Use of a plant extract for the preparation of a medicament for inhibiting canine parvovirus

Abstract

This invention discloses the application of plant extracts in the preparation of drugs that inhibit canine parvovirus, belonging to the field of biomedical technology. The plant extract is at least one selected from phlorizin, neomethylhesperidin-dihydrochalcone, tribulus terrestris saponin, stevia glycoside, soy isoflavones, berberine hydrochloride, diosgenin, and silymarin. Through in-depth research on various plant extracts, this invention has found that phlorizin, neomethylhesperidin-dihydrochalcone, tribulus terrestris saponin, stevia glycoside, soy isoflavones, berberine hydrochloride, diosgenin, and silymarin all exhibit significant inhibitory effects on canine parvovirus, with soy isoflavones showing particularly outstanding effects. This research provides a novel natural drug option for the prevention and treatment of canine parvovirus. This invention not only expands the application scope of natural plant extracts in the veterinary medicine field but also provides important technical support for the development of novel, highly effective, safe, and side-effect-free drug formulations for the prevention and treatment of canine parvovirus.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of plant extracts in the preparation of drugs that inhibit canine parvovirus. Background Technology

[0002] Plant extracts refer to a class of active substances derived from plants that possess one or more biological functions. There are numerous types, with the most frequently reported natural plant extracts including essential oils, saponins, alkaloids, polysaccharides, polyphenols, and flavonoids. Studies have shown that natural plant extracts possess various biological activities, such as antioxidant, antibacterial, antiviral, anti-inflammatory, performance-enhancing, and immune-boosting effects. Compared to traditional antibacterial drugs, plant extracts offer advantages such as safety, low toxicity, less burden on the organism, broad spectrum, lower risk of drug resistance, and synergistic effects with multiple antibiotics. With advancements in technology and in-depth research, the diminishing efficacy of serum and biological agents, and the urgent need for antibiotic reduction and replacement, natural plant extracts are receiving increasing attention, and their various effects are gradually being discovered. While plant extracts are widely used in livestock farming, their application in companion animals and small animal diseases remains limited, and their efficacy and mechanisms of action are still unclear.

[0003] This invention aims to screen plant extracts from plant extracts such as flavonoids, phenols, and saponins that have inhibitory effects on canine parvovirus, and to study their mechanisms of action, in order to provide a reference for the application of plant extracts in the field of disease prevention and control in pet dogs. Summary of the Invention

[0004] The purpose of this invention is to provide the application of plant extracts in the preparation of drugs that inhibit canine parvovirus, thereby solving the problems existing in the prior art. This invention has found that plant extracts—phlorizin, neomethylhesperidin-dihydrochalcone, tribulus terrestris saponins, stevia glycosides, soy isoflavones, berberine hydrochloride, diosgenin, and silymarin—all have significant inhibitory effects on canine parvovirus and can therefore be used in the preparation of drugs that inhibit canine parvovirus.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of plant extracts in the preparation of drugs that inhibit canine parvovirus, wherein the plant extracts are at least one selected from phloretin, neomethylhesperidin-dihydrochalcone, tribulus terrestris saponins, stevia glycosides, soy isoflavones, berberine hydrochloride, diosgenin, and silymarin.

[0007] Furthermore, the plant extract is soy isoflavones.

[0008] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0009] Furthermore, the excipients include solubilizers, binders, disintegrants, fillers, stabilizers, preservatives, coating materials, and / or fragrances.

[0010] Furthermore, the dosage form of the drug is granules, tablets, capsules, powders, pills, injections, or oral liquids.

[0011] The present invention also provides a drug for inhibiting canine parvovirus, the active ingredient of which includes plant extracts;

[0012] The plant extract is at least one of the following: phloretin, neomethylhesperidin-dihydrochalcone, tribulus saponin, stevia glycoside, soy isoflavones, berberine hydrochloride, diosgenin, and silymarin.

[0013] Furthermore, the plant extract is soy isoflavones.

[0014] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0015] Furthermore, the excipients include solubilizers, binders, disintegrants, fillers, stabilizers, preservatives, coating materials, and / or fragrances.

[0016] Furthermore, the dosage form of the drug is granules, tablets, capsules, powders, pills, injections, or oral liquids.

[0017] The present invention discloses the following technical effects:

[0018] This invention, through in-depth research on various plant extracts, has discovered that phlorizin, neomethylhesperidin-dihydrochalcone, tribulus terrestris saponins, stevia glycosides, soy isoflavones, berberine hydrochloride, diosgenin, and silymarin all exhibit significant inhibitory effects on canine parvovirus, with soy isoflavones showing particularly outstanding effects. This research provides a novel natural drug option for the prevention and treatment of canine parvovirus.

[0019] Compared to traditional chemically synthesized drugs, these plant extracts, derived from natural plants, offer advantages such as low toxicity and good biocompatibility, effectively reducing side effects on animals and providing greater safety. This invention not only expands the application scope of natural plant extracts in the veterinary pharmaceutical field but also provides crucial technical support for developing novel, highly effective, safe, and side-effect-free drug formulations for the prevention and treatment of canine parvovirus. It is expected to become an ideal alternative to traditional chemical drugs in the future, driving the veterinary pharmaceutical field towards a greener and more environmentally friendly direction. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The image shows the cytotoxicity test results of Tribulus terrestris saponins.

[0022] Figure 2 The image shows the cytotoxicity test results of phloretin.

[0023] Figure 3 The image shows the cytotoxicity test results for caffeic acid.

[0024] Figure 4 The image shows the cytotoxicity test results for chlorogenic acid.

[0025] Figure 5 The image shows the cytotoxicity test results of diosgenin.

[0026] Figure 6 The image shows the cytotoxicity test results of silymarin.

[0027] Figure 7 The image shows the cytotoxicity test results of betaine.

[0028] Figure 8 The image shows the cytotoxicity test results of berberine hydrochloride.

[0029] Figure 9 The image shows the cytotoxicity test results of neomethylhesperidin-dihydrochalcone.

[0030] Figure 10 The image shows the cytotoxicity test results of soy isoflavones.

[0031] Figure 11 The image shows the cytotoxicity test results for isochlorogenic acid.

[0032] Figure 12 The image shows the cytotoxicity test results of ginsenosides.

[0033] Figure 13 This is a graph showing the results of conventional PCR amplification of CPV; where M: Marker; 1: Positive control; 2: CPV sample;

[0034] Figure 14 The image shows the results of conventional PCR validation using primers for CPV's real-time PCR; where M: Marker; 1: Positive control; 2: CPV sample;

[0035] Figure 15A preliminary screening result of the inhibitory effect of various plant extracts on CPV;

[0036] Figure 16 Growth curves of CPV in F81 for different treatment groups;

[0037] Figure 17 The image shows the results of CPV viral load detection 4 hours after F81 cell infection;

[0038] Figure 18 The graph shows the results of CPV viral load detection 8 hours after F81 cell infection;

[0039] Figure 19 The graph shows the results of CPV viral load detection 12 hours after F81 cell infection;

[0040] Figure 20 The graph shows the results of CPV viral load detection 18 hours after F81 cell infection;

[0041] Figure 21 The graph shows the results of CPV viral load detection 24 hours after F81 cell infection;

[0042] Figure 22 The graph shows the results of CPV viral load detection 36 hours after F81 cell infection;

[0043] Figure 23 The graph shows the results of CPV viral load detection 48 hours after F81 cell infection;

[0044] Figure 24 The graph shows the results of CPV viral load detection 54 hours after F81 cell infection. Detailed Implementation

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0048] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0049] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0050] Example 1

[0051] 1. Experimental Materials

[0052] 1.1 Cells and Viruses

[0053] Primary feline embryonic kidney cells (F81) and canine parvovirus (CPV) were provided by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0054] 1.2 Main reagents (see Table 1)

[0055] Table 1 Main Reagents

[0056]

[0057]

[0058] 1.3 Main Instruments (see Table 2)

[0059] Table 2 Main Reagents

[0060]

[0061] 2. Experimental Methods

[0062] 2.1 Cell Culture

[0063] (1) Cell resuscitation

[0064] Remove the F81 cell cryovials quickly from liquid nitrogen and immediately place them in a 37°C water bath, shaking them constantly to thaw the cell suspension as quickly as possible. Wipe the cryovials with 75% alcohol swabs, open the caps in a biosafety cabinet, aspirate the cell suspension, centrifuge at 1000 rpm for 5 minutes, and discard the cryopreservation solution. Prepare for cell culture.

[0065] (2) Cell Culture

[0066] The centrifuged cells were resuspended in DMEM medium containing 10% fetal bovine serum (FBS) and inoculated into T25cm cells. 2 Place the cell culture flask in a cell culture incubator (37℃, 5% CO2), observe the cell growth status on the second day, and passage the cells when they have basically formed a monolayer.

[0067] 2.2 Virus Culture

[0068] When the monolayer of F81 cells reached 80%, the culture medium was discarded and the cells were washed once with PBS. The CPV strain stored in the -80℃ freezer was taken out, thawed on ice, and 100 μL was inoculated into the cell culture bottle. The cell culture bottle was then placed in a cell culture incubator for 1 hour to allow for virus adsorption. After 1 hour, DMEM medium with 10% FBS was added, and the cell culture bottle was placed in the cell culture incubator again for 72 hours to observe the cytopathic effect.

[0069] 2.3 CPV half-maximal tissue culture infectious dose (TCID) 50 Determination of )

[0070] F81 cells were seeded into 96-well plates and, when they reached 90% confluence, the cell culture medium was discarded, and the cells were washed once with PBS. The CPV stock solution was serially diluted 10-fold with serum-free DMEM medium to achieve a viral dilution of 10. 0 -10 -9 Add 100 μL of virus solution of each dilution to each well, with 8 replicates per dilution. After completion, incubate the 96-well plate at 37°C and 5% CO2 for 72 hours. Observe the cytopathic effect (CPE) and calculate the median tissue culture infectious dose (TCID) of the virus according to the Reed-Muench formula. 50 ).

[0071] 2.4 CCK-8 assay for drug cytotoxicity against F81 cells

[0072] Plant extracts were dissolved in DMSO and filtered through a 0.22 μm filter to prepare a 1000 mg / L stock solution. The plant extract solutions were serially diluted twofold using DMEM cell culture medium to obtain final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.13, and 1.56 mg / L. 10 6 F81 cell suspension (number of cells / mL) was seeded into 96-well plates. Once the cells reached confluence and formed a monolayer, the culture medium was discarded, and 100 μL of drug solution at different concentrations was added to each well, with three replicates for each concentration. The 96-well plates with drug solution were incubated at 37°C and 5% CO2 for 48 h. Afterward, the culture medium was discarded, the plates were rinsed once with PBS, and then 10 μL of CCK-8 reagent was added to each well. The plates were then returned to the incubator for 1 h. The OD values ​​were measured using a microplate reader. 450nm The absorbance of each well was measured at the specified wavelength to calculate the toxicity of different drug concentrations to F81 cells.

[0073] 2.5 Real-time quantitative PCR detection of the effect of plant extracts on virus expression

[0074] 2.5.1 Establishment of CPV absolute fluorescence quantitative method

[0075] (1) Primer design

[0076] Based on the canine parvovirus VP2 gene sequence published in NCBI GenBank, a pair of primers was designed using Premier 6.0 software and synthesized by Beijing Ruibo Kexing Gene Technology Co., Ltd. The expected PCR amplification product length is 846bp.

[0077] The primer sequences are as follows:

[0078] Upstream primer CPV-24-F: 5'-GGATGGGTGGGAAATCACAGC-3' (SEQ ID NO.1);

[0079] Downstream primer CPV-24-R: 5'-ATAACCAACCTCAGCTGGTC-3' (SEQ ID NO.2).

[0080] (2) Extraction of CPV virus genes

[0081] Following the previous steps for culturing the virus, the diseased cells were harvested, and after three freeze-thaw cycles, viral DNA was extracted according to the kit's operating procedures. The concentration was then detected using a nucleic acid protein analyzer, and the cells were immediately stored at -40°C for later use.

[0082] (3) Establishment of standard positive templates for gene plasmids:

[0083] Using the DNA extracted in step (2) as a template, amplification was performed according to the standard PCR reaction system and conditions. PCR reaction system: 10 μL Prime Star Mix (10×), 1 μL upstream primer, 1 μL downstream primer, 1 μL DNA template, 7 μL RNase-free H2O.

[0084] The PCR reaction conditions were set as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 15 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 5 min.

[0085] After amplification, 5 μL of the reaction solution was subjected to 1% agarose gel electrophoresis. The gel containing the amplified product was excised, and the target DNA fragment was recovered using a gel extraction kit. The recovered target DNA fragment was ligated into the pTOPO vector and transformed into DH5α competent cells. The cells were cultured overnight on LB agar containing ampicillin. Single colonies were picked, identified by PCR, and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Positive plasmids with correct sequencing results can be used for subsequent experiments.

[0086] (4) Primer and probe design

[0087] The sequences obtained from sequencing were imported into NCBI for BLAST alignment. Primers were then designed using Beacon Designer 8.14 software based on the CPV sequences published in NCBI GenBank. The reaction system was confirmed and the sequences were then synthesized by Beijing Ruibo Kexing Gene Technology Co., Ltd. The expected length of the PCR amplification product is 84bp.

[0088] The primer sequences are as follows:

[0089] Upstream primer CPV 66Q-F: 5'-GGCTTTAGATGATACTCATG-3' (SEQ ID NO.3);

[0090] Downstream primer CPV 66Q-R: 5'-CCTGGATTAAACCAAACTC-3' (SEQ ID NO.4);

[0091] Probe: 5'-CATCAACCAATGACCAAGGTGTTACAA-3' (SEQ ID NO.5).

[0092] Using the DNA extracted in step (2) as a template, a qRT-PCR reaction was performed. The reaction system was as follows: 10 μL of Premix EX Taq (Probe qRT-PCR) (2×), 0.4 μL of upstream primer, 0.4 μL of downstream primer, 0.8 μL of probe, 0.2 μL of ROX ReferenceDye II (50×), 2 μL of DNA template, and 6.2 μL of RNase-free H2O.

[0093] The experiment was conducted using an Applied Biosystems 7500 Real-Time PCR System. The reaction conditions were a standard two-step amplification procedure, set as follows: 98℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 34 s, for 40 cycles.

[0094] (5) Preparation of standard curve

[0095] Perform 10-fold serial dilutions on the obtained positive plasmids, and take 10... -5 10 -6 10 -7 10 -8 10 -9 10 -10 10 -11 Seven consecutive serial dilutions were used as templates for qRT-PCR, with ddH2O as a negative control. A standard curve was constructed by plotting the logarithm of the template standard copy number on the x-axis and the Ct value of the amplification curve on the y-axis. The equation of the standard curve and the correlation coefficient R were calculated. 2 Amplification efficiency E.

[0096] 2.5.2 Preliminary screening of the inhibitory effects of various plant extracts on CPV:

[0097] Based on previous cytotoxicity tests of various plant extracts against F81 cells, 12.5 mg / L was selected as the initial screening concentration. F81 cells were seeded in 6-well cell culture plates. When the cell abundance reached 90%, the culture medium was discarded, the cells were washed three times, and then 100 μL of 100 TCID45 solution was inoculated. 50 The CPV virus solution was placed in an incubator for 1 hour to adsorb, and then 12.5 mg / L of each plant extract solution was added. Each group was repeated three times.

[0098] The cell culture plates of each group were placed in a 5% CO2, 37°C cell culture incubator and cultured for 48 hours. After that, the supernatant was discarded, and the cells were collected to extract DNA for reverse transcription. qRT-PCR was performed on each group of samples to detect the CPVVP2 gene, and the CPV copy number in each sample was calculated according to the standard curve formula.

[0099] Based on the results, soy isoflavones showed the best inhibitory effect on CPV, so subsequent experiments will focus on soy isoflavones.

[0100] 2.5.3 Effect of soybean isoflavones on the growth curve of CPV in F81:

[0101] F81 cells were seeded in 6-well cell culture plates. When the cell abundance reached 90%, the culture medium was discarded, and the cells were washed three times before being inoculated with 100 μL of 100 TCID45 solution. 50 The CPV virus solution was placed in an incubator for 1 hour for adsorption. Then, different concentrations of plant extract solution were added simultaneously to achieve final concentrations of 12.5 mg / L, 6.25 mg / L, and 3.125 mg / L, respectively. A virus control group without the drug was also set up, and each group was performed in triplicate.

[0102] The cell culture plates of the above groups were placed in a 5% CO2, 37℃ cell culture incubator, and the supernatant was discarded and the cells were collected at 4h, 8h, 12h, 18h, 24h, 36h, and 48h, respectively, to extract DNA for reverse transcription. The obtained cDNA samples were subjected to qRT-PCR to detect the CPV VP2 gene in each sample, and the CPV copy number in each sample was calculated according to the standard curve formula to plot the virus growth curve under the influence of the drug.

[0103] 3. Experimental Results

[0104] 3.1 Median Infectious Value of Virus in Tissue Culture (TCID) 50 The measurement results of )

[0105] CPV virus solutions at different dilutions were added to cell culture plates containing a monolayer of F81 cells and incubated at 37°C in a 5% CO2 incubator for 48 hours. Cytopathic effect (CPE) was observed and recorded, and the results are shown in Table 3. TCID was calculated using the Reed-Muench formula. 50 The value is 10 6.48 / 100μL.

[0106] Table 3 Virus titer determination

[0107]

[0108] 3.2 Results of CCK-8 assay for drug cytotoxicity against F81 cells

[0109] The effects of different concentrations of various plant extracts on F81 cell viability were detected using the CCK-8 assay. The results are as follows: Figures 1-12 As shown in the figure. The results indicated that at concentrations of 12.5 mg / L and below, the plant extracts exhibited mild cytotoxicity to cells.

[0110] 3.3 Real-time quantitative PCR detection of the effect of plant extracts on virus expression

[0111] 3.3.1 Establishment of the CPV absolute fluorescence quantitative method:

[0112] (1) Construction of CPV recombinant positive plasmid:

[0113] The CPV gene fragment amplified using a standard PCR reaction system was approximately 846 bp in size. Figure 13 The size of the amplified product matched the expected size, indicating that the target gene fragment was correctly amplified. After gel recovery, the amplified product was ligated into the Adley pTOPO-TA vector. The positive plasmid with correct sequencing was named pTOPO-CPV. The concentration of the extracted plasmid was determined using a nucleic acid protein detector, and it was 168 ng / μL with an OD of [missing value]. 260 / 0D 280 The concentration is 1.99, which meets the purity requirements. The standard concentration, converted to copy number using the formula, is 5.9 × 10⁻⁹. 10 copies / μL.

[0114] (2) Design of CPV absolute fluorescence quantitative primers and probes:

[0115] Based on the Taq probe-based quantitative PCR system, probes and primers were designed. Primers were validated by conventional PCR, and the product was singular in size, 84 bp. Figure 14 This is in line with expectations.

[0116] (3) Preparation of CPV absolute fluorescence quantitative standard curve:

[0117] 10 positive standards were selected. -2 10 -3 10 -4 10 -5 10 -6 10 -7 and 10 -8 Seven consecutive serial dilutions were used as reaction templates, with ddH2O as a negative control. The logarithm of the copy number of the template standard was used as the x-axis, and the Ct value of the amplification curve was used as the y-axis to establish a standard curve versus standard curve.

[0118] The results showed that in 10 -5 -10 -8 Within the dilution range, a good linear relationship is observed.

[0119] Correlation coefficient R of standard curve 2 =0.998, amplification efficiency E=93.8%, which meets the requirements and can be used for quantitative detection of CPV.

[0120] 3.4 Preliminary screening results of the inhibitory effects of various plant extracts on CPV

[0121] Based on the results of cytotoxicity tests, this invention conducts preliminary screening of antiviral effects using various plant extracts. The test results are shown below. Figure 15 The results showed that phlorizin, caffeic acid, neomethylhesperidin-dihydrochalcone, chlorogenic acid, tribulus terrestris saponins, stevia glycosides, isochlorogenic acid, ginsenosides, soy isoflavones, berberine hydrochloride, diosgenin, and silymarin all had significant anti-canine parvovirus effects, among which soy isoflavones were the most significant. Therefore, soy isoflavones were selected for further testing in this invention.

[0122] 3.5 Effect of plant extracts on the growth curve of CPV in F81:

[0123] Cells from different concentrations of soybean isoflavone treatment groups and virus-infected groups were collected at 4h, 8h, 12h, 18h, 24h, 36h, 48h, and 54h, respectively. DNA was extracted using an Adley virus DNA extraction kit, and the DNA was immediately reverse transcribed into cDNA. Purity was assessed using a nucleic acid protein analyzer, and the CPV copy number in each sample was detected using the previously established absolute fluorescence quantitative PCR method. Growth curves were plotted, and the overall trend and comparisons at various time points are shown below. Figure 16 As shown.

[0124] like Figures 17-24 As shown, in the virus control group, the copy number of CPV gradually increased from 4 to 24 hours after virus infection of F81 cells, reached a peak between 24 and 36 hours, and gradually decreased from 36 to 48 hours. This may be because the massive proliferation of viral particles during this stage caused a large number of cell deaths, affecting the further proliferation of the virus. However, after treatment with soy isoflavones, the proliferation of CPV in F81 cells was significantly inhibited after virus infection.

[0125] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of a plant extract for the preparation of a medicament for inhibiting canine parvovirus, characterized in that, The plant extract is dioscin.

2. Use according to claim 1, characterized in that, The medicine includes a pharmaceutically acceptable excipient.

3. Use according to claim 2, characterized in that, The excipient includes a co-solvent, a binder, a disintegrant, a filler, a stabilizer, a preservative, a coating material, and / or a fragrance.

4. Use according to claim 2, characterized in that, The dosage form of the medicine is a granule, a tablet, a capsule, a powder, a pill, an injection, or an oral liquid.

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