV N protein expression; the effect of inhibiting PEDV is verified by using Co+post-treatment mode, alo can obviously improve CPE (C) caused by PEDV, and the more red circles in the figure indicate that the more severe the CPE.

3. Flow cytometry detection results of aloperine reduced PEDV infected cells

According to the early investigation of the action time of matrine for inhibiting PEDV and the action effect of different concentrations of matrine, the effects of different concentrations of matrine and ribavirin serving as a control medicament on the proportion of infected cells after virus infection are detected by using a flow cytometry, wherein the concentrations of the matrine are 12.5, 6.25 and 3.13 mug/mL, and the ribavirin serving as the control medicament is 50 mug/mL. The results are shown in FIG. 3: compared with the positive control group (PEDV-0.1), the other groups have extremely remarkable difference of the ratio of the infected cells (P < 0.001) at the treatment time of 12, 24 and 36 hours, the number of the infected cells and the concentration of the aloperine show concentration dependency, and the larger the concentration of the aloperine is in a dosage range, the smaller the ratio of the infected cells is. Meanwhile, as the contamination time increases, the contamination cell ratio of the positive control group (PEDV-0.1) is continuously increased, the contamination cell ratio is increased by nearly 1 time every 12 hours, and 36 hours are nearly 80 percent. By observing the effect of ribavirin as a control drug, the ribavirin group (Rib-50 mug/mL) and the aloperine-containing dose group (Alo-6.25 mug/mL) are basically equivalent in the ratio level of reduction of toxic cells at 12 and 24 hours, but the effect of ribavirin group at 36 hours is weakened, and the ratio of toxic cells is higher than that of aloperine-containing low dose group (Alo-3.13 mug/mL), so that the effect time of aloperine for inhibiting PEDV is longer, the effect of aloperine-containing high dose aloperine group (Alo-12.5 mug/mL) for inhibiting PEDV is always in a stable state, and the effect is not reduced with the time.

As shown in fig. 3, different concentrations of Alo and Rib at 50 μg/mL were used to treat PEDV-infected cells, after 12, 24, and 36 hours incubation, FITC-PEDV-N antibodies were collected after cell permeabilization, FITC positive cell numbers were detected by flow cytometry (a), data statistics analysis was performed (B), NS indicated no statistical difference compared to model group (i.e., PEDV-0.1), P indicated statistical difference, P indicated significant difference; * P represents the difference is very significant.

4. Aloperine reduces the relative expression level of PEDV N protein

The influence of different concentrations of aloperine and ribavirin serving as a control medicament after virus infection on the expression of virus N protein is detected by an indirect immunofluorescence method, wherein the concentrations of aloperine are 12.5, 6.25 and 3.13 mug/mL, and the concentration of ribavirin serving as the control medicament is 50 mug/mL. The results are shown in FIG. 4 (A): the fluorescence patterns in each time phase are subjected to semi-quantitative analysis, and the positive control group is subjected to normalization treatment and then is compared with other groups, wherein the relative expression amount of N protein is extremely obvious (P < 0.001) in the other groups at 12, 24 and 36h treatment time, and the relative expression amount of N protein and the concentration of aloperine show concentration dependency in the 12h and 24h treatment time, and the higher the concentration of aloperine is, the lower the relative expression amount of N protein is in the dosage range. As can be seen from the fluorescence graph, the expression of PEDV N protein in the positive control group gradually increases from 12h to 36h, namely the green area in the graph gradually expands; by observing the effect of ribavirin in a control drug group, the effect of ribavirin (Rib-50 mug/mL) and aloperine in a dosage group (Alo-6.25 mug/mL) on reducing the expression level of virus N protein in 12 hours and 24 hours is basically equivalent, and the effect of ribavirin in 36 hours and aloperine in the dosage group is weakened, and the expression level of N protein is even with that of aloperine in a low dosage group (Alo-3.13 mug/mL), so that the effect of inhibiting the expression level of virus N protein in the middle and low dosage aloperine groups and the ribavirin in the low dosage group is gradually weakened along with the time extension; the high-dose aloperine group (Alo-12.5 mug/mL) has longer action time for inhibiting the expression of PEDV N protein, the effect is not reduced with the time, and the capability of inhibiting the PEDV of the high-dose aloperine group is always in a stable state in 12, 24 and 36 hours.

According to the results of detecting the expression level of PEDV N protein and performing semi-quantitative analysis according to an indirect immunofluorescence test, the PEDV N protein expression is subjected to more accurate western blot analysis on the influence of aloperine, and the results are shown in FIG. 4 (B): after gray analysis of Western blot results, the relative expression amounts of N proteins of the high-dose aloperine groups are reduced and are extremely remarkably different (P < 0.001) compared with the positive control groups at 12, 24 and 36h treatment time, the medium-dose aloperine groups have unstable trend, are extremely remarkably different (P < 0.001) compared with the positive control groups at 12 and 36h treatment time, are remarkably different (P < 0.01) at 24h treatment time, the low-dose aloperine groups also have unstable trend, and are not different from the positive control groups at 12 and 24h treatment time, and are remarkably different at 36 h; the relative expression amounts of N proteins at medium and low doses do not show stable differences in statistics, but can be found by a western blot image, the relative expression amounts of N proteins are reduced to a certain extent. The ribavirin group as a control drug showed a similar trend as that of the intermediate-dose aloperine group, and the relative expression amount of N protein showed a significant difference (P < 0.01) compared with the positive control group at 12h and 24h treatment time, and showed a very significant difference (P < 0.001) at 36 h. Therefore, the high-dose aloperin group has obvious advantages in reducing the relative expression quantity of the viral N protein, and has stable data and shows the same results as FCM and IFA detection.

As shown in fig. 4, PEDV-infected cells were treated with Alo at different concentrations and Rib at 50 μg/mL, after incubation for 12, 24, 36h, the cells were collected for incubation with PEDV-N antibodies, fluorescent microscopy after FITC secondary incubation was observed and pictures were taken for semi-quantitative analysis (a), blue fluorescence represented DAPI-stained nuclei and green fluorescence represented FITC-stained PEDV N protein; collecting cells after the same treatment, extracting proteins, performing Western Blot analysis, incubating an HRP secondary antibody, and then collecting images (B) by a protein molecular imaging system, and performing gray analysis on WB strips by using an Image J and making a graph; NS indicates no statistical difference, P indicates statistical difference, and P indicates significant difference compared to the model group (i.e., PEDV-0.1); * P represents the difference is very significant.

5. Matrine inhibits PEDV N and ORF3 protein gene mRNA transcription results

Early results have shown that aloperin can significantly reduce the expression level of viral proteins, and subsequent analysis of mRNA transcription levels of viral N and ORF3 genes is performed using a real-time fluorescent quantitative PCR method, with the N gene mRNA transcription results shown in fig. 5 (a): the N gene mRNA transcription levels were reduced and exhibited very significant differences (P < 0.001) for the aloperine dose groups compared to the positive control group at 12, 24 and 36h treatment time; the control drug ribavirin group showed very significant differences in mRNA transcription level of the N gene at 24h treatment time (P < 0.001) and positive control group, whereas no differences were shown at 12h and 36h treatment time, which may be related to the mechanism of action of ribavirin in inhibiting viral replication. The PEDV ORF3 protein has the function of regulating the output of PEDV particles, the mRNA transcription result of the ORF3 gene is shown as a figure 5 (B), the mRNA transcription of the ORF3 gene shows different trends of N genes, the mRNA transcription level of the ORF3 gene of each dosage group of aloperine is reduced under the treatment time of 12, 24 and 36 hours, the high dosage aloperine group shows extremely significant difference (P < 0.001) with the positive control group in each time group, the medium and low dosage aloperine groups are unstable, the medium dosage group shows extremely significant difference (P < 0.001) in 12 hours and 36 hours, and the 24 hours show significant difference (P < 0.01); the low dose group showed very significant differences at 12h, 36h (P < 0.001), no difference at 24h (P > 0.05); ribavirin group as control drug can reduce mRNA transcription level of ORF3 gene in 12h and 36h, and has very significant difference (P < 0.001) in 12h compared with positive control group, difference (P < 0.05) in 36h compared with positive control group, and level in 24h basically compared with positive control group, and no difference (P > 0.05) compared with positive control group.

As shown in fig. 5, the PEDV-infected cells were treated with Alo and Rib at different concentrations of 50 μg/mL, and after 12, 24, 36 hours incubation, total RNA was extracted after cell lysis was collected, real-time fluorescent quantitative PCR assays were performed using PEDV N (a) and ORF3 (B) -specific primers and data processing analysis was performed after normalizing the model set; NS indicates no statistical difference, P indicates the presence of a statistical difference, and P indicates a significant difference compared to the model group (i.e., PEDV); * P represents the difference is very significant.

6. CC of aloperine on Vero cells ₅₀ And its IC for PEDV ₅₀ And SI (information and information)

CC for aloperine on Vero cells based on data of early cytotoxicity and indirect immunofluorescence ₅₀ And IC for inhibiting PEDV ₅₀ The data were fitted and the results are shown in fig. 6: in the 95% confidence interval, the CC50 value of aloperine on Vero cells gradually decreases along with the time, the value is reduced from 56.70-63.07 mug/mL for 12h to 36.67-42.64 mug/mL for 36h, the IC50 value is not greatly changed between 12h and 24h and is 2.70-4.10 mug/mL, and the increase is more for 36 h; from the above data, the selection index SI of aloperine on Vero cells for PEDV at three times was calculated and the results are shown in the table: over time, SI showed a decreasing trend from 15.09-21.69 for 12h to 6.87-16.35 for 36 h.

As shown in FIG. 6, the effect of Alo, CCK-8 method, on Vero cell viability was determined by resetting different concentrations, non-linear fitting was performed using Graphpad Prism, and CC was calculated at 95% confidence interval ₅₀ A value (A); based on the inhibition data of Alo on PEDV N protein expression in IFA results, nonlinear fitting was performed using Graphpad Prism, and IC50 value (B) of Alo on PEDV at 95% confidence interval was calculated.

TABLE 9 inhibition selection index of Alo against PEDV in Vero cells

7. Effect of aloperine on PEDV replication cycle results

Earlier studies have demonstrated the inhibitory effect of aloperin on PEDV, and subsequent studies have been conducted on the effect of aloperin on the life cycle of PEDV from four stages of virus adsorption (Attachment), invasion (Entry), replication (Replication) and Assembly (Assembly), the course of the study is shown in fig. 7, with different concentrations of aloperin and ribavirin as control agents intervening in different life cycles of the virus, while negative and positive controls are set, and the expression level of PEDV N protein is detected using Western blot method, and the results are shown in fig. 8 (a): according to the gray level analysis result, it can be found that, compared with the positive control group, the aloperine in the high-dose group shows a certain degree of virus inhibition effect in 4 stages of the virus life cycle, the expression quantity of virus N protein is different (P < 0.05), wherein the relative expression quantity difference of N protein in the high-dose aloperine group in the replication stage is obvious (P < 0.01); besides the high-dose aloperine group, the relative expression amount of N protein in the replication phase of the medium-dose aloperine group also shows a significant difference (P < 0.01) from that of the positive control group, which indicates that the inhibition effect of aloperine in the viral replication phase is obvious. The relative expression levels of N proteins of the other dose groups of aloperine and the ribavirin group serving as a control drug at other stages of the virus life cycle are not different (P > 0.05), but each group can be found to reduce the relative expression levels of the N proteins of the virus to different degrees, and no statistical difference exists in data.

According to the above results, the high dose aloperin group can inhibit the expression of viral N protein at various stages of the viral life cycle, so that the high dose aloperin can inhibit the transcription level of PEDV gene mRNA, and the transcription level of PEDV N and ORF3 gene mRNA is detected by using RT-qPCR method, and the result is shown in FIG. 8 (B): compared with the positive control group, the high-dose aloperin has extremely remarkable difference (P < 0.001) in the transcription level of N and ORF3 gene mRNA in the virus entering, replicating and assembling stages, wherein the mRNA in the replicating stage is most obviously reduced, and the mRNA in the adsorbing stage is not different (P > 0.05), but at the same time, the high-dose aloperin also reduces the transcription level of N and ORF3 gene mRNA in the adsorbing stage, but has no statistical difference in data.

Adsorption is the first step of PEDV infection, and different concentrations of Alo and 50 μg/mL were mixed with PEDV dilutions with moi=0.1 to pre-chilled cells for 2h, virus particles not adsorbed on the cells were washed off, PEDV dilutions were added to incubate for 24h, and cell extract proteins were collected.

After PEDV adsorption is completed, an invasion process is started. Different concentrations of Alo and control drugs were used to interfere with PEDV invasion cell stages. After cell pre-cooling, the cells were incubated with PEDV with moi=0.1 at 4 ℃, virus particles not adsorbed to the cell surface were washed off with pre-cooled PBS, transferred to a 37 ℃ incubator, and virus invasion was started. Alo was added at various concentrations at the beginning of the shift temperature to effect, and after 2h of effect, alo was washed off, PEDV diluent was added to culture for 24h, and then cell extract protein was collected.

For the influence of Alo on the replication process of PEDV nucleic acid, different concentrations of Alo and control drugs are added 4h after PEDV infection, the medicine liquid is washed off after 8h of intervention, PEDV diluent is added for continuous culture until 24h, and cell extract proteins are collected. For the assembly and release process of the virus, different concentrations of Alo and control drugs were added 12h after virus infection for intervention until 24h to collect cell extract proteins.

As shown in fig. 8, cells were harvested after treatment according to the PEDV different stage Alo intervention assay design to extract cells and viral proteins, PEDV N protein primary and HRP secondary antibodies were incubated, and images (a) were collected and plotted using a protein molecular imaging system for gray scale analysis; cell and PEDV RNA were extracted by a similar procedure, RT-qPCR was performed using PEDV specific primers, data was analyzed and mapped; NS indicates no statistical difference, P indicates the presence of a statistical difference, and P indicates a significant difference compared to the model group (i.e., PEDV); * P represents the difference is very significant.

EXAMPLE 3 analysis of the results of the dehydrocornine study

1. Effect of dehydroevodiamine on Vero CCL81 and IPEC-J2 cell viability

Vero CCL81 and IPEC-J2 cell lines are cell lines commonly used in PEDV research, so in the example, toxicity tests of dehydroevodiamine (DHED) are carried out on the two cell lines, and the two cell lines are respectively processed for 12, 24 and 48 hours, and the measured absorbance values are calculated according to a formula to obtain cell activity data after different processing times, so that the results are shown in FIG. 9, and when the concentration of the dehydroevodiamine is 6.25 mug/mL on Vero cells, the cell activities of 12, 24 and 48 hours are above 90%, so that the maximum safe concentration of the dehydroevodiamine on the Vero cells is determined to be 6.25 mug/mL; on IPEC-J2 cells, when the concentration of dehydroevodiamine is 12.5 mug/mL, the cell activities of 12, 24 and 48 hours are all over 90 percent, so that the maximum safe concentration of dehydroevodiamine on IPEC-J2 cells is determined to be 12.5 mug/mL; in combination with cytotoxicity data of dehydroevodiamine on both cells, 6.25 μg/mL was finally selected as the maximum concentration for the subsequent antiviral assay.

As shown in fig. 9, after DHED treatment of cells 12, 24, 36h at different concentrations, absorbance at 450nm was detected using CCK-8 method, vero CCL81 (a) and IPEC-J2 (B) cell viability maps were plotted and analyzed after calculation according to cell viability formula, NS indicated no statistical difference compared to the blank group (i.e. concentration of 0), P indicated a statistical difference, P indicated a significant difference; * P represents the difference is very significant.

2. Time of action result of dehydroevodiamine for inhibiting PEDV virus

The maximum safe concentration of dehydroevodiamine, which has been determined by cytotoxicity test, was investigated for the duration of action of dehydroevodiamine against PEDV at this concentration, and the test procedure is shown in fig. 10 (a), and the investigation of different dosing times was performed separately: 2 hours before virus inoculation (Pre-treatment), simultaneous incubation of drug and virus (Co-treatment), post-treatment and full-course administration (Whole-treatment); as shown in FIG. 10 (B), the Pre-treatment process can not inhibit the expression of PEDV N protein, and Co-treatment and Post-treatment both inhibit the expression of PEDV N protein to a certain extent, and compared with the white-treatment, the Post-treatment has the strongest effect, so that the drug addition time of the subsequent test adopts the Co-treatment+post-treatment mode. As shown in fig. 10 (C), vero cells were inoculated with PEDV with moi=0.1, and after treatment with Co-treatment + Post-treatment for 48 hours with different concentrations of dehydroevodiamine (6.25, 3.13, 1.56 μg/mL), the cells of PEDV exhibited significant cytopathy compared with Control, and cells were greatly wrinkled and shed, and the test group with 1.56 μg/mL of dehydroevodiamine was able to significantly improve the situation, and cells of the 6.25 μg/mL test group showed little cytopathy as the cell state gradually returned to normal with increasing concentration.

As shown in fig. 10, DHED was designed for the experiment of the effect of inhibiting PEDV at different addition times (a); after PEDV inoculation and administration according to the experimental design, collecting cells 24h to analyze PEDV N protein expression (B) and comparing the influence of different administration times on PEDV N protein expression; the effect of inhibiting PEDV is verified by using Co+post-treatment mode, the DHED can obviously improve CPE (C) caused by PEDV, and the more red circles in the figure indicate that the more severe the CPE.

3. Dehydroevodiamine reduces TCID of PEDV infected cells ₅₀ Results

According to the previous investigation of the action time of inhibiting PEDV by dehydroevodiamine and the action effect of different concentrations of dehydroevodiamine, the virus TCID under different treatment times of the dehydroevodiamine is carried out ₅₀ Is measured. The results are shown in FIG. 11, where dehydroevodiamine was treated for 12h, compared to the positive control, virus TCID ₅₀ There was little difference in the decrease, and the high dose dehydroevodiamine group (6.25. Mu.g/mL) and the control drug ribavirin group (100. Mu.g/mL) showed a difference (P)<0.05 A) is provided; dehydroevodiamine treatment for 24h, high dose of virus TCID of dehydroevodiamine group (6.25 μg/mL) and control drug ribavirin group compared with positive control ₅₀ The difference in drop was very significant (P<0.001 Dehydroevodiamine is stronger than ribavirin Lin Zuoyong); medium dose dehydroevodiamine group (3.13. Mu.g/mL) virus TCID ₅₀ The drop off showed a difference. Dehydroevodiamine treatment for 48h, removing high dose virus TCID of dehydroevodiamine group ₅₀ The difference in drop was very significant (P<0.001 Other groups were not different, indicating that the high dose dehydroevodiamine was more durable compared to ribavirin.

As shown in FIG. 11, each group of viruses was collected for virus TCID using IFA ₅₀ The assay, NS, indicates no statistical difference, P indicates significant difference compared to the model group (i.e., PEDV-0.1)The method comprises the steps of carrying out a first treatment on the surface of the * P represents the difference is very significant.

4. Flow cytometry detection result for reducing PEDV infected cells by dehydroevodiamine

According to the action time of the early dehydroevodiamine and the virus TCID ₅₀ As a result, the effect of different concentrations of dehydroevodiamine and ribavirin as a control drug on the proportion of infected cells after virus infection is detected by using a flow cytometry method, wherein the concentrations of the dehydroevodiamine are 6.25, 3.13 and 1.56 mug/mL, and the concentrations of the ribavirin as a control drug are 100 mug/mL. The results are shown in FIG. 12: compared with the positive control group (PEDV-0.1), the high and medium dose dehydroevodiamine groups have extremely obvious ratio difference of infected cells under the treatment time of 12, 24 and 48 hours (P) <0.001 The difference of the dehydroevodiamine group at low dose (1.56 mug/mL) under 12h treatment time is very obvious (P)<0.001 The effects of 24 hours and 48 hours are obviously weakened. As the contamination time increases, the ratio of the contaminated cells of the positive control group (PEDV-0.1) increases, the ratio of the contaminated cells of 24 hours approaches 100%, peaks have been reached, and the periods of 48 hours and 24 hours are almost equal. By observing the effect of ribavirin as a control drug, the effect of ribavirin groups (Rib-100 mug/mL) is obvious between high and medium dose dehydroevodiamine groups on the aspect of reducing the proportion of contaminated cells in the treatment time of 12 hours and 24 hours, but the effect of ribavirin groups is weakened in the 48-hour treatment time, and the proportion of contaminated cells is higher than that of the dehydroevodiamine low dose groups, so that the effect time of inhibiting PEDV by dehydroevodiamine is longer, the capability of inhibiting PEDV by the high dose dehydroevodiamine groups is always in a stable state, and the effect is not reduced with the time.

As shown in fig. 12, DHED at different concentrations and Rib at 100 μg/mL treated PEDV infected cells, after 12, 24, 36h incubation, FITC-PEDV-N antibody was collected after cell permeabilization, FITC positive cell numbers were detected by flow cytometry (a), data statistics analysis was performed (B), NS indicated no statistical difference compared to model group (i.e., PEDV-0.1), P indicated statistical difference, P indicated significant difference; * P represents the difference is very significant.

5. Dehydroevodiamine reduces relative expression level of PEDV N protein

The influence of different concentrations of dehydroevodiamine and ribavirin serving as a control medicament after virus infection on the expression of virus N protein is detected by an indirect immunofluorescence method, wherein the concentration of the dehydroevodiamine is 6.25, 3.13 and 1.56 mug/mL, and the concentration of the ribavirin serving as the control medicament is 100 mug/mL. The results are shown in fig. 13 (a): the fluorescence images in each time phase are subjected to semi-quantitative analysis, the positive control group is subjected to normalization treatment and then is compared with other groups, the relative expression quantity of N protein is obviously reduced in the other groups under the treatment time of 12 hours, the difference between the high and medium dose dehydroevodiamine groups and the positive control group under the treatment time of 12 hours is obvious (P < 0.01), the treatment difference between the high and medium dose dehydroevodiamine groups and the positive control group under the treatment time of 24 hours is extremely obvious (P < 0.001), and the fluorescence images are obviously changed. Whereas the low dose dehydroevodiamine group only showed very significant differences at 24h treatment time (P < 0.001), only at 12 and 36h treatment time. As can be seen from the fluorescence graph, the expression of the N protein of the PEDV of the positive control group gradually increases from 12h to 24h, namely the green area of the graph gradually expands, which is unified with the detection result of the flow cytometry, and the 24h and the 48h are close to each other and keep the level. The effect of ribavirin in the control medicine group is observed, the effect of ribavirin in the ribavirin group (Rib-100 mu g/mL) is obvious in reducing the expression level of virus N protein in 12, 24 and 48 hours of treatment time, and the effect is extremely obvious (P < 0.001) compared with the positive control, but the effect is obviously lower than that of high-dose dehydroevodiamine from 24 hours, so that the effect of inhibiting the expression level of virus N protein is gradually weakened when the low-dose dehydroevodiamine group and the ribavirin group are prolonged along with time; the action time of inhibiting the N protein expression of the PEDV by the high and medium dosage dehydroevodiamine groups is longer, the effect is not reduced due to the extension of time, and the PEDV inhibition capability of the high and medium dosage dehydroevodiamine groups is always in a stable state in 12, 24 and 48 hours.

According to the results of detecting the N protein expression amount of PEDV and performing semi-quantitative analysis according to an indirect immunofluorescence test, the N protein expression of PEDV is subjected to more accurate western blot analysis on the influence of dehydroevodiamine, and the results are shown in FIG. 13 (B): after gray analysis is carried out on Western blot results, the relative expression quantity of N proteins of a high-dose dehydroevodiamine group is reduced in 12, 24 and 48h treatment time, and compared with a positive control group, the N proteins are extremely obviously different in 12h and 24h treatment time (P < 0.001), and the difference in 48h treatment time is obvious (P < 0.01); the relative expression quantity of N protein in the medium-dose dehydroevodiamine group is reduced in the treatment time of 12, 24 and 48 hours, compared with the positive control group, the N protein shows a significant difference (P < 0.01) in 12 hours, a very significant difference (P < 0.001) in 24 hours, and no difference (P > 0.05) in 48 hours; according to a western blot image, the relative expression amount of the N protein of the low-dose dehydroevodiamine group is reduced in the treatment time of 12, 24 and 48 hours, but no difference exists compared with a positive control group; the relative expression quantity of N protein is reduced to a certain extent in the control medicament ribavirin group in 12, 24 and 48h treatment time, wherein the relative expression quantity of N protein is extremely obviously different (P < 0.001) compared with that of the positive control group in the 12h and 24h treatment time, and no difference (P > 0.05) appears in 48h, which indicates that the ribavirin cannot inhibit the expression of viral N protein in 48 h. From the results, the high-dose dehydroevodiamine group has obvious advantages in reducing the relative expression quantity of the viral N protein, has lasting effect and is superior to the ribavirin serving as a control drug, and the results also correspond to the same trend of FCM and IFA detection.

As shown in fig. 13, PEDV-infected cells were treated with DHED at different concentrations and Rib at 100 μg/mL, after incubation for 12, 24, 36h, the cells were collected for incubation with PEDV-N antibodies, fluorescent microscopy after FITC secondary incubation was observed and pictures were taken for semi-quantitative analysis (a), blue fluorescence represented DAPI-stained nuclei and green fluorescence represented FITC-stained PEDV N protein; collecting cells after the same treatment, extracting proteins, performing Western Blot analysis, incubating an HRP secondary antibody, and then collecting images (B) by a protein molecular imaging system, and performing gray analysis on WB strips by using an Image J and making a graph; NS indicates no statistical difference, P indicates statistical difference, and P indicates significant difference compared to the model group (i.e., PEDV-0.1); * P represents the difference is very significant.

6. N and ORF3 gene mRNA transcription results of PEDV inhibited by dehydroevodiamine

The previous results have shown that dehydroevodiamine can significantly reduce the expression level of viral proteins, and the subsequent analysis of the mRNA transcription level of viral N and ORF3 genes is performed using a real-time fluorescent quantitative PCR method, with the N gene mRNA transcription results shown in fig. 14 (a): the high dose dehydroevodiamine group and the control drug ribavirin group showed reduced levels of transcription of the N gene mRNA and exhibited very significant differences (P < 0.001) compared to the positive control group at 12, 24, and 48h treatment times, wherein the high, medium, and low dose dehydroevodiamine groups all exhibited very significant differences (P < 0.001) at 24h treatment times; at the treatment time of 12h and 48h, the N gene mRNA transcription level of the medium and dosage dehydroevodiamine groups is also reduced, but compared with 24h, the effect is poorer, the peak value is probably not reached in 12h due to virus proliferation, and the excessive viral load is probably caused in 48 h. The PEDV ORF3 protein has the function of regulating the output of PEDV particles, the mRNA transcription result of the ORF3 gene is shown as a figure 14 (B), the mRNA transcription of the ORF3 gene shows the trend that the N gene is basically the same, the mRNA transcription level of the ORF3 gene of each dosage group of dehydroevodiamine is reduced under the treatment time of 12, 24 and 48 hours, the high dosage of dehydroevodiamine group shows extremely obvious difference (P < 0.001) with the positive control group in each time group, the medium and low dosage of dehydroevodiamine group is unstable, the medium dosage group shows extremely obvious difference (P < 0.001) in 12 hours and 24 hours, and the no difference (P > 0.05) is shown in 48 hours; the low dose group exhibited very significant differences at 24h, 48h (P < 0.001), and 12h exhibited significant differences (P < 0.01); the ribavirin group as a control drug can reduce the mRNA transcription level of the ORF3 gene in 12h and 24h, and the difference is obvious (P < 0.01), but at the treatment time of 48h, the mRNA transcription level of the ORF3 gene is increased, and the mRNA transcription level is extremely obvious (P < 0.001) compared with that of a positive control group, so that the effect of ribavirin in 48h is not obvious, and the effect is consistent with the Western blot result.

As shown in fig. 14, PEDV-infected cells were treated with DHED at different concentrations and Rib at 100 μg/mL, after 12, 24, 36h incubation, total RNA was extracted after cell lysis collected, real-time fluorescent quantitative PCR assays were performed using PEDV N (a) and ORF3 (B) -specific primers and data processing analysis was performed after normalization of the model set; NS indicates no statistical difference, P indicates the presence of a statistical difference, and P indicates a significant difference compared to the model group (i.e., PEDV); * P represents the difference is very significant.

7. CC of dehydroevodiamine on Vero cells ₅₀ And its IC for PEDV ₅₀ And SI (information and information)

CC for dehydroevodiamine on Vero cells based on data of early cytotoxicity and indirect immunofluorescence ₅₀ And IC for inhibiting PEDV ₅₀ The data were fitted and the results are shown in fig. 15: CC of dehydroevodiamine on Vero cells with time extension at 95% confidence interval ₅₀ The value gradually decreases from 14.15-17.36 mug/mL for 12h to 11.60-12.25 mug/mL for 48h, and IC ₅₀ The value is not greatly changed between 12h and 24h, and is increased to 3.087-4.037 mug/mL in 48h between 2.4-3.6 mug/mL; from the above data, the index of selection SI of dehydroevodiamine on PEDV in Vero cells was calculated over three times and the results are shown in tables 3-10: as time increases, the value of SI decreases.

TABLE 10 inhibition selection index of DHED on PEDV in Vero cells (95% confidence interval)

As shown in FIG. 15, the effect of the CCK-8 method on Vero cell viability was determined by resetting DHED at different concentrations, performing a nonlinear fit using Graphpad Prism, and calculating CC at 95% confidence interval ₅₀ A value (A); based on the inhibition data of Alo on PEDV N protein expression in IFA results, nonlinear fitting was performed using Graphpad Prism, and IC of DHED on PEDV at 95% confidence interval was calculated ₅₀ Value (B).

Effect of 1.4.8 dehydroevodiamine on the replication cycle of PEDV

Earlier studies have demonstrated the inhibitory effect of dehydroevodiamine on PEDV, the subsequent studies were conducted on the effect of dehydroevodiamine on the life cycle of PEDV, from four stages of virus adsorption (Attachment), invasion (Entry), replication (Replication) and Assembly (Assembly), the study procedure is shown in fig. 7, the intervention of dehydroevodiamine and control drug ribavirin was conducted at different concentrations during different life cycles of virus, while negative and positive controls were set, and the N protein expression level of PEDV was detected using Western blot method, the results are shown in fig. 16 (a): according to the gray level analysis result, compared with the positive control group, the dehydroevodiamine in the high dose group has stronger virus inhibition effect in the replication stage of the virus life cycle, the virus N protein expression quantity difference is extremely obvious (P < 0.001), and the virus N protein expression quantity in other stages is reduced but has no statistical difference (P > 0.05); in addition to the high-dose dehydroevodiamine group, the N protein relative expression of the medium-dose dehydroevodiamine group in the replication stage also shows a reduced trend, but no statistical difference (P > 0.05), and in other stages of the virus life cycle, the N protein relative expression is also reduced to a certain extent, but no statistical difference (P > 0.05). The low-dose dehydroevodiamine group has no obvious trend of reducing the relative expression amount of N protein at each stage of the virus life cycle and no statistical difference (P > 0.05). The relative expression of N protein in the control drug ribavirin group is obviously reduced and shows variability (P < 0.05) in the virus replication stage, but the effect is not obvious in other stages, and no statistical difference (P > 0.05) exists.

According to the above results, the high dose of dehydroevodiamine group can obviously inhibit the expression of viral N protein in the replication stage of the viral life cycle, so that the subsequent determination of the transcription level of PEDV mRNA by using the high dose of dehydroevodiamine is carried out, and the transcription level of PEDV N and ORF3 gene mRNA is detected by using an RT-qPCR method, and the result is shown in FIG. 16 (B): compared with the positive control group, the high-dose dehydroevodiamine shows extremely remarkable difference (P < 0.001) in the transcription level of N and ORF3 gene mRNA in the virus entry, replication and assembly stages, wherein the mRNA in the replication stage has the most obvious trend, but at the same time, the high-dose dehydroevodiamine can not reduce the transcription level of the virus gene in the adsorption stage, and no difference occurs (P > 0.05).

As shown in fig. 16, cells were harvested after treatment according to the PEDV different stage Alo intervention assay design to extract cells and viral proteins, and images (a) were collected and plotted for gray scale analysis using a protein molecular imaging system after incubation of PEDV N protein primary and HRP secondary antibodies; extracting cells and PEDV RNA, performing RT-qPCR by using PEDV specific primers, analyzing data and mapping; NS indicates no statistical difference, P indicates the presence of a statistical difference, and P indicates a significant difference compared to the model group (i.e., PEDV); * P represents the difference is very significant.

Example 4 analysis of results of combination of Sophocarpidine, dehydroevodiamine and ribavirin

1. anti-PEDV for aloperine, dehydroevodiamine and ribavirin Lin Lian

According to the results of the early-stage test, the combination of the aloperine, the dehydroevodiamine and the ribavirin is used for resisting PEDV, the relative expression quantity of PEDV N protein is measured, and the results are shown in figure 17, compared with a positive control group, the relative expression quantity of PEDV N protein in other groups is reduced, wherein the effects of the medium-dose and low-dose aloperine groups and the medium-dose dehydroevodiamine groups are very remarkable (P<0.001 Unified with the earlier results, and further, the medium-dose aloperine group has the best effect, the 25 mug/mL ribavirin+medium-dose aloperine group is next to the medium-dose dehydroevodiamine group, but the 25 mug/mL ribavirin+medium-dose aloperine and medium-dose dehydroevodiamine group have little difference; 25 μg/mL ribavirin + medium dose sophocarpidine group and 25 μg/mL ribavirin + medium dose dehydroevodiamine group also showed very significant effects (P)<0.001 A) is provided; the 25 μg/mL ribavirin+low dose matrine group and the 25 μg/mL ribavirin+low dose dehydroevodiamine group also showed significant differences (P)<0.01 However, the effect was worse than that of aloperine alone at low dose, which suggests 25μg/mLRibavirin cannot combine with low-dose aloperine and low-dose dehydroevodiamine monomer to exert an antiviral effect which is obviously stronger than that of low-dose aloperine alone; while the single use of ribavirin and low-dose dehydroevodiamine can reduce the relative expression of PEDV N protein, the effect is not obviously different (P)>0.05 The difference between the single functions and effects is not great; wherein 25 mug/mL ribavirin and low-dose dehydroevodiamine can be combined to obviously reduce the relative expression quantity (P) of PEDV N protein<0.01 The results also demonstrate that 25 μg/mL ribavirin and low dose dehydroevodiamine are combinedThe effect is better than that of two medicines singly. Based on the above results, in the drug combination test, 25 μg/mL ribavirin combined with two monomers at different concentrations can exert antiviral effects stronger or weaker than those of two alkaloids alone, obviously, the effect is optimal, and other western medicine components such as ribavirin cannot obtain obvious effects stronger than those of the aloperine and the dehydroevodiamine, and even the effects of the combination of the two monomers after the combination of the ribavirin with the aloperine monomer and the dehydroevodiamine monomer are needed to be further explored.

As shown in fig. 17, three drugs with different concentrations are combined with each other to treat infected cells, and protein extraction is performed after the cells are collected, and Westen Blot analysis is performed; NS indicates no statistical difference compared to the model group (i.e., PEDV), indicating significant difference (×p < 0.01; P < 0.001).

2. Combination of aloperine and dehydroevodiamine for resisting PEDV

In summary, the above results show that the aloperine can inhibit the expression of viral N protein in each stage of the virus life cycle, and the dehydroevodiamine has more obvious effect in the virus replication stage, so that the antiviral test of combination medication of aloperine and dehydroevodiamine for 24 hours is carried out, the antiviral effect of high-concentration aloperine and dehydroevodiamine is taken as a reference, and the results are shown in fig. 18 (A), compared with the positive control group, the other groups can reduce the relative expression quantity of PEDV N protein, and the effect is extremely obvious (P < 0.001); meanwhile, the effect of the drug combination of Alo-6.25+DHED-6.25 and Alo-3.13+DHED-3.13 on inhibiting PEDV protein is found to be almost the same as that of the high-concentration aloperine group, and the statistical analysis is not different. The antiviral effects of the medium-concentration aloperine and dehydroevodiamine are taken as references, and the results are shown in the figure 18 (B), and compared with a positive control group, the relative expression quantity of PEDV N protein can be reduced in other groups, and the effect is extremely remarkable (P < 0.001); meanwhile, the effect of inhibiting PEDV protein by the drug combination of dehydroevodiamine and aloperine (Alo-3.13+DHED-3.13 and Alo-1.56+DHED-1.56) is almost the same as that of the high-concentration dehydroevodiamine group, and the statistical analysis is not different, so that the results show that the drug dosage can be reduced by using two monomers together, and the same effect can be obtained.

As shown in fig. 18, two monomers with different concentrations are combined with each other to treat infected cells, and protein extraction is performed after the cells are collected, and Westen Blot analysis is performed; NS indicates no statistical difference compared to the model group (i.e., PEDV), indicating significant differences (×p < 0.01; P < 0.001), and two groups compared to each other have been shown in the figure.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that will occur to those skilled in the art are included within the invention without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An alkaloid preparation, which is characterized by comprising aloperine and evodiamine,

the evodiamine is dehydroevodiamine,

the mass concentration ratio of the aloperine to the dehydroevodiamine is 1:1,

the preparation has the activity of resisting/inhibiting porcine epidemic diarrhea virus.

2. The alkaloid preparation according to claim 1, wherein,

the effective concentration of the aloperine is 1 mu g/mL to 25 mu g/mL;

the effective concentration of the dehydroevodiamine is 1 mu g/mL to 10 mu g/mL.

3. The alkaloid preparation according to claim 2, wherein the mass ratio of aloperine and dehydroevodiamine is the ratio of the effective weights of the above concentration ratios.

4. Use of an alkaloid formulation according to any of the claims 1-3 for the preparation of a medicament against porcine epidemic diarrhea virus.