CN116808015A - Application of chickpea sprout extract A in preparation of medicines for resisting porcine epidemic diarrhea virus infection - Google Patents

Application of chickpea sprout extract A in preparation of medicines for resisting porcine epidemic diarrhea virus infection Download PDF

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CN116808015A
CN116808015A CN202310469375.1A CN202310469375A CN116808015A CN 116808015 A CN116808015 A CN 116808015A CN 202310469375 A CN202310469375 A CN 202310469375A CN 116808015 A CN116808015 A CN 116808015A
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epidemic diarrhea
porcine epidemic
diarrhea virus
biochanin
preparation
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CN202310469375.1A
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陈建新
程梓钰
林琪胜
曾振灵
黄显会
龙飞翔
苏国明
何应敏
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South China Agricultural University
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South China Agricultural University
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Abstract

The application discloses application of chickpea sprout essence A in preparation of a medicament for resisting porcine epidemic diarrhea virus infection. Pharmacological tests show that the chickpea sprout extract A can effectively inhibit proliferation of porcine epidemic diarrhea virus in cells and piglets, and can be used as a medicinal component to prepare a preparation by auxiliary agents for preventing and treating diseases caused by porcine epidemic diarrhea virus infection.

Description

Application of chickpea sprout extract A in preparation of medicines for resisting porcine epidemic diarrhea virus infection
Technical Field
The application belongs to the technical field of medicines, and particularly relates to application of biochanin A in medicines for resisting porcine epidemic diarrhea virus infection.
Background
Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is an infectious disease of acute diarrhea and vomiting, dehydration and high mortality in piglets caused by infection with porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV). PEDV is a single stranded positive strand RNA virus with an encapsulated RNA genome size of about 28kb. The genome comprises, in order, a 5 'end cap, a 5' untranslated region (UTR), seven open reading frames, a 3 'untranslated region (UTR), and a 3' terminal polyadenylation (polyA) tail. Seven open reading frames encode four structural proteins: s protein (spike protein), M protein (membrane protein), E protein (envelope protein) and N protein (nucleocapsid protein), and pp1a, pp1ab and ORF3 proteins. PEDV primarily infects porcine small intestine epithelial cells and can cause severe mucosal atrophy and malabsorption. The virus can infect pig groups of all ages, but is most sensitive to piglets in lactation, and the death rate of piglets in one week age is up to 100% because the organs and tissues of the piglets are not good. The main transmission route of PEDV is faecal-oral transmission. After PEDV infection in newborn pigs, the virus is excreted with the feces and causes acute viremia, and severe atrophic enteritis occurs in jejunum and ileum.
At present, the prevention and treatment of PED mainly takes biosafety prevention and control measures and vaccination, but due to the wide variety of genotypes of PEDV and extremely rapid virus mutation, especially the high variability of the main target S protein of a neutralizing antibody, new variant strains frequently occur, so that the vaccine has serious hysteresis, therefore, the development of effective broad-spectrum anti-PEDV medicaments for treating PED is needed to complement the loss caused when the vaccine cannot be protected.
Biochanin A (BCA) is an isoflavone compound which is present in red clover, cabbage, alfalfa and many other herbs, and is extracted from various natural plants such as rosewood leaves, rosewood leaves and chickpeas. In recent years, the research on the chickpea sprout extract A mainly focuses on the antioxidation and anti-inflammatory effects of the chickpea sprout extract A, and also has research on the chickpea sprout extract A, which can improve the insulin sensitivity of type II diabetics and control hyperglycemia. Research on the effect of Guan Ying-mouth bean sprout essence A on resisting porcine epidemic diarrhea virus infection and application of the bean sprout essence A in preventing and treating porcine epidemic diarrhea virus infection are not reported.
Disclosure of Invention
The present application is directed to solving, at least to some extent, one of the problems in the related art. Therefore, the application aims to provide the application of the chickpea sprout essence A in preparing medicines for resisting porcine epidemic diarrhea virus infection.
The chickpea sprout extract A is obtained by the following method: (1) Pulverizing semen Ciceris Arietini seeds to 50-100 mesh, wetting with acid water of pH4-6, adding biological enzyme, and hydrolyzing at 35-40deg.C to obtain enzymolysis product. (2) Extracting the enzymolysis material with ethanol under reflux for 2-3 times, and recovering ethanol from the extractive solution to obtain crude extract. (3) Feeding the crude extract into supercritical CO 2 Injecting entrainer into extraction kettle, and introducing CO 2 Extracting under 20-55MPa at 30-55deg.C for 1-2 hr, collecting extract, and volatilizing solvent to obtain crude extract. (4) Dissolving the crude extract obtained by extraction with absolute ethyl alcohol, mixing with 100-200 mesh polyamide, stirring, volatilizing ethanol to obtain a sample, loading into pretreated polyamide column chromatography, gradient eluting with petroleum ether-ethyl acetate (10:1, 9:1, 4:1), detecting by TLC, collecting biochanin A fraction, concentrating, crystallizing, and recrystallizing with acetone to obtain white crystal powder, namely biochanin A.
The medicine also contains pharmaceutically acceptable auxiliary materials; the dosage forms of the medicine are tablets, capsules, granules, powder, syrup, oral liquid or injection.
The chickpea sprout essence A has antiviral activity on porcine epidemic diarrhea virus.
The chickpea sprout extract A can inhibit the synthesis of N protein in the porcine epidemic diarrhea virus, and the concentration of the chickpea sprout extract A for reducing the expression level of N protein in the porcine epidemic diarrhea virus is 7.5-30 mu M.
The chickpea sprout extract A can inhibit the transcription of RNA in porcine epidemic diarrhea virus.
The concentration of the chickpea sprout extract A for inhibiting RNA transcription in the porcine epidemic diarrhea virus is 7.5-30 mu M.
The chickpea sprout extract A can inhibit the replication of progeny viruses in porcine epidemic diarrhea viruses.
The concentration of the chickpea sprout extract A for inhibiting the replication of progeny viruses in the porcine epidemic diarrhea virus is 7.5-30 mu M.
The chickpea sprout essence A has an inhibiting effect on replication of porcine epidemic diarrhea virus in piglets.
The use of the chickpea sprout A of the present application for the preparation of a medicament against porcine epidemic diarrhea virus infection, but not limited to, the use of an effective amount of the compound of the present application for preventing or treating porcine epidemic diarrhea virus-induced disease, alleviating symptoms of porcine epidemic diarrhea virus-induced disease or delaying the progression of porcine epidemic diarrhea virus-induced disease, is described.
The medicine also contains pharmaceutically acceptable auxiliary materials. The auxiliary materials can be carriers, excipients, diluents, vehicles and the like.
The application has the following beneficial effects: the application discovers a new application of the chickpea sprout essence A in inhibiting porcine epidemic diarrhea virus. On Vero cells, half-effective concentration of biochanin a on PEDV (EC 50 ) The value was 6.9. Mu.M. Meanwhile, the chickpea sprout extract A can effectively reduce the virus titer of PEDV in Vero cells, and the virus inhibition rate reaches more than 90% after 48 hours of administration under the highest administration dosage of 30 mu M. In addition, 8mg/kg of biochanin A is fed to the newborn piglet artificially infected by the PEDV, and can obviously inhibit the replication of the PEDV in the digestive tract of the piglet. The chickpea sprout essence A of the application has remarkable inhibition effect on porcine epidemic diarrhea virus and has application value for clinical treatment of PEDV infection.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description, serve to explain the principles of the application.
In order to more clearly illustrate the embodiments of the application or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, and it will be obvious to a person skilled in the art that other drawings can be obtained from these drawings without inventive effort.
In the accompanying drawings:
FIG. 1 is a graph showing the evaluation of the cytotoxicity of biochanin A against Vero cells in example 1 (CC 50 ) And its anti-PEDV viral activity on cells (EC 50 ) Is a statistical graph of (1);
FIG. 2 is an immunofluorescence graph of the immunofluorescence assay of example 2 for the effect of various concentrations of biochanin A on the reduction of the expression level of PEDV virus N protein in cells;
FIG. 3 is a Western Blot analysis of the inhibition of PEDV virus N protein synthesis in cells by varying concentrations of biochanin A in example 3;
FIG. 4 is a data statistical chart of the relative expression amounts of mRNA corresponding to the inhibitory effect of qRT-PCR analysis of different concentrations of biochanin A on proliferation of PEDV RNA in cells in example 4;
FIG. 5 is a statistical plot of the viral titers of various concentrations of biochanin A analyzed by the end point dilution method of example 5 for inhibition of PEDV progeny virus in cells.
FIG. 6 is a statistical chart of viral mRNAs corresponding to the inhibition of PEDV replication by qRT-PCR analysis of biochanin A in piglets in example 6.
Detailed Description
The following examples are illustrative of the application and are not intended to limit the scope of the application. Modifications and substitutions to methods, procedures, or conditions of the present application may be made without departing from the spirit and nature of the application and are intended to be within the scope of the present application. Unless otherwise indicated, the experimental materials, reagents, instruments, etc. used in the examples of the present application are commercially available; all technical means in the embodiments of the present application are conventional means well known to those skilled in the art unless specifically indicated.
Example 1: evaluation of the cytotoxicity of Cinnamomum chickpea sprout extract A on Vero cells (CC 50 ) And its anti-PEDV viral activity on cells (EC 50 )。
The density was 1.5X10 5 Vero cells at a concentration of 1.87-240. Mu.M were seeded in 96-well plates at 100. Mu.L per well, washed twice with PBS after they had grown to a monolayer, and compound was diluted twice with DMEM maintenance solution containing 2% FBS, and a 1.87-240. Mu.M concentration gradient of biochanin A (BCA) drug group, a solvent control group containing 4%DMSO, and a blank control group were set at 100. Mu.L per well. At 37℃with 5% CO 2 After 48h incubation in a constant temperature incubator, the supernatant was discarded, and 100. Mu.L per well of MTT solution, 0.5mg/mL, was added and incubated at 37℃in the absence of light. After 4 hours, stopping incubation, discarding the supernatant, adding 150 mu L of DMSO into each well, and oscillating for 10 minutes by a low-speed oscillator to fully dissolve formazan crystals; the OD was measured at 570nm using a full wavelength microplate reader and the cell viability was calculated. Half-cell Cytotoxicity Concentration (CC) was calculated by nonlinear regression function using GraphPad Prism 9.0 software 50 )。
The calculation formula is as follows:
as above, after Vero cells grew to a monolayer, PBS was washed twice, compound was diluted with DMEM maintenance solution containing 2% fbs at a double ratio, BCA drug groups were set up at 3.75-480 μm for a total of 8 concentration gradients, solvent control group containing 8%o DMSO and blank control group, and 100 μl per well. At 37℃with 5% CO 2 After culturing in a constant temperature incubator for 48 hours, the culturing is terminated. After observing the cell morphology, IFA detection was performed with 4% paraformaldehyde fixation and observed using a fluorescent inverted microscope and photographed for recording. Fluorescence intensity (blue fluorescence and red fluorescence) of each well was quantified using Image J software, DMSO-treated control group was set to 100%, and each of the other groups was compared with DMSO-treated group, and half-Effective Concentration (EC) was determined by the quantified cytoprotection of the drug-treated group 50 ) Values and passed using GraphPad Prism 9.0 softwareA nonlinear regression function.
The calculation formula is as follows:
as shown in FIG. 1, the medicine biochanin A of the application has antiviral activity to PEDV virus and EC thereof 50 The value was 6.9. Mu.M. After the chickpea sprout extract A acts on Vero cells for 48 hours, the cytotoxicity index CC of chickpea sprout extract A 50 A value of 270.6. Mu.M suggests that biochanin A is of low toxicity to Vero cells.
Example 2: immunofluorescence assay was performed to analyze the inhibition of PEDV virus N protein synthesis in cells by varying concentrations of biochanin a.
Spreading Vero cells in 96-well plate, washing with PBS twice, diluting virus with DMEM maintenance solution containing 2% FBS to 100TCID 50 Setting a cell blank control group, and placing at 37deg.C and 5% CO 2 After incubation for 2h in a constant temperature incubator, PBS was washed twice to remove unbound virus particles, and a blank control group, a solvent control group and a BCA drug group containing different concentrations of 100. Mu.L per well were set. After 48h of incubation, the supernatant was discarded, the incubation was terminated and fixed with 4% paraformaldehyde at room temperature, 150 μl per well. After 15min, PBS was washed three times, 50. Mu.L of 0.3% Triton-X100 solution was added to each well, and incubated at room temperature; after 10min, PBS was washed three times, 100. Mu.L of 2% BSA solution was added to each well and incubated at 37 ℃; after 1h, PBS was gently washed three times, 50. Mu.L of Anti-PEDV-Anti-body (1:1000 dilution) was added to each well and incubated overnight at 4 ℃; washing with PBS for three times, 5min each time, adding 50 μl of AlexaFluor 568 labeled goat anti-mouse lgG (H+L) (ab 175473) in dark place into each well, and incubating at 37deg.C; after 1h, PBS was washed three times, each for 5min, 100. Mu.L of DAPI (300 nM) solution was added to each well in the dark and incubated at room temperature. After 5min, the cells were washed three times with PBS, each time for 5min, observed with a fluorescent inverted microscope and photographed for recording.
The test result is shown in figure 2, and the medicine chickpea sprout essence A of the application obviously reduces the expression quantity of PEDV N protein in Vero cells within the concentration range of 7.5-30 mu M, and shows good quantity-effect relationship.
Example 3: western Blot analysis of inhibition of PEDV N protein synthesis in cells by different concentrations of biochanin A.
Vero cells were seeded in 6-well plates at 2mL per well and after growing the cells to a monolayer the following experiments were performed. The steps of infection of PEDV with Vero cells and addition of the drug were the same as in example 1, and after 48 hours incubation in the incubator, the culture was terminated, the supernatant was discarded, and the solution was washed 2 times with PBS. The cell culture plate was placed on ice, 120. Mu.L/well of RIPA lysate was added, the solution was aspirated into a centrifuge tube after repeated pipetting, centrifuged at 13000 rpm for 20min, and the supernatant was aspirated into another clean tube for further use. After the protein concentration of each sample was measured by the BCA method, bands of PEDV N protein and the internal reference protein GAPDH were detected by Western Blot.
The test result is shown in figure 3, and the medicine chickpea sprout essence A has remarkable inhibition effect on PEDV N protein synthesis in Vero cells in the concentration range of 7.5-30 mu M, and has good dose-effect relationship.
Example 4: qRT-PCR analysis of the inhibition of proliferation of PEDV RNA in cells by varying concentrations of biochanin A.
The steps of infection of PEDV by Vero cells and addition of the drug were the same as in example 1, the culture was terminated after incubation in an incubator for 48 hours, and after observation of the cell morphology, the cell plates were repeatedly freeze-thawed 3 times at-80 ℃ and 4 ℃ to allow the cells to be sufficiently lysed so that the virus in the cells was completely released into the cell supernatant, and the supernatant from each well was collected. The collected cell supernatant was subjected to total RNA extraction using the procedure recommended by the total RNA rapid extraction kit (Shanghai Fei Biotechnology Co., ltd.). Performing reverse transcription immediately after RNA extraction, taking cDNA as a template, taking beta-action as an internal reference gene, and detecting the copy number of PEDV N gene by Real Time PCR; the change in N mRNA was evaluated by reference to a virus control group.
Primer sequences upstream and downstream of PEDV N gene:
PEDV-N-F：5'-CGCAAAGACTGAACCCACTAATTT-3'
PEDV-N-R：5'-TTGCCTCTGTTGTTACTTGGAGAT-3'
the primer sequence of the upstream and downstream of the beta-action gene:
β-Actin-F：5'-GGACTTCGAGCAGGAGATGG-3'
β-Actin-R：5'-AGGAAGGAGGGCTGGAAGAG-3'
the test results are shown in FIG. 4, wherein the Mock group represents a blank control group, the DMSO group represents a virus infection control group, and the BCA group represents a biochanin A treatment group; the ordinate indicates the relative expression amount of NmRNA in PEDV. In the figure, P <0.05 compared to the virus-infected control group; * P <0.01; * P <0.001, indicating significant differences. As shown in the test result in figure 4, the medicine biochanin A has remarkable inhibition effect on RNA transcription of PEDV in Vero cells in the concentration range of 7.5-30 mu M, and has good dose-effect relationship. At the highest administration dose of 30 mu M, the virus inhibition rate reaches 100% after 48 hours of administration.
Example 5: the end point dilution method analyzes the inhibition of PEDV progeny virus by different concentrations of biochanin A in cells.
Spreading Vero cells in 24-well plate, washing with PBS twice, and adding 100TCID diluted with maintaining solution except for the blank control group and maintaining solution containing 2% FBS 50 PEDV virus solution 100. Mu.L per well at 37deg.C in 5% CO 2 Culturing in a constant temperature incubator. After 2h, PBS was washed twice to remove unbound virions, the drug-treated group was added with the corresponding compound diluted with a double ratio of DMEM maintenance solution of 2% FBS, and the blank control group and the solvent control group were replaced with fresh DMEM maintenance solution, 500 μl per well, and the culture was continued in an incubator. After 48h, the culture was terminated, the cell plates were placed in a-80℃refrigerator and a 4℃refrigerator to freeze-thaw 3 times, so that the virus was sufficiently released into the supernatant, 120. Mu.L of the freeze-thaw solution per tube was collected, and 3-4 replicates were collected for subsequent experiments.
The density was 1.5X10 5 the/mL Vero cells were seeded in 96-well plates at 100. Mu.L per well, washed twice with PBS after the monolayer was grown, 10-fold dilution of the sample stock with DMEM maintenance medium was performed, 100. Mu.L per well was added to the cell plates, and 4-well replicates were set. After 48h incubation, the mixture is placed under an inverted microscope for observationCytopathic status was examined and recorded, and wells with CPE were recorded as positive wells.
The test results are shown in FIG. 5, wherein the Mock group represents a blank control group, the DMSO group represents a virus infection control group, and the BCA group represents a biochanin A treatment group; the ordinate indicates the viral titer. In the figure, P <0.05 compared to the virus-infected control group; * P <0.01; * P <0.001, indicating significant differences. The test result is shown in figure 5, and the medicine chickpea sprout essence A has remarkable inhibition effect on the replication of PEDV progeny virus in Vero cells in the concentration range of 7.5-30 mu M, and has good dose-effect relationship.
Example 6: qRT-PCR analysis of inhibition of PEDV replication by Cicer biochanin A in piglets
The 9 newborn piglets of 3 days of age were randomly divided into 3 groups, namely a PEDV challenge group, a biochanin a treatment group and a negative control group. Each piglet was housed in separate cages and tested after 1d adaptation. PEDV challenge group and biochanin a treatment group were orally administered PEDV challenge, negative control group was orally administered PBS. When diarrhea occurs in piglets, 8.0mg/kg of biochanin A is used for treatment, and the administration is orally carried out 2 times a day for 4 days. After the toxicity is removed, the fecal samples of the piglets in the test group are collected at fixed time every day and stored in a refrigerator at-80 ℃. 0.1g of fecal sample was weighed, 1mL of PBS was added, mixed well, centrifuged (12000 rpm,10min,4 ℃ C.), fecal supernatant was collected, and the virus mRNA level was detected by qRT PCR method.
The results of the experiment are shown in FIG. 6, and the ordinate indicates the logarithmic value of the initial copy number of RNA in PEDV, namely PEDV RNA (log) 10 cobies/reactions), the ordinate indicates the days after infection, the PBS group represents the negative control group, the DMSO group represents the virus infection control group, and the BCA group represents the biochanin a-treated group. In the figure, P compared to the virus-infected control group<0.05；**，P<0.01；***，P<0.001, indicating significant differences. As shown in fig. 6, compared with DMSO group, oral administration of biochanin a significantly reduced PEDV viral load in the feces of infected piglets, suggesting that biochanin a has inhibitory effect on PEDV replication in piglets.
It is to be understood that the above examples only represent preferred embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the application; it should be noted that, for a person skilled in the art, the above technical features can be freely combined, and several variations and modifications can be made without departing from the scope of the application; therefore, all changes and modifications that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims (6)

1. Application of chickpea sprout extract A in preparing medicine for resisting porcine epidemic diarrhea virus infection is provided.
2. The use of biochanin a according to claim 1 for the preparation of a medicament for the treatment of porcine epidemic diarrhea virus infection, wherein said biochanin a has antiviral activity against porcine epidemic diarrhea virus.
3. The use of biochanin a according to claim 1 for the preparation of a medicament for the treatment of porcine epidemic diarrhea virus infection, wherein said biochanin a is capable of inhibiting replication of progeny virus in porcine epidemic diarrhea virus.
4. The use of biochanin a according to claim 3 for the preparation of a medicament for the treatment of porcine epidemic diarrhea virus infection, wherein biochanin a is capable of inhibiting replication of progeny virus in porcine epidemic diarrhea virus at a concentration of 7.5-30 μm.
5. The use of biochanin a according to claim 1 for the preparation of a medicament for the treatment of porcine epidemic diarrhea virus infection, wherein said biochanin a has an inhibitory effect on replication of porcine epidemic diarrhea virus in piglets.
6. The use of biochanin a according to claim 1 for the preparation of a medicament for the treatment of porcine epidemic diarrhea virus infection, wherein the medicament further comprises pharmaceutically acceptable excipients; the dosage forms of the medicine are tablets, capsules, granules, powder, syrup, oral liquid or injection.
CN202310469375.1A 2023-04-27 2023-04-27 Application of chickpea sprout extract A in preparation of medicines for resisting porcine epidemic diarrhea virus infection Pending CN116808015A (en)

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