CN117752693A - Application of galli extract in preparation of medicines for preventing and treating porcine epidemic diarrhea - Google Patents

Abstract

The invention provides application of a galli extract (TGE) in preparation of a medicament for preventing and treating porcine epidemic diarrhea, and belongs to the technical field of veterinary medicines. The invention provides that the Gallic acid extract (comprising meta-digallic acid (BA), ellagic Acid (EA) and Gallic Acid (GA)) has the effect of treating porcine epidemic diarrhea for the first time, and the meta-digallic acid, the Gallic acid or the ellagic acid is obtained by first screening and is a potential inhibitor of porcine epidemic diarrhea virus 3C-like protease (3 CLpro), and the replication of the porcine epidemic diarrhea virus can be effectively blocked by inhibiting the function of 3CLpro. The invention provides the effect of the gallic extract on regulating the intestinal flora of pigs suffering from porcine epidemic diarrhea for the first time.

Description

Application of galli extract in preparation of medicines for preventing and treating porcine epidemic diarrhea

Technical Field

The invention belongs to the technical field of veterinary medicines, and particularly relates to application of a galli extract in preparation of a medicine for preventing and treating porcine epidemic diarrhea.

Background

Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is an infectious intestinal disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrheavirus, PEDV). PEDV is an enveloped, positive single stranded RNA virus of the subfamily coronaviridae and belongs to the alpha coronavirus (alphacorenavirus). PEDV affects piglets more severely than adult pigs and is generally manifested by symptoms such as vomiting, diarrhea in the fluid, dehydration, anorexia and weight loss. Faecal transmission is the primary transmission mode of PEDV. The most critical method for preventing and controlling PED is vaccination. Live attenuated and inactivated vaccines against PEDV are currently available. However, there is currently no effective control method due to the extreme unpredictability of viruses, the weakness of vaccines, and the uniqueness of intestinal immunity. In addition to vaccine immunization, chemically synthesized drugs, hormones, antibiotics, etc. have also been used to relieve diarrhea symptoms. Effective antiviral drugs are urgently needed to remedy the lack of immunoprotection in vaccines. The plant components have the advantages of no drug residue and no pollution, and become a popular antiviral research direction at present.

The botanical drug has wide research prospect in the treatment of diseases due to the overall comprehensive regulation and control advantages of wide sources, various components, unique structure, small toxic and side effects, low drug resistance and the like. In recent years, more and more researches show that Chinese herbal medicines have the activity of resisting PEDV infection, and some natural compounds and Chinese herbal medicine compounds have good application potential. Substances that have been shown to successfully inhibit PEDV replication include quercetin, quercetin-7-rhamnoside (Q7R), aloe vera extract and the like. However, plant-derived natural products have relatively shallow studies of anti-PEDV activity, and their activity screening is mainly at the cellular level, animal studies are few, and use of a sufficient sample size is not clinically performed. Furthermore, the active ingredient and the mechanism of action of the active ingredient are not pointed out in the report of the extract of natural medicine. In summary, no effective anti-PEDV drug has been developed yet, and development of an effective antiviral drug is urgently required.

The gallnut is a dried insect gall produced by larvae of the insect galla nutraceae-tinctoria Oliv. Of the family gallinaceae, which are parasitic on shoots of the plant galli nutraceutica Quercus infectoria Oliv. Of the family Fagaceae. Among them, the total of gallic polyphenols is 15 in 3, and the gallic tannins composed of 1-7 galloylglucoses are most. At present, no research on the effect of the gallic extract on preventing and treating porcine epidemic diarrhea exists.

Disclosure of Invention

In view of the above, the invention aims to provide application of the gallic acid extract in preparing medicines for preventing and treating porcine epidemic diarrhea.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides application of a galli extract in preparing a medicament for preventing and treating porcine epidemic diarrhea.

The invention also provides application of the gallic acid extract in preparing a product for regulating pig intestinal flora.

The invention also provides application of the gallic acid extract in preparing a porcine epidemic diarrhea virus 3C-like protease (3 CLpro) inhibitor.

The invention also provides a medicament for preventing and treating porcine epidemic diarrhea or regulating porcine intestinal flora, and the active ingredient of the medicament comprises a gallic extract.

The invention also provides a porcine epidemic diarrhea virus 3CLpro inhibitor, and the active ingredient of the inhibitor comprises a gallic extract.

Preferably, the gallic extract is a gallic water extract.

Preferably, the gallic extract comprises gallic acid, ellagic acid or meta-digallic acid.

Preferably, the preparation method of the galli extract comprises the following steps: mixing Galla Turcica with water, boiling, concentrating under reduced pressure to paste, and lyophilizing to obtain Galla Turcica extract.

The invention has the beneficial effects that:

the invention provides the effect of the gallic acid extract (comprising Gallic Acid (GA), ellagic Acid (EA) and meta-digallic acid (BA)) for treating the porcine epidemic diarrhea for the first time, and the potential inhibitors of PEDV3CLpro, which are obtained by screening BA, EA and GA for the first time, can effectively block the replication of the porcine epidemic diarrhea virus by inhibiting the function of 3CLpro. The invention provides the effect of the gallic extract on regulating the intestinal flora of pigs suffering from porcine epidemic diarrhea for the first time.

Drawings

FIG. 1 shows TGE to improve survival rate of sick piglets, wherein A is a schematic diagram of an experimental scheme; b is the diarrhea index of pedv+tge group over time, the values shown are mean ± SD, n=102. Significant differences compared to day one are expressed as p <0.05, p <0.01, p < 0.001; c is a survival curve and HR (Hazard Ratio) is the relative value calculated using the COX proportional-risk regression model to quantify the overall difference between the two survival curves. Smaller HR indicates lower risk of mortality after treatment. The values shown are mean ± SD, n (PEDV) =101, n (pedv+tge) =102, the significant difference being expressed as p <0.001 compared to the PEDV group;

FIG. 2 shows the TGE-dependent diarrhea index and organ index, wherein A is the course of treatment of piglets; b is weight change; c is diarrhea index change; d is cardiac index; e is liver index; f is spleen index; g is the lung index; h is kidney index, the numerical value is represented by mean ± SD, statistical significance adopts single factor analysis of variance, p <0.01, p <0.001, ns have no significant difference;

FIG. 3 is a box plot of Alpha diversity for Chao 1, observed_ species, shannon index and Simpson index, respectively, for TGE treatment to alter intestinal microbiota diversity; e is an inter-group difference box graph; f is an NMDS diagram; values are expressed as mean ± SD, n=4, significant differences compared to PEDV groups expressed as p <0.05, < p <0.01, < p < 0.001;

FIG. 4 is a graph showing the analysis of the classification composition of the intestinal microbiota significantly altered by TGE treatment, A and C being the phylum and genus levels respectively, B and D being the thermal graph of the composition of the species at the phylum and genus levels respectively, E being the effect of TGE on the levels of representative phylum, F being the effect of TGE on the levels of representative harmful bacteria, G being the effect of TGE on the levels of representative beneficial bacteria, H being the Venn graph; values are expressed as mean ± SD, n=4, significant differences compared to PEDV groups expressed as p <0.05, < p <0.01, < p < 0.001;

FIG. 5 shows LEfSe analysis of dominant bacteria of each group, wherein A is the difference between the PEDV group and PEDV+TGE, B is the difference between the PEDV group and PEDV+EN, C is the difference between the PEDV group and CG group, the left graph in A, B, C shows LEfSe analysis of corresponding dominant bacteria of two groups, and the right graph shows the difference bacteria with LDA score of greater than 4 of the corresponding groups;

FIG. 6 is a flow chart of an experiment in which TGE inhibits replication of PEDV in Vero E6, wherein A is TGE inhibits PEDV; b is CCK8 method to measure cell activity; c is the detection of viral N gene level by qPCR; d is the collection of supernatants for virus titration (TCID 50) assay, the values are expressed as mean ± SD, n=3, significant differences are expressed as p <0.05, p <0.01, p <0.001 compared to PEDV group;

FIG. 7 shows immobilization of BL21-pET-28a-3CLpro protein and screening of active components, wherein A is expression and purification of BL-21 cells transformed with recombinant plasmid pET-28a-3CLpro by SDS-PAGE, wherein columns 1, 2, 3 and 4 are wash samples for protein purification and columns 5, 6, 7 and 8 are wash samples for protein purification; b gel electrophoresis of the concentrated protein; c is the response of pET-28a-3CLpro protein after dilution in sodium acetate buffer with different pH values; d is a sensor diagram showing immobilization of BL21-pET-28a-3CLpro protein at pH 4.5 by amino coupling; e is a response signal of 5 pre-screening concentrations;

FIG. 8 is a total ion flow diagram (TIC) of the three compounds identified for LC-MS, wherein A, B and C are TGE, D, E, F are EIC, primary and secondary mass spectra of BA, EA and GA in TGE, respectively, G, H, I are EIC, primary and secondary mass spectra of BA, EA and GA in TGE recovered sample, J, K, L are EIC, primary and secondary mass spectra of BA, EA and GA standard, respectively;

FIG. 9 shows the better affinity results for three compounds with 3CLpro, wherein A and B are a series of concentration fit curves and affinity test plots for GC-376 and BL21-pET-28a-3CLpro, respectively, C and D are a series of concentration fit curves and affinity test plots for BA and BL21-pET-28a-3CLpro, respectively, E and F are a series of concentration fit curves and affinity test plots for EA and BL21-pET-28a-3CLpro, respectively, and G and H are a series of concentration fit curves and affinity test plots for BA and BL21-pET-28a-3CLpro, respectively, wherein GC376 is an inhibitor of 3 CLpro;

FIG. 10 shows the molecular docking results of a-D for GC376, BA, EA and GA, respectively, for the small molecule targeting PEDV3CLpro active pocket, E for RMSD of three compounds with 3CLpro active pocket, F for distance of three compounds from 3CLpro active pocket, G for number of hydrogen bond interactions of three compounds with 3CLpro active pocket, 3CLpro model based on co-crystal structure of 3CLpro dimer (PDB: 4 XFQ);

fig. 11 shows three compounds inhibiting replication of PEDV in Vero E6, wherein A, C and E are the CCK8 assay cell viability results of BA, EA and GA, respectively, B, D, F are the results of BA, EA and GA treatment of Vero E6 cells, using qPCR to detect viral N gene level, values expressed as mean ± SD, n=3, significant differences compared to PEDV group expressed as p <0.05, p <0.01, p < 0.001;

fig. 12 is a flowchart of the technical idea of the present invention.

Detailed Description

The invention provides application of a galli extract in preparing a medicament for preventing and treating porcine epidemic diarrhea, and in preparing a product for regulating porcine intestinal flora and preparing a PEDV3CLpro inhibitor.

In the present invention, the gallic extract is preferably a gallic water extract, and the gallic extract preferably contains one or more of gallic acid, ellagic acid and meta-digallic acid. In the present invention, the gallic extract is preferably prepared by the steps of: mixing Galla Turcica with water, boiling, concentrating under reduced pressure to paste, and lyophilizing to obtain Galla Turcica extract.

The specific source of the gallnut is not particularly limited in the present invention, and the water is preferably distilled water. The volume ratio of the weight of the gallnut to the water is preferably 1g:3mL, the number of boiling times is preferably 3, and the time of each boiling is preferably 2h. In the present invention, the temperature of the reduced pressure concentration is preferably 60 to 70℃and the reduced pressure concentration is preferably reduced to 0.08 to 0.1MPa by using a rotary evaporator. In the present invention, the conditions for the freeze-drying are preferably-90℃and 3-4Pa for 8-10 hours.

The invention also provides a medicament for preventing and treating porcine epidemic diarrhea or regulating porcine intestinal flora, and the active ingredient of the medicament comprises a gallic extract.

In the medicament of the invention, the gallic extract is preferably a gallic water extract, and the gallic extract preferably comprises one or more of BA, EA and GA. In the medicine of the present invention, the preparation method of the galli extract is the same as that described above, and will not be described in detail herein.

The invention also provides a PEDV3CLpro inhibitor, the active ingredient of which comprises a gallic extract, more preferably one or more of BA, EA and/or GA.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In the following examples, conventional methods are used unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1

The preparation method of the Galla Turcica extract comprises the following steps: galling (purchased from Xinjiang Ansal pharmaceutical Co., ltd.) was crushed, and the powder was sieved through a 80 mesh sieve, and the powder was sieved. Accurately weighing 60.0g of sieved powder, adding 180mL of distilled water, boiling for 3 times, filtering the extract with 4 layers of sterile gauze after each extraction is finished, and continuing to use the precipitate for extraction, finally mixing 3 times of filtrate (namely the extract) for subsequent decompression concentration, decompressing and concentrating the filtrate into paste (the filtrate is added into a 1000mL round bottom flask in a divided manner, decompressing to 0.08Mpa by a rotary evaporator until all filtrate is concentrated into paste at the temperature of 60 ℃), freeze-drying (the concentrated paste extract is respectively paved in dry and clean glass dishes with the diameter of 10mm and the thickness of 1 mm), completely wrapping the culture dishes with a sealing film, then tying a plurality of small holes on the sealing film by using a clean syringe needle, horizontally placing the culture dishes in a refrigerator at the temperature of minus 20 ℃ overnight, freezing and drying the next day, wherein the condition of the freezing and drying is minus 90 ℃ and the freezing and drying for 8 h), and obtaining the powder after the crushing, namely the total nutgall extract (TGE).

Example 2

Experiment of the effectiveness of TGE obtained in example 1 in treating porcine epidemic diarrhea

The test adopts a single-factor test design, and selects a Du X long X big ternary hybrid to naturally infect piglets. Two groups were provided, PEDV and pedv+tge, respectively. The PEDV group randomly selected 9 litter (101 head) 1-6 day old sick piglets, which were not treated with additional treatment other than normal daily diet. The pedv+tge group randomly selected 9 litter (102 head) 1-6 day old diseased piglets. TGE lavage (300 mg/kg) 3 times daily was administered for 4 consecutive days. Both groups were given breast feeding to piglets, and after the end of the administration, the mental state, vomiting and diarrhea conditions of the piglets were continuously observed, and vital signs of the piglets were evaluated (observation of diarrhea degree, mental state, vomiting degree and survival rate of the piglets were performed during 3-10 days). The piglet survived until day 10 had good mental status, no vomiting and normal faeces. The administration and observation procedure described above is shown in fig. 1A.

The results suggest that TGE can improve diarrhea symptoms in affected piglets (see fig. 1B) and increase survival of piglets (see fig. 1C).

Example 3

In vivo test of TGE obtained in example 1 for treatment of porcine epidemic diarrhea

The test adopts a single factor test design, 18 piglets which are naturally infected by 6-8 day old Du X long X big ternary hybridization with similar weight are selected, and the male and female piglets are respectively divided into 3 groups at half random. Simultaneously 6 healthy piglets of the same age (6-8 days old) were selected, each group being repeated 6 times. The following groups of piglets were individually raised. The piglets in the PEDV+TGE group were given TGE 300mg/kg for gastric lavage. The piglets of the negative control group (PEDV group) were fed the same dose of physiological saline as pedv+tge group. The piglets of PEDV+EN group are drenched with enrofloxacin 7mg/kg. The healthy control group (CG group) was given the same dose of physiological saline as pedv+tge group. The test designs of the above groups are shown in table 1.

TABLE 1 in vivo test designs for different groups

Each group of piglets was intragastrically 3 times daily, and the weight of the piglets and the condition of excrement were recorded before the intragastric administration in the morning and evening, respectively. The fecal scoring criteria were as follows: 0: normal; 1: soft; 2: soft stool with water sample; 3: watery feces with small amounts of solids; 4: watery diarrhea. The experimental period was 10 days, the adaptation period was 2 days before the experiment, the continuous administration was carried out for 4 days from 3 to 6 days, and the other treatments were the same, and the treatment process is shown in fig. 2A. After 12h of fasting on day 10, 4 piglets per group were sacrificed for a section observation and organ quality was recorded. The intestinal contents were taken for subsequent analysis.

The results showed that PEDV group developed symptoms characterized by acute vomiting and watery diarrhea, 2 piglets died, while none of the other groups died, with lower incidence of vomiting and diarrhea (fig. 2C). The weight gain of pedv+tge piglets was also higher than PEDV (fig. 2B). The organ indexes show that after administration, the organ coefficients of piglets can be effectively improved (fig. 2D-2H), the spleen coefficients of the sick piglets are obviously improved by the PEDV+EN group (fig. 2F), and meanwhile, the lung coefficients of the sick piglets are obviously improved by the PEDV+TGE group and the PEDV+EN group (fig. 2G), so that other diseases have no obvious difference. In conclusion, TGE has a certain protection effect on piglet PEDV infection.

Example 4

TGE obtained in example 1 alters the intestinal flora structure of sick pigs

The test group design was the same as in example 3. The specific group is as follows: PEDV group (negative control group, i.e. diseased piglet, group without drug intervention); (2) Pedv+tge group (group of gallic extract obtained in example 1 administration); (3) Pedv+en group (enrofloxacin-administered positive drug treatment group) (4) CG group (healthy, uninfected virus group). Intestinal contents were sampled using sterile swabs and rapidly transferred to liquid nitrogen for delivery to Shanghai Paenox Biotechnology Inc. for 16S rRNA sequencing. Total genomic DNA samples were extracted and stored at-20℃prior to further analysis, followed by determination of the amount and quality of extracted DNA. The bacterial 16S rRNA gene V3-V4 region was PCR amplified using forward primer 338F (5 '-barcode+ ACTCCTACGGGAGGCAGCA-3') (SEQ ID NO. 1) and reverse primer 806R (5 '-GGACTACHVGGGTWTCTAAT-3', wherein H represents A/T/C, V represents G/A/C, W represents A/T) (SEQ ID NO. 2) and a sample-specific 7bp barcode was included into the primers for multiplex sequencing. Finally, the 2 nd pair of terminal (250 bp) sequencing was performed using the illuminaNovaSeq platform and NovaSeq 6000SP kit (500 cycles). Sequence data analysis was performed using QIIME2 and R software package (v3.2.0).

The results showed that there was no significant difference in the alpha diversity of the intestinal flora of piglets in the pedv+tge group from the PEDV group (figures 3A, 3B, 3C and 3D). But there was significant inter-group variability (fig. 3E). A non-metric multidimensional scaling (NMDS) analysis with a strength coefficient (Stress) of 0.132 showed significant separation between groups (fig. 3F).

Furthermore, at the gate level, pedv+tge group significantly increased the abundance of bacteroides gates in infected piglets, decreasing the abundance of proteus gates (fig. 4A, 4B and 4E). At the genus level, TGE has a tendency to reduce the abundance of lactobacillus, shigella and truffle (fig. 4C, 4D and 4F). Also, the abundance of faecalis, rogowski, prasuvorexant and prasugrel can be increased, but no significant differences were found (fig. 4G). In the Venn plot of ASV/OTU abundance table, the total ASV/OTU for the PEDV group was found to be 1649, the PEDV+EN group was found to be 3354, and the number of PEDV+TGE groups reached 5449, which is closer to 5158 for the CG group. This suggests that TGE and EN can increase the number of ASV/OTUs (fig. 4H).

The specific groups of bacteria were determined using a LEfSe analysis with LDA score > 4.0. As a result, as shown in fig. 5, each node represents a different classification group, and a white node represents no significant difference in classification properties; the taxonomic branching diagram shows the classification grade relation from door to genus (from inner circle to outer circle) main classification units in the sample community; node size corresponds to the average relative abundance of the taxa; the hollow nodes represent classification units with insignificant differences among groups, and the blue and red nodes indicate that the classification units represent significant differences among groups and have higher abundance in the group samples represented by the color; letters identify the names of the taxa that differ significantly from group to group, as can be seen in fig. 5, pedv+tge group shows a higher content of beneficial bacteroides, prasuvorexant and bacillus bacteria compared to PEDV group (fig. 5A), which is consistent with that shown in CG group (5C). In comparison of pedv+en with PEDV, PEDV group showed higher levels of proteus, enterobacteria and shigella associated with intestinal disorders, whereas pedv+en group did not show changes in levels of beneficial intestinal bacteria (fig. 5B).

The above results indicate that intestinal flora analysis suggests that TGE can modulate flora structure to alter piglet intestinal flora.

Example 5

TGE vs. Vero E6 cytotoxicity test performed in example 1

After trypsin digestion of Vero E6 cells, the cells were grown at 1.5X10 ⁶ personal/mLDensity was resuspended and plated in 96-well plates (100. Mu.L/well) leaving a column without plating. The cells were cultured until they were completely confluent, the medium was aspirated, and washed 3 times with PBS. The incubation was replaced with DMEM medium containing different concentrations of standard compound (1.56, 3.125, 6.25, 12.5, 25, 50, 100 and 200 μm) and 8 replicates were set per concentration. At 37 ℃,5% CO ₂ After 24h incubation under conditions, the absorbance of Vero E6 cells at 450nm was determined by adding DMEM-formulated 10% cck8 solution (100 μl/well) and incubating for 1h, thereby calculating cell viability.

The results indicated that the highest concentration of TGE for Vero E6 at 80% or more was 25 μg/mL (FIG. 6B).

Example 6

qPCR detection of changes in PEDV N Gene in Vero E6 cells treated with TGE obtained in example 1 at different concentrations

In 3 6-well cell culture plates according to 2X 10 ⁶ cell/mL cell concentration 1mL cell suspension was inoculated at 37℃with 5% CO ₂ Culturing under conditions until the cells are fully confluent. The medium in the 6-well plate was discarded and washed 3 times with PBS, and PEDV (0.01 MOI) was inoculated into CO, respectively, in the wells inoculated with cells except the first well ₂ Incubate in incubator for 1h. After 1h, the six-hole plate is taken out, the virus liquid is sucked away, and PBS is used for washing 3 times. After 4 hours of virus inoculation, each well was sequentially added fresh DMEM medium at 37℃and 5% CO at a concentration of 0 μg/mL of the gallic extract (blank group without virus inoculation), 0 μg/mL (negative group with virus inoculation), 1.56 μg/mL, 6.25 μg/mL, 25 μg/mL and 12.5 μM GC376 ₂ The conditions were cultured for 10 hours, respectively. The above process is shown in fig. 6A. After 10 hours, the six-hole plate is taken out, and the freezing and thawing are repeated three times under the normal temperature condition of minus 80 ℃. Transferring the cell sap after freeze thawing into a centrifuge tube, centrifuging at 8000r/min for 10min to obtain a supernatant, and transferring the supernatant into a new centrifuge tube. Total RNA was extracted from Vero E6 cells using a UNlQ-10 column Trizol total RNA isolation kit (Bio, china). Reverse transcription to cDNA was performed using the ReverteAid first strand cDNA Synthesis kit (Siemens, USA) according to the manufacturer's instructions. Quantitative qPCR was performed using perfectstart@green qPCR SuperMix (full gold, china). The following primers PEDV N (Forward-5'-GTCTGACAACAGCGGCAAAA-3' (SEQ ID NO. 3), reverse-5' -)TTTCGCCCTTGGGAATTCTC-3'(SEQ ID NO.4))；GAPDH(Forward-5'-AGGTCGGAGTCAACGGATTT-3'(SEQ ID NO.5)，Reverse-5'-TAGTTGAGGTCAATGAAGGG-3'(SEQ ID NO.6))。

The results showed that TGE showed a dose-dependent inhibition of PEDV proliferation and a significant inhibition capacity at 1.56 μg/mL (fig. 6D).

Example 7

Detection of TCID of Vero E6 cells treated with TGE at different concentrations by Reed-Muench method ₅₀ Variation of (2)

In 3 6-well cell culture plates according to 2X 10 ⁶ cell/mL cell concentration 1mL cell suspension was inoculated at 37℃with 5% CO ₂ Culturing under conditions until the cells are fully confluent. The medium in the 6-well plate was discarded and washed 3 times with PBS, and PEDV (0.01 MOI) was inoculated into CO, respectively, in the wells inoculated with cells except the first well ₂ Incubate in incubator for 1h. Taking out, sucking out virus liquid, and washing with PBS for 3 times. After 4 hours of virus inoculation, each well was treated with a nutrient solution having a concentration of gallic acid extract of 0. Mu.g/mL (blank group without virus inoculation), 0. Mu.g/mL (negative control group with virus inoculation), 1.56. Mu.g/mL, 6.25. Mu.g/mL, 25. Mu.g/mL and 12.5. Mu.M GC376, in this order, at 37℃and 5% CO ₂ The conditions were cultured for 10 hours, respectively. After 10 hours, the culture plate is taken out, and freeze thawing is repeated for 3 times at the temperature of-80 ℃ and normal temperature. Transferring the cell sap after freeze thawing into a centrifuge tube, centrifuging at 8000r/min for 10min, taking the supernatant, and transferring into a new centrifuge tube. According to 1X 10 ⁶ cell/mL cell concentration in 96-well plate, 100. Mu.L per well, at 37℃in 5% CO ₂ Culturing under conditions until the cells are fully confluent. The 96-well plate with the grown cells was washed 3 times with PBS, the collected samples were diluted with nutrient solution by 10-fold for 9 gradients, each gradient was repeated for 8, the diluted samples were added to the corresponding cell wells, 100. Mu.L per well, and empty cell controls were set. Finally at 37 ℃ and 5% CO ₂ Culturing for 4-5d under conditions, observing and recording wells with CPE (referring to the cell degeneration generated after infection of tissue culture cells by the virus) daily, and calculating statistical results according to the Reed-Muench method.

The results suggest that TGE showed a dose-dependent inhibition of PEDV proliferation and a significant inhibition capacity at 1.56 μg/mL (fig. 6C).

Example 8

Construction of recombinant plasmid and purification of recombinant protein

To construct the PEDV3CLpro prokaryotic expression plasmid, the 3CLpro protein sequence (amino acids shown in SEQ ID No. 7) was downloaded from NCBI (PDB: 4xfq_a). Determining the restriction enzyme site as NCOI/XHOI, connecting the target gene with an expression vector pET-28a (+) to obtain a recombinant plasmid pET-28a-3CLpro, and transforming the recombinant plasmid pET-28a-3CLpro into BL21 (DE 3) pLysS Chemically Competent Cell cells to obtain BL21-pET-28a-3CLpro. isopropyl-beta-D-thiopyran galactoside (IPTG) induced expression recombinant protein, purifying by His-tag protein purification kit (denaturation-resistant formulation) (Biyun, china) and obtaining the band of purified product on SDS-PAGE gel electrophoresis. The results showed that the 3CLpro protein was successfully expressed in BL21 (fig. 7A-7B).

Screening of PEDV3CLpro inhibitors from TGE using Biacore T200

Sodium acetate at pH 4.0, 4.5, 5.0 and 5.5 was used as dilution buffer for pre-coupling detection of 3CLpro protein to determine the optimal pH 4.5 for BL21-pET-28a-3CLpro protein (FIG. 7C). Protein immobilization was performed using sodium acetate at pH 4.5. The recombinant 3CLpro protein was immobilized on the detection channel of CM5 chip (fig. 7D). Responsive signal tests were performed at different concentrations, and finally subsequent fishing was performed at a concentration of 10 μg/mL (fig. 7E). The lyophilized extract was crushed, the powder was passed through a 50 mesh sieve, the extract powder (1.00 g) was accurately weighed, diluted with PBS (1. Mu.g/mL), and filtered through a 0.22 μm filter. The sample was run on a 3CLpro immobilized chip with a contact time of 60s and a separation time of 180s. The herbal extracts were flowed on the 3 CLpro-immobilized chip surface for 180s, the flow rate was maintained at 5 μl/min, the incubation time was 20s, the recovery solution volume was 2 μl, and the total recovery volume per cycle was 20 μl for 10 μl of precipitation solvent. The TGE extract was recovered for 15 cycles. The recovered sample was dried with nitrogen, dissolved with 100. Mu.L of 80% methanol, and then centrifuged at 8000r/min for 5min and filtered with a 0.22 μm filter. The supernatant was transferred to a glass vial with microinsertion for LC-MS detection. Firstly, TGE flows through the surface of a CM5 chip for fixing target proteins, the ligand combined with the target proteins is reserved in a protein pocket due to affinity, and unbound components flow out along with a buffer solution; (ii) Then, the cleaning reagent cleans the sample injection needle and the pipeline system under the condition of not flowing through the sample cell, so that the residual components are prevented from interfering the components recovered in the next step; (iii) Then, a recovery reagent is injected to the CM5 chip surface of the immobilized target protein, so that the ligand bound to the chip surface is dissociated; (iv) Finally, the circulation direction of the micro-flow control is changed, and the recovery reagent containing the ligand flows into the deposition reagent to be collected, and 5 times of repetition is a recovery cycle. The cleaning reagent is double distilled water, the recovery reagent is 0.5% formic acid aqueous solution, the deposition solvent is 50mM ammonium bicarbonate solution, and the setting parameters of ligand fishing are as follows: the sample loading time of the traditional Chinese medicine extract sample is 180s, the flow rate is 5 mu L/min, the volume of the recovery solution is 2 mu L, the incubation time is 20s, the deposition solvent is 10 mu L, and the theoretical recovery total volume of one cycle is 20 mu L. The cleaning reagent is double distilled water, 0.5% formic acid water solution is used as recovery liquid, and the precipitation solvent is 50mM ammonium bicarbonate.

LC-MS analysis of SPR recovered samples

Chromatographic separation was performed on Eclipse Plus C18 (. Phi.4.6X100 mm,3.5 μm) at 30 ℃. The sample was measured using mobile phases of different gradients of 0.1% aqueous formic acid (v/v) (A) and methanol (B), with a sample loading of 10. Mu.L. The elution procedure is 5% B0-5 min, 5-95% B5-20 min,95% B20-25 min,5% B25.5-30 min, and the flow rate is 0.5mL/min. Mass spectrometry was performed using 15/30/45% Normalized Collision Energy (NCE), data dependent MS/MS Top 10. The MS scanning adopts an anion mode, and the m/z scanning range is 100-1000. The control was tested under the same conditions. Last all data taken Thermo Scientific FreeStyle ^TM 1.7. And (5) processing software. LC-MS results are shown in FIG. 8, for the screening of ligands binding to 3CLpro in TGE, and ion flow diagrams (Total Ion Chromatography, TIC) in TGE recovery are shown in FIGS. 8 (A), 8 (B) and 8 (C). Ion signal (R can be detected in TGE total extract _t ＝10.55,m/z＝321.0254[M-H] ^- )(R _t ＝11.90,m/z＝300.9992[M-H] ^- )(R _t ＝4.74,m/z＝169.0131[M-H] ^- ) And their secondary mass spectra (fig. 8d,8e and 8F).At the same time, an ion signal (R _t ＝10.55,m/z＝321.0123[M-H] ^- )(R _t ＝11.89,m/z＝300.9993[M-H] ^- )(R _t ＝4.74,m/z＝169.0131[M-H] ^- ) And their secondary mass spectra (fig. 8g,8h and 8I). The compounds were presumed to be BA, EA and GA by mass spectrometry. Standards of three compounds were analyzed under the same mass spectrometry conditions (R _t ＝10.55,m/z＝321.0252[M-H]-)(R _t ＝11.92,m/z＝300.9994[M-H]-)(R _t ＝4.76,m/z＝169.0132[M-H] ^- ) (FIGS. 8J,8K and 8L) retention time and mass fragmentation results were consistent with the ion signal detected in the TGE recovered samples, so BA, EA and GA were identified as candidate compounds, indicating that the present invention successfully screens 3 possible inhibitors of PEDV3CLpro from TGE, BA, EA and GA, respectively.

Affinity verification (affinity verification)

Standards of GC376, BA, EA and GA were dissolved in PBS to make a 128 μm stock solution. Then re-diluted to solutions of different concentrations (2. Mu.M, 4. Mu.M, 8. Mu.M, 16. Mu.M, 32. Mu.M and 64. Mu.M) by passing through a 0.22 μm filter. The sample solution was then injected into the sensor chip using PBS as running buffer. The sample was injected through the surface of the 3CLpro immobilized chip at a flow rate of 30. Mu.L/min. The contact time and separation time were 60s and 120s, respectively. According to the 1:1 binding model, kinetic constants including association constant (ka), dissociation constant (KD) and affinity (KD, kd=kd/ka) were calculated using software. SPR detection results show that BA, EA and GA have better affinity with 3CLpro protein (KD values are 4.920, 4.417 and 4.745. Mu.M respectively). In this test, GC376 was used as a positive control with a KD of 2.244. Mu.M (FIG. 9).

Computer simulation docking

The X-ray crystal structure of the PEDV3CLpro dimer (PDB: 4 XFQ) was downloaded from https:// www.rcsb.org/. Website. The 3D structure of all small molecule compounds was downloaded from PubChem. The homology model of PEDV3CLpro with the compound was constructed using Autodock vina 4 software. A grid of 52 x 56 x 50 grid points was drawn covering the active site and binding pocket of PEDV 3CLpro. Then, half is set with x=0.4, y=1.9, and z=0.5 as the center of the binding siteThe diameter isAnd (5) carrying out molecular butt joint. A total of 9 docking conformations were obtained and the conformation with the lowest binding energy was selected for analysis of the interaction of the compound with PEDV 3CLpro. And finally, carrying out visual drawing on the optimal conformation result by using PyMOL software.

The results showed that binding energies of BA (binding energy = -9.6 kJ/mol), EA (binding energy = -10.1 kJ/mol) and GA (binding energy = -7.4 kJ/mol) (B, C, D in fig. 10) were all lower than-7 kJ/mol, indicating that GA, EA and BA can bind to 3CLpro, and the simulated docking results of GC376 as a positive control are shown in fig. 10A. Thus, BA, EA and GA were submitted for further biological evaluation.

Molecular dynamics simulation

The 4 complexes were selected for further Molecular Dynamics (MD) analysis. MD uses newton physics to simulate atomic motion in solvation systems, a precise calculation method that simulates protein-drug interactions. To determine the results of the above described docking model, 0.1ms molecular dynamics simulations were performed to evaluate the stability of the receptor and ligand by Root Mean Square Deviation (RMSD), hydrogen bond distance and number between different compounds and active pockets of protein. Semi-flexible docking of proteins with small molecules was performed using autodock vina software, and the results obtained were all simulated in Gromacs-2022.3. All simulations used periodic boundary conditions to keep the particle count constant. Thereafter, 50000 steps of energy minimization were performed on the system, and 100ps of NVT and NPT ensembles were performed on the energy minimization system. The cut-off distance of the non-bond interaction is set toThe long-range electrostatic force is calculated using a particle grid Ewald (PME) summation method. The temperature was maintained at 298K by V-rescale with a coupling constant of 0.1ps. The Berendsen pressure was maintained at 1bar and the lincs algorithm was used to constrain hydrogen bonding. The time step is set to 2fs. Finally, 100ns simulation is carried out on the whole model system, and data analysis is carried out by using a simulation track file.

The results showed that the RMSD values for all the composites were around 0.2 (fig. 10E). The distance between EA-3CLpro and the 3CLpro pocket is closer to GC376-3CLpro (FIG. 10F). The number of hydrogen bonds was measured in the MD simulations to better capture the polar interactions between molecules. All compounds are able to form hydrogen bonds with 3CLpro, which may contribute to the stability of the complex. The EA-3CLpro complex has a similar number of hydrogen bonds to GC376-3CLpro (FIG. 10G). This may mean that EA binds more tightly to 3CLpro than to the other two sets of complexes. In summary, all 3 compounds were able to form stable complexes with 3CLpro.

Example 9

Test for cytotoxicity of 3 Compounds BA, EA and GA obtained in example 8 on Vero E6 the specific assay was as in example 5.

The results showed that cell viability was higher than 80% for 100. Mu.M BA (FIG. 11A), 25. Mu.M EA (FIG. 11C) and 50. Mu.M GA (FIG. 11E)). Thus, in the following experiments, 1.56, 6.25 and 25. Mu.M BA, EA and GA were used, respectively, to inhibit PEDV proliferation. GC376 served as positive control.

qPCR detection of changes in PEDV N gene after treatment of Vero E6 cells with 3 compounds at different concentrations was performed in the same manner as in example 6.

The results show that 12.5 μm GC376 significantly inhibited PEDV proliferation. Both GA and EA are dose dependent inhibiting proliferation of PEDV. BA and EA showed extremely significant inhibitory capacity at 1.56 μm (B and D in fig. 11), and GA also showed significant inhibitory capacity at 6.25 μm (fig. 11F). EA has been shown to play an important role in the antiviral effect of TGE.

The general technical concept of the invention is shown in figure 12, and the invention proves that TGE can effectively treat diarrhea of piglets caused by PEDV infection for the first time. In addition, TGE can also improve the structure of intestinal flora of affected piglets. Time analysis showed that TGE inhibited mainly PEDV replication during the viral propagation phase. The active ingredient against PEDV3CLpro in TGE was screened using Surface Plasmon Resonance (SPR) technique. Three active ingredients, BA, EA and GA, are obtained. Wherein BA was first proposed to inhibit the activity of PEDV 3CLpro. Molecular docking and kinetic modeling experiments showed that BA binds to the active site of PEDV 3CLpro. SPR analysis showed that it has binding affinity to PEDV 3CLpro. The study of the present invention shows that TGE can improve the clinical symptoms and intestinal flora structure of PED, and its active ingredient inhibits PEDV proliferation by inhibiting the activity of 3CLpro.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (8)

1. Application of Galla Turcica extract in preparing medicine for preventing and treating porcine epidemic diarrhea is provided.

2. Application of Galla Turcica extract in preparing product for regulating intestinal flora of pig is provided.

3. Application of Galla Turcica extract in preparing 3C-like protease inhibitor for porcine epidemic diarrhea virus is provided.

4. A medicament for preventing and treating porcine epidemic diarrhea or regulating porcine intestinal flora, wherein the active ingredient of the medicament comprises a gallic acid extract.

5. A porcine epidemic diarrhea virus 3C-like protease inhibitor, wherein the active ingredient of the inhibitor comprises a gallic extract.

6. The use according to any one of claims 1-3 or the medicament according to claim 4 or the inhibitor according to claim 5, wherein the gallic extract is an aqueous gallic extract.

7. The use according to any one of claims 1-3 or the medicament according to claim 4 or the inhibitor according to claim 5, wherein the gallic extract comprises gallic acid, ellagic acid or meta-digallic acid.

8. The use according to any one of claims 1 to 3 or the medicament according to claim 4 or the inhibitor according to claim 5, characterized in that the preparation method of the gallic extract comprises the following steps: mixing Galla Turcica with water, boiling, concentrating under reduced pressure to paste, and lyophilizing to obtain Galla Turcica extract.