Application of karanjin in preparation of anti-Seneca virus medicine

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an application of karanjin in preparation of anti-Seneca virus medicines. The karanjin is separated from seeds of pongamia pinnata, and the purity of the karanjin reaches 99% or above; the effective concentration range of the karanjin in the Seneca virus resisting medicine is 1 to 20 [mu] M. The invention also provides a product for treating Seneca virus. The product comprises the active ingredient, namely the karanjin. It is found for the first time that the karanjin has the anti-Seneca virus effect, the karanjin serves as a natural product, toxicity to host cells is low, toxic and side effects are small, the source is relatively wide, the karanjin is applied to preparation of the anti-SVV medicine, and a new choice is provided for treatment of SVV infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of phellopterin in preparation of anti-Seneca virus drugs. BACKGROUND

[0002] Seneca Valley virus (SVV) is a small RNA virus belonging to the Senecavirus genus of the Picornaviridae family, and has a linear, non-segmented, single-stranded positive-sense RNA genome of about 7.3 kb in length. The structural composition includes a 5' untranslated region, a large open reading frame (ORF), a 3' untranslated region, and a poly(A) tail at the end. The large open reading frame encodes a single protein precursor, which is initially processed into a leader (L) protein and P1, P2, and P3 precursor proteins. Subsequently, the precursor protein is further cleaved into four structural proteins (VP1, VP2, VP3, and VP4), three non-structural proteins (2A, 2B, and 2C), and another four non-structural proteins (3A, 3B, 3C, and 3D). The virus can cause vesicular disease in pigs, and the clinical manifestations include the appearance of blisters and ulcers on the oral and nasal parts, hooves, and other parts of pigs. In severe cases, it can cause lameness and growth retardation in pigs, causing significant economic losses to the pig industry. In addition, SVV can also infect other animals, posing a certain public health risk.

[0003] At present, there is no specific treatment drug for SVV infection, and the main clinical treatments are symptomatic treatment and preventive measures, such as strengthening biological safety and vaccination. However, the development and application of vaccines have certain limitations, such as the possibility of vaccine failure due to rapid virus variation. Therefore, it is of great practical significance to find safe and effective anti-SVV drugs. SUMMARY

[0004] The application solves the problem that there is no specific treatment drug for SVV infection at present, and provides application of phellopterin in preparation of anti-Seneca virus drugs, filling the gap of specific treatment drugs for SVV infection.

[0005] The technical solution claimed in the application is as follows:

[0006] The application provides application of phellopterin in preparation of anti-Seneca virus drugs.

[0007] In the above application, the phellopterin is separated from the seeds of Phellodendron amurense, and has a purity of more than 99%.

[0008] In the above application, the anti-Seneca virus drug includes an active ingredient, and the active ingredient includes phellopterin.

[0009] In the application, the effective concentration of the phlomisoside in the anti-Senecavirus drug is 1-20 μM.

[0010] In the application, the anti-Senecavirus drug comprises pharmaceutically acceptable excipients.

[0011] In the application, the dosage form of the anti-Senecavirus drug comprises injection, oral liquid, pill, powder, ointment, tablet, granule, powder or capsule.

[0012] In the application, the administration route of the anti-Senecavirus drug comprises intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0013] The application also provides a preparation for treating Senecavirus, which comprises active ingredient phlomisoside isolated from the seeds of Phyllanthus emblica, the purity of which is more than 99%, and the effective concentration of the phlomisoside is 1-20 μM.

[0014] Beneficial effects:

[0015] The application provides an application of phlomisoside in preparing an anti-Senecavirus drug, and the application first discovers that phlomisoside has the effect of resisting Senecavirus, can play the antiviral effect by inhibiting the replication of SVV, and interferes with the protein expression of the virus; phlomisoside, as a natural product, has low toxicity to host cells, small toxic and side effects, and a relatively wide source, and the application of phlomisoside in preparing an anti-SVV drug provides a new choice for the treatment of SVV infection, and solves the problem that there is no specific treatment drug for SVV infection. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Fig. 1 shows the effect of phlomisoside on cell viability, wherein the left graph shows the effect of phlomisoside on BHK-21 cell viability, and from left to right, the control, phlomisoside with a concentration of 1 μM, phlomisoside with a concentration of 10 μM, and phlomisoside with a concentration of 20 μM are shown; and the right graph shows the effect of phlomisoside on PK-15 cell viability, and from left to right, the control, phlomisoside with a concentration of 1 μM, phlomisoside with a concentration of 10 μM, and phlomisoside with a concentration of 20 μM are shown.

[0017] Figure 2Figure of the effect of the phloroglucinol on the replication of the recombinant virus (rSVV-eGFP) in the BHK-21 cells in the embodiment of the present application; from left to right, the effect of the phloroglucinol with the concentration of 0 μM, 1 μM, 10 μM and 20 μM on the replication of the recombinant virus (rSVV-eGFP) is represented; DAPI is a kind of blue fluorescent nucleic acid dye, which can specifically bind to double-stranded DNA in the nucleus, and does not react with cytoplasmic components, and is used to indicate the number of cells (i.e. the upper and lower figures are the same sample, the green fluorescent intensity of rSVV-eGFP is used to indicate the replication level of the virus, and the blue fluorescent point represents the total cell number of the sample), by combining the upper and lower figures, the proportion of green fluorescence in blue fluorescence can be used to judge whether the recombinant virus is affected by the drug treatment.

[0018] Figure 3 Figure of the effect of the phloroglucinol on the expression of the viral protein VP3; the left figure is the result figure of the effect of the phloroglucinol on the expression of the viral protein VP3 after treating the BHK-21 cells, from left to right, the blank group without virus and drug, the control group containing virus and only containing DMSO solvent, the group containing virus and the phloroglucinol with the concentration of 1 μM, the group containing virus and the phloroglucinol with the concentration of 10 μM, and the group containing virus and the phloroglucinol with the concentration of 20 μM are represented; the right figure is the result figure of the effect of the phloroglucinol on the expression of the viral protein VP3 after treating the PK-15 cells, from left to right, the blank group without virus and drug, the control group containing virus and only containing DMSO solvent, the group containing virus and the phloroglucinol with the concentration of 1 μM, the group containing virus and the phloroglucinol with the concentration of 10 μM, and the group containing virus and the phloroglucinol with the concentration of 20 μM are represented.

[0019] Figure 4 Figure of the effect of the phloroglucinol with different concentrations on the replication level of the SVV in different cells in the embodiment of the present application; the left figure represents the effect of the phloroglucinol on the viral titer after treating the BHK-21 cells, from left to right, the DMSO control group, the phloroglucinol with the concentration of 1 μM, the phloroglucinol with the concentration of 10 μM, and the phloroglucinol with the concentration of 20 μM are represented; the right figure represents the effect of the phloroglucinol on the viral titer after treating the PK-15 cells, from left to right, the DMSO control group, the phloroglucinol with the concentration of 1 μM, the phloroglucinol with the concentration of 10 μM, and the phloroglucinol with the concentration of 20 μM are represented.

[0020] Figure 5Figure 1 shows the results of the expression of viral protein (VP3) at different time points after SVV infection of different cells with or without 10 μM meliacins treatment; the left panel shows the results of the detection of the expression level of viral protein at different time points after SVV infection of BHK-21 cells with or without 10 μM meliacins treatment by Western blotting: from left to right, the groups are the virus infection group without drug treatment (the infection time is 0 h, 9 h, and 12 h, respectively) and the virus infection group with 10 μM meliacins treatment (the infection time is 0 h, 9 h, and 12 h, respectively); the right panel shows the results of the detection of the expression level of viral protein at different time points after SVV infection of PK-15 cells with or without 10 μM meliacins treatment by Western blotting: from left to right, the groups are the virus infection group without drug treatment (the infection time is 0 h, 9 h, and 12 h, respectively) and the virus infection group with 10 μM meliacins treatment (the infection time is 0 h, 9 h, and 12 h, respectively).

[0021] Figure 6 Figure 2 shows the results of the viral replication level at different time points after SVV infection of different cells with or without 10 μM meliacins treatment; the left panel shows the viral titer at different time points after SVV infection of BHK-21 cells with or without 10 μM meliacins treatment: the red line represents the virus infection group with drug treatment, and the SVV infection time from left to right is 3 h, 6 h, 9 h, and 12 h; the blue line represents the virus infection group without drug treatment (only containing the solvent DMSO), and the SVV infection time from left to right is 3 h, 6 h, 9 h, and 12 h; the right panel shows the viral titer at different time points after SVV infection of PK-15 cells with or without 10 μM meliacins treatment; the red line represents the virus infection group with drug treatment, and the SVV infection time from left to right is 3 h, 6 h, 9 h, and 12 h; the blue line represents the virus infection group without drug treatment (only containing the solvent DMSO), and the SVV infection time from left to right is 3 h, 6 h, 9 h, and 12 h. DETAILED DESCRIPTION

[0022] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0023] Unless otherwise specified, the components described in the present application can be obtained by routine purchase by those skilled in the art.

[0024] Embodiment 1

[0025] This embodiment provides a use of meliacins in the preparation of an anti-senecavirus virus drug.

[0026] In specific embodiments of the present application, the phlorizin is isolated from the seeds of P. koenigii, and has a purity of 99% or more.

[0027] Preferably, the anti-Senecavirus drug comprises an active ingredient, which comprises phlorizin.

[0028] Preferably, the effective concentration of phlorizin in the anti-Senecavirus drug ranges from 1 to 20 μM.

[0029] Preferably, the anti-Senecavirus drug comprises a pharmaceutically acceptable excipient.

[0030] Preferably, the dosage form of the anti-Senecavirus drug comprises injection, oral liquid, pill, powder, paste, tablet, granule, powder or capsule.

[0031] Preferably, the administration route of the anti-Senecavirus drug comprises intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0032] Any act of using phlorizin to prepare an anti-Senecavirus drug, and / or, any act of making phlorizin into an anti-Senecavirus product, and / or, any act of putting phlorizin into a product package labeled for anti-Senecavirus use, any act of selling, offering for sale, using, producing, preparing a product containing phlorizin and having anti-Senecavirus effect, falls within the scope of protection of the present application.

[0033] Example 2

[0034] The present example provides a product for treating Senecavirus, which comprises an active ingredient phlorizin.

[0035] In specific embodiments of the present application, the phlorizin is isolated from the seeds of P. koenigii, and has a purity of 99% or more.

[0036] In specific embodiments of the present application, the effective concentration of phlorizin ranges from 1 to 20 μM.

[0037] Experimental Example Verification experiment of phlorizin against Senecavirus

[0038] 1. Experimental materials and methods

[0039] 1.1 Experimental materials

[0040] 1.1.1 Cells and viruses

[0041] BHK-21 (hamster kidney cell), PK-15 (porcine kidney cell) were preserved in our laboratory, and SVV CHhb17 strain was isolated in our laboratory.

[0042] 1.1.2 Main reagents

[0043] The main reagents used in the present application and the sources of the reagents are shown in Table 1.

[0044] Table 1. Main reagents

[0045] Reagent Source DMEM medium Thermo Fisher Scientific (China) Co., Ltd. Fetal bovine serum Thermo Fisher Scientific (China) Co., Ltd. Trypsin Amresco, Inc. Goat anti-mouse IgG (H+L) HRP ZSGB-BIO Goat anti-rabbit IgG (H+L) HRP ZSGB-BIO FITC labeled goat anti-mouse IgG (H+L) ZSGB-BIO Protein Marker Thermo Fisher Scientific (China) Co., Ltd. SDS-PAGE Buffer (5×) Kangwei Century Biotechnology Co., Ltd. Methanol Fu Yu Fine Chemical Co., Ltd. Skim milk powder Solabio Technology Co., Ltd. Tris Base Solabio Technology Co., Ltd. Proteose peptone Solabio Technology Co., Ltd. Yeast extract Solabio Technology Co., Ltd. Agarose Solabio Technology Co., Ltd. Sodium dodecyl sulfate (SDS) Solabio Technology Co., Ltd. Transfection reagent lip2000 MCE Biological Science and Technology Co., Ltd. RNA extraction kit Yixing Biotechnology Co., Ltd. Reverse transcription kit Lambolide Biotechnology Co., Ltd. 50×TAE buffer Solabio Technology Co., Ltd. Green fluorescent nucleic acid dye Solabio Technology Co., Ltd. Nitrocellulose membrane (NC membrane) Bio-Rad Laboratories, Inc. Ethidium bromide Thermo Fisher Scientific (China) Co., Ltd. ECL reaction solution Lambolide Trading Co., Ltd. 5× Loading Buffer Solabio Technology Co., Ltd. SDS-PAGE gel preparation kit Yazhen Biopharmaceutical Co., Ltd.

[0046] 1.1.3 Main instruments

[0047] The main experimental instruments used in the present application and their manufacturers and regions are shown in Table 2.

[0048] Table 2. Main experimental instruments

[0049] Equipment name Manufacturer Country / region Carbon dioxide incubator Thermo Fisher Scientific USA Clean bench Thermo Fisher Scientific USA Electric heating constant temperature water tank Shanghai Bosen Industrial Co., Ltd. China Low temperature centrifuge Eppendorf Germany Micropipette Eppendorf Germany Gel imaging system Tian Neng China Waste liquid pump, constant temperature shaker Kylin-Bell Lab Instruments China Biological safety cabinet Beijing Mingyuan Laboratory Furniture Manufacturing Co., Ltd. China Electronic balance Mettler Toledo Switzerland Constant temperature metal bath Hangzhou Auson Instruments Co., Ltd. China Pipette Eppendorf Germany Inverted microscope Nikon Japan Microwave oven Galanz Group Co., Ltd. China Freezing and refrigeration box Haier Group China Ice maker Haier Group China

[0050] 1.1.4 Preparation of solutions

[0051] The main chemical reagents used in the present application and their ingredient composition and total volume are shown in Table 3.

[0052] Table 3. Main chemical reagents and their ingredient composition and total volume

[0053] Reagent name Component A Component B Component C Component D Total volume 10% DMEM complete medium 445 mL DMEM medium 50 mL FBS 5 mL double antibody solution (100×) 500 mL 2% DMEM maintenance medium 485 mL DMEM medium 10 mL FBS 5 mL double antibody solution (100×) 500 mL 0.25% trypsin 0.25 g trypsin PBS 100 mL 10× electrophoresis buffer 30 g Tris 188 g glycine 10 g SDS Deionized water 1 L 10x PBS solution 80 g NaCl 2 g KC1 14.4 g anhydrous Na2HP04 2.4 g KH2P04 1 L 10x PBST solution Tween-20 10x PBS Deionized water 1 L Transfer buffer 5.8 g Tris 2.9 g glycine 0.37 g SDS Deionized water 1 L

[0054] 1.2 Method

[0055] 1.2.1 Cell culture

[0056] In the present study, BHK-21 and PK-15 cells were mainly used as host cells for SVV infection.

[0057] 1.2.2 Cell recovery

[0058] Prepare a 37℃ constant temperature water bath in advance, quickly put the cells into it for thawing, and control the whole process for about 1 minute until the cryopreserved solution is completely melted. Then centrifuge the cells at 1000 rpm for 3 minutes, carefully aspirate the supernatant with a pipette, supplement 5 mL of culture medium and mix well by blowing. Move the cell suspension into a culture bottle, slowly shake to distribute the cells evenly, and then place it in the incubator for culture.

[0059] 1.2.3 Cell passage

[0060] Subculture when cell density reaches 90% and above: discard old medium, take phosphate buffer solution (PBS) gently add cell hole to wash cells, then use 0.25% trypsin digestion; when the naked eye can observe that the cell falls off, discard the supernatant, pat the cell bottle side wall to make the cell into a sandy state, and then add an appropriate amount of medium to blow evenly, and transfer to the cell hole plate for standby.

[0061] 1.2.4 Cell freezing

[0062] Centrifugal collection of cells in centrifugal tube, add the prepared cell freezing solution (10% DMSO solution and 90% fetal bovine serum), first placed in-80℃ refrigerator for 24 h, then moved to liquid nitrogen tank for long-term storage.

[0063] 2. Toxicity experiment of phloretin on SVV infected cells

[0064] (1) Cell culture: PK-15 cells and BHK-21 cells suitable for SVV infection were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37℃, 5% CO2.

[0065] (2) Drug treatment: PK-15 cells were treated with different concentrations (1 μM, 10 μM, 20 μM) of phloretin for 48 h.

[0066] (3) Cell viability detection: MTT method was used, and after incubating the cells with MTT reagent, the absorbance was measured at 490 nm, and the cell viability was calculated. The results showed that, as shown in Figure 1 there was no significant difference between the phloretin at the concentration range of 1-20 μM and the control group. It showed that phloretin had no obvious effect on the activity of the two kinds of cells in this concentration range.

[0067] 3. In vitro experiment of phloretin inhibiting SVV replication

[0068] (1) Phloretin inhibits the replication of recombinant virus (rSVV-eGFP): PK-15 cells were inoculated in a 24-well plate and cultured overnight, then infected with eGFP labeled SVV recombinant virus (rSVV-eGFP) at a multiplicity of infection (MOI=5) for 1 h, then 1 μM, 10 μM, 20 μM concentration of phloretin was added, and the control group (infected with SVV, only containing solvent DMSO) was set, and cultured at 37℃, 5% CO2 for 12 h, then observed the effect of different concentrations of phloretin on SVV replication by fluorescence microscope. The results showed that, as shown in Figure 2Compared with the control group, different concentrations of phlorizin significantly inhibited the replication of rSVV-eGFP, and the inhibition showed a dose-dependent manner.

[0069] (2) Analysis of viral (SVV) gene expression inhibited by phlorizin: PK-15 cells and BHK-21 cells were respectively plated in 24-well plates with DMEM medium containing 10% fetal bovine serum and cultured at 37 ℃, 5% CO2. When the cell density just reached the confluence of single-layer fullness (100%), SVV was inoculated at a dose of MOI=5, and after 1 h of infection, the culture medium was discarded, and the cells were washed twice with PBS, and then DMEM medium (containing 2% FBS) containing different concentrations (1 μM, 10 μM, 20 μM) of phlorizin was added for continuous culture for 12 h. At the same time, a virus infection group control (only containing DMSO solvent) and a cell blank group (without virus and drug) were set up, and then the cell samples were collected for gene expression analysis by Western blot. The results showed that, as shown in Figure 3 , different concentrations of phlorizin could inhibit the expression of viral protein VP3 (VP3 was used to indicate viral protein expression, and β-actin was used as an internal reference protein), and the inhibition showed a dose-dependent manner.

[0070] (3) Detection of viral titer by TCID50 experiment: PK-15 cells and BHK-21 cells were respectively plated in 24-well plates with DMEM medium containing 10% fetal bovine serum and cultured at 37 ℃, 5% CO2. When the cell density just reached the confluence of single-layer fullness (100%), SVV was inoculated at a dose of MOI=5, and after 1 h of infection, the culture medium was discarded, and the cells were washed twice with PBS, and then DMEM medium (containing 2% FBS) containing different concentrations (1 μM, 10 μM, 20 μM) of phlorizin was added for continuous culture for 12 h. At the same time, a virus infection group control (only containing DMSO solvent) was set up, and then the cell supernatant was collected for determination of SVV titer by TCID50 experiment. The results showed that, as shown in Figure 4 , compared with the control group, the SVV titers of 1 μM, 10 μM, and 20 μM phlorizin treatment groups were reduced by about 10 times, 1000 times, and 10000 times, respectively, and similar results were obtained in two kinds of cells, indicating that phlorizin could dose-dependently inhibit the replication of SVV.

[0071] (4) Time course analysis of inhibition of SVV replication by phloretin: PK-15 cells and BHK-21 cells were plated in 24-well plates in DMEM medium containing 10% fetal bovine serum and incubated at 37 °C in 5% CO2. When the cell density reached 100% confluence, SVV was inoculated at an MOI of 5, and after 1 h, the medium was discarded, and the cells were washed with PBS and then supplemented with DMEM containing 10 μM phloretin (containing 2% FBS). Subsequently, cell samples were collected at 3 h, 6 h, 9 h, and 12 h after infection, and the expression of viral VP3 protein and viral titer were analyzed by Western blot and TCID50, respectively. The results showed that compared with the control group, phloretin significantly inhibited the expression of VP3 protein at 9 h after viral infection ( Figure 5 ). In addition, TCID50 results showed that phloretin significantly inhibited the replication of SVV at an early stage (6 h) Figure 6 ).

[0072] In summary, phloretin can effectively inhibit the replication of SVV and has good anti-SVV activity, and can be used for the preparation of anti-SVV drugs.

[0073] The above examples are only used to illustrate the technical solutions of the present application and not to limit the present application. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. Use of phlorizin in the preparation of a drug for resisting Senecavirus.

2. Use according to claim 1, characterized in that, The phlorizin is separated from the seeds of Phyllanthus emblica, and has a purity of more than 99%.

3. Use according to claim 1 or 2, characterized in that, The drug for resisting Senecavirus comprises an active component, and the active component comprises phlorizin.

4. Use according to claim 3, characterized in that, An effective concentration of the phlorizin in the drug for resisting Senecavirus ranges from 1 to 20 μM.

5. Use according to claim 4, characterized in that, The drug for resisting Senecavirus comprises pharmaceutically acceptable excipients.

6. Use according to claim 5, characterized in that, Dosage forms of the drug for resisting Senecavirus include injections, oral liquids, pills, powders, ointments, tablets, granules, powders, or capsules.

7. Use according to claim 6, characterized in that, Administration routes of the drug for resisting Senecavirus include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

8. A preparation for the treatment of a Senecavirus, characterized in that, The preparation comprises the active component phlorizin.

9. The article of claim 8, wherein, The phlorizin is separated from the seeds of Phyllanthus emblica, and has a purity of more than 99%.

10. The article of claim 8, wherein, An effective concentration of the phlorizin ranges from 1 to 20 μM.

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