Use of meliatoxine in the preparation of a drug against a senecavirus

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of phyllangin in preparation of anti-senecavirus virus (SVV) drugs. The phyllangin is separated from seeds of phyllanthus emblica, and the purity of the phyllangin reaches more than 99%. The effective concentration of the phyllangin in the anti-SVV drug is in the range of 1-20 muM. The application further provides a preparation for treating SVV, and the preparation comprises an active ingredient of phyllangin. The application firstly finds that the phyllangin has the effect of resisting SVV, the phyllangin as a natural product has low cytotoxicity to host cells, has small toxic and side effects, and has a relatively wide source, and the application of the phyllangin to preparation of anti-SVV drugs provides a new choice for treatment of SVV infection.

Description

Application of hydroflavin in the preparation of anti-Seneca virus drugs Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of hydroquinone in the preparation of anti-Seneca virus drugs. Background Technology

[0002] Seneca Valley virus (SVV) is a small RNA virus belonging to the genus Senecavirus in the family Picornaviridae. Its genome is a linear, non-segmented, single-stranded, positive-sense RNA, approximately 7.3 kb in length. Its structure includes a 5' untranslated region, a large open reading frame (ORF), a 3' untranslated region, and a terminal poly(A) tail. The ORF encodes a single protein precursor, which is initially processed into a leader (L) protein and three precursor proteins (P1, P2, and P3). Subsequently, the precursor proteins are further cleaved into four structural proteins (VP1, VP2, VP3, VP4), three non-structural proteins (2A, 2B, 2C), and four more non-structural proteins (3A, 3B, 3C, 3D). This virus can cause vesicular disease in pigs, clinically manifesting as vesicles and ulcers on the snout, hooves, and other areas. In severe cases, it can lead to lameness and growth retardation, causing significant economic losses to the pig farming industry. In addition, SVV can infect other animals, posing a certain potential public health risk.

[0003] Currently, there is no specific treatment for SVV infection. Clinically, treatment mainly involves symptomatic relief and preventative measures, such as enhanced biosafety and vaccination. However, vaccine development and application have limitations, such as the rapid mutation of the virus potentially rendering the vaccine ineffective. Therefore, finding safe and effective anti-SVV drugs is of significant practical importance. Summary of the Invention

[0004] This invention addresses the problem that there are currently no effective treatments for SVV infection by providing an application of hydroquinone in the preparation of anti-Seneca virus drugs, thus filling the gap in the availability of effective treatments for SVV infection.

[0005] The technical solution claimed by this invention is as follows:

[0006] This invention provides the application of hydroflavin in the preparation of anti-Seneca virus drugs.

[0007] In the above applications, the hydroquinone is isolated from the seeds of hydroquinone, and its purity is over 99%.

[0008] In the above applications, the anti-Seneca virus drug includes an active ingredient, which includes hydroquinone.

[0009] In the above applications, the effective concentration range of the hydroflavin in the anti-Seneca virus drug is 1-20 μM.

[0010] In the above applications, the anti-Seneca virus drug includes pharmaceutically acceptable excipients.

[0011] In the above applications, the dosage forms of the anti-Seneca virus drug include: injections, oral liquids, pills, powders, ointments, tablets, granules, powders, or capsules.

[0012] In the above applications, the routes of administration of the anti-Seneca virus drug include: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

[0013] The present invention also provides a product for treating Seneca virus, the product comprising the active ingredient hydroquinone, the hydroquinone being isolated from the seeds of hydroquinone and having a purity of over 99%; the effective concentration range of the hydroquinone is 1-20 μM.

[0014] Beneficial effects:

[0015] This invention provides the application of hydroquinone in the preparation of anti-Seneca virus drugs. This invention is the first to discover that hydroquinone has an anti-Seneca virus effect, which can exert its antiviral effect by inhibiting SVV replication and interfering with viral protein expression. As a natural product, hydroquinone has low toxicity to host cells, few side effects, and relatively wide sources. Its application in the preparation of anti-SVV drugs provides a new option for the treatment of SVV infection and solves the problem that there are no specific drugs for the treatment of SVV infection. Attached Figure Description

[0016] Figure 1 shows the effect of hydroquinone on cell viability in the embodiments of the present invention; wherein: the left figure shows the effect of hydroquinone on BHK-21 cell viability, from left to right representing control, hydroquinone at a concentration of 1 μM, hydroquinone at a concentration of 10 μM, and hydroquinone at a concentration of 20 μM; the right figure shows the effect of hydroquinone on PK-15 cell viability, from left to right representing control, hydroquinone at a concentration of 1 μM, hydroquinone at a concentration of 10 μM, and hydroquinone at a concentration of 20 μM.

[0017] Figure 2 shows the effect of hydroquinone on the replication of recombinant virus (rSVV-eGFP) in BHK-21 cells in this embodiment of the invention; from left to right, it represents the effect of hydroquinone concentrations of 0 μM, 1 μM, 10 μM, and 20 μM on the replication of recombinant virus (rSVV-eGFP); DAPI is a blue fluorescent nucleic acid dye that can specifically bind to double-stranded DNA in the cell nucleus and does not interact with cytoplasmic components. It is used to indicate the number of cells (i.e., the upper and lower figures are of the same sample, the green fluorescence intensity of rSVV-eGFP is used to indicate the viral replication level, and the blue fluorescent dots represent the total number of cells in the sample). Combining the upper and lower figures, the proportion of green fluorescence in blue fluorescence can be used to determine whether the recombinant virus is affected by drug treatment.

[0018] Figure 3 shows the effect of hydroquinone on the expression of viral protein VP3 in the embodiments of the present invention. The left figure shows the effect of hydroquinone treatment on the expression of viral protein VP3 in BHK-21 cells. From left to right, the results are: blank group without virus and drug, control group with virus and only DMSO solvent, virus and 1 μM hydroquinone, virus and 10 μM hydroquinone, and virus and 20 μM hydroquinone. The right figure shows the effect of hydroquinone treatment on the expression of viral protein VP3 in PK-15 cells. From left to right, the results are: blank group without virus and drug, control group with virus and only DMSO solvent, virus and 1 μM hydroquinone, virus and 10 μM hydroquinone, and virus and 20 μM hydroquinone.

[0019] Figure 4 shows the effect of different concentrations of water-based quercetin on the replication level of SVV in different cells in the embodiments of the present invention. The left figure shows the effect of water-based quercetin treatment on the viral titer of BHK-21 cells, from left to right: DMSO control group, 1 μM water-based quercetin, 10 μM water-based quercetin, and 20 μM water-based quercetin. The right figure shows the effect of water-based quercetin treatment on the viral titer of PK-15 cells, from left to right: DMSO control group, 1 μM water-based quercetin, 10 μM water-based quercetin, and 20 μM water-based quercetin.

[0020] Figure 5 shows the expression results of viral protein (VP3) at different time points after SVV infection of different cells treated with or without 10 μM water-based quercetin in the embodiments of the present invention. The left figure shows the results of Western blot detection of viral protein expression levels at different time points after SVV infection of BHK-21 cells treated with or without quercetin: from left to right, the drug-free virus infection group (infection time 0 h, 9 h, 12 h) and the virus infection group treated with 10 μM water-based quercetin (infection time 0 h, 9 h, 12 h) are shown. The right figure shows the results of Western blot detection of viral protein expression levels at different time points after SVV infection of PK-15 cells treated with or without quercetin: from left to right, the drug-free virus infection group (infection time 0 h, 9 h, 12 h) and the virus infection group treated with 10 μM water-based quercetin (infection time 0 h, 9 h, 12 h) are shown.

[0021] Figure 6 shows the statistical results of viral replication levels at different time points after SVV infection of different cells treated with or without 10 μM quercetin in the embodiments of the present invention. The left figure shows the viral titer at different time points after SVV infection of BHK-21 cells treated with or without quercetin. The red line represents the drug-treated group, with SVV infection times from left to right being 3 h, 6 h, 9 h, and 12 h. The blue line represents the untreated group (containing only DMSO solvent), with SVV infection times from left to right being 3 h, 6 h, 9 h, and 12 h. The right figure shows the viral titer at different time points after SVV infection of PK-15 cells treated with or without quercetin. The red line represents the drug-treated group, with SVV infection times from left to right being 3 h, 6 h, 9 h, and 12 h. The blue line represents the untreated group (containing only DMSO solvent), with SVV infection times from left to right being 3 h, 6 h, 9 h, and 12 h. Detailed Implementation

[0022] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Unless otherwise specified, all components described in this invention are readily available to those skilled in the art.

[0024] Group 1 Examples

[0025] This set of examples provides an application of hydroflavin in the preparation of anti-Seneca virus drugs.

[0026] In a specific embodiment of the present invention, the hydroflavin is isolated from the seeds of hydroflavin, and its purity is above 99%.

[0027] Preferably, the anti-Seneca virus drug comprises an active ingredient, which includes hydroquinone.

[0028] Preferably, the effective concentration range of the hydroflavin in the anti-Seneca virus drug is 1-20 μM.

[0029] Preferably, the anti-Seneca virus drug includes pharmaceutically acceptable excipients.

[0030] Preferably, the dosage form of the anti-Seneca virus drug includes: injection, oral liquid, pill, powder, ointment, tablet, granule, powder or capsule.

[0031] Preferably, the route of administration of the anti-Seneca virus drug includes: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

[0032] According to the teachings of this invention, any act of preparing an anti-Seneca virus drug using hydroquinone, and / or any act of making an anti-Seneca virus product from hydroquinone, and / or any act of placing hydroquinone in a product packaging box labeled with anti-Seneca virus use, and any act of selling, offering for sale, using, producing, or preparing a product containing hydroquinone and having anti-Seneca virus activity, falls within the protection scope of this invention.

[0033] Group 2 Examples

[0034] This set of embodiments provides a product for treating Seneca virus, the product comprising the active ingredient hydroquinone.

[0035] In a specific embodiment of the present invention, the hydroflavin is isolated from the seeds of hydroflavin, and its purity is above 99%.

[0036] In a specific embodiment of the present invention, the effective concentration range of the aqueous chlorophyll is 1-20 μM.

[0037] Experimental Verification Experiment of Water-based Cortexin against Seneca Virus

[0038] 1. Experimental Materials and Methods

[0039] 1.1 Experimental Materials

[0040] 1.1.1 Cells and Viruses

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

[0042] 1.1.2 Main Reagents

[0043] The main reagents used in this invention and their sources are shown in Table 1.

[0044] Table 1. Main Reagents

[0045] Reagent sources: DMEM culture medium, Thermo Fisher Scientific (China) Co., Ltd., fetal bovine serum, Thermo Fisher Scientific (China) Co., Ltd., pancreatic enzymes, Amresco Co., Ltd., goat anti-mouse IgG (H+L), HRP, Zhongshan Jinqiao Biotechnology Co., Ltd., goat anti-rabbit IgG (H+L), HRP, Zhongshan Jinqiao Biotechnology Co., Ltd., FITC-labeled goat anti-mouse IgG (H+L), Zhongshan Jinqiao Biotechnology Co., Ltd., protein marker, Thermo Fisher Scientific (China) Co., Ltd., SDS-PAGE Buffer (5×), Kangwei Century Biotechnology Co., Ltd., methanol, Fuyu Fine Chemical Co., Ltd., skim milk powder, Solarbio Technology Co., Ltd., tris(hydroxymethyl)aminomethane (Tris) Base) Solarbio Science & Technology Co., Ltd. Peptone Solarbio Science & Technology Co., Ltd. Yeast Extract Solarbio Science & Technology Co., Ltd. Agarose Solarbio Science & Technology Co., Ltd. Sodium Dodecyl Sulfate (SDS) Solarbio Science & Technology Co., Ltd. Transfection Reagent lip2000 MCE Biotechnology Co., Ltd. RNA Extraction Kit Yisheng Biotechnology Co., Ltd. Reverse Transcription Kit Lamborghini Biotechnology Co., Ltd. 50× TAE Buffer Solarbio Science & Technology Co., Ltd. Green Fluorescent Nucleic Acid Dye Solarbio Science & Technology Co., Ltd. Nitrocellulose Membrane (NC Membrane) Bio-Rad Life Sciences Co., Ltd. Ethidium Bromide Thermo Fisher Scientific (China) Co., Ltd. ECL Reaction Solution Lamborghini Trading Co., Ltd. 5× Loading Buffer Solarbio Science & Technology Co., Ltd. SDS-PAGE Gel Preparation Kit Yamei Biopharmaceutical Technology Co., Ltd. surface

[0046] 1.1.3 Main Instruments

[0047] The main experimental instruments used in this invention, along with their manufacturers and their regions, are shown in Table 2.

[0048] Table 2. Main Test Instruments

[0049] Equipment Name | Manufacturer | Country / Region | CO2 Incubator | Thermo Fisher Scientific (USA) | Clean Bench | Thermo Fisher Scientific (USA) | Thermo Fisher Scientific (USA) | Thermo Fisher Scientific (USA) | Electric Heating Constant Temperature Water Bath | Shanghai Boxun Industrial Co., Ltd. | China | Low Temperature Centrifuge | Eppendorf (Germany) | Micropipette | Eppendorf (Germany) | Gel Imaging System | Tianneng Co., Ltd. | China | Waste Liquid Pump, Thermostatic Shaker | Kylin-Bell Lab Instruments | China | Biosafety Cabinet | Beijing Mingyuan Laboratory Furniture Co., Ltd. | China | Electronic Balance | Mettler Toledo | Switzerland | Thermostatic Metal Bath | Hangzhou Aosheng Instrument Co., Ltd. | China | Pipette | Eppendorf (Germany) | Inverted Microscope | Nikon | Japan | Microwave Oven | Guangdong Galanz Group Co., Ltd. | China | Refrigeration Box | Haier Group | China | Ice Maker | Haier Group | China surface

[0050] 1.1.4 Solution Preparation

[0051] The main chemical reagents used in this invention, their composition and total volume are shown in Table 3.

[0052] Table 3. Main chemical reagents, their 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 | Antibiotic Solution (100×) 500 mL 2% DMEM Maintenance Medium 485 mL | DMEM Medium 10 mL | FBS 5 mL | Antibiotic 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 | 10× PBS Solution 80 g | NaCl 2 g | KCl 14.4 g | Anhydrous Na2HPO4 2.4 g | KH2PO4 1 L | 10× PBS T Solution Tween-20 | 10× PBS Deionized Water 1 L | Transfer Buffer 5.8 g | Tris 2.9 g | Glycine 0.37 g | SDS Deionized Water 1 L surface

[0054] 1.2 Methods

[0055] 1.2.1 Cell Culture

[0056] This study primarily used BHK-21 and PK-15 cells as host cells for SVV infection.

[0057] 1.2.2 Cell resuscitation

[0058] Prepare a 37°C constant temperature water bath in advance, and quickly place the cells in it to thaw, controlling the process to about 1 minute until the cryopreservation solution is completely thawed. Then, centrifuge the cells horizontally at a low speed of 1000 rpm for 3 minutes, carefully aspirate the supernatant with a pipette, add culture medium to 5 mL and mix well by pipetting. Transfer the cell suspension to a culture flask, gently shake to distribute the cells evenly, and then place it in an incubator for culture.

[0059] 1.2.3 Cell passage

[0060] When the cell density reaches 90% or higher, passage is performed: discard the old culture medium, gently add phosphate-buffered saline (PBS) to the cell wells to wash the cells, and then digest with 0.25% trypsin; when cells are observed to detach with the naked eye, discard the supernatant, gently tap the side wall of the cell flask to make the cells detach in a flowing sand-like manner, add an appropriate amount of culture medium, pipette evenly, and transfer to cell culture plates for later use.

[0061] 1.2.4 Cell cryopreservation

[0062] Cells were collected by centrifugation into centrifuge tubes, and freshly prepared cell cryopreservation solution (10% DMSO solution and 90% fetal bovine serum) was added. The cells were first placed in a -80 ℃ freezer for 24 h, and then transferred to a liquid nitrogen tank for long-term storage.

[0063] 2. Toxicity experiment of hydroquinone against SVV-infected cells

[0064] (1) Cell culture: PK-15 cells and BHK-21 cells suitable for SVV infection were cultured in Durbeco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2.

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

[0066] (3) Cell viability assay: The MTT assay was used. After incubating cells with MTT reagent, the absorbance was measured at 490 nm, and cell viability was calculated. The results showed, as shown in Figure 1, that hydroquinone had no significant effect on the activity of PK-15 cells and BHK-21 cells compared with the control group within the concentration range of 1-20 μM. This indicates that hydroquinone had no significant effect on the activity of either cell type within this concentration range.

[0067] 3. In vitro experiment on the inhibition of SVV replication by chloroquine

[0068] (1) Inhibition of recombinant virus (rSVV-eGFP) replication by hydroquinone: PK-15 cells were seeded in 24-well plates and cultured overnight. Cells were then infected with eGFP-labeled recombinant SVV virus (rSVV-eGFP) at a multiplicity of infection (MOI=5) for 1 h. Hydroquinone at concentrations of 1 μM, 10 μM, and 20 μM was then added, with a control group (infected with SVV, containing only DMSO solvent) included. Cells were cultured at 37℃ and 5% CO2 for 12 h. The effect of different concentrations of hydroquinone on SVV replication was then observed using fluorescence microscopy. The results, as shown in Figure 2, compared with the control group, showed that different concentrations of hydroquinone significantly inhibited rSVV-eGFP replication, and this inhibition was dose-dependent.

[0069] (2) Gene expression analysis of salivarius-inhibited virus (SVV): PK-15 cells and BHK-21 cells were seeded in 24-well plates with DMEM medium containing 10% fetal bovine serum and cultured at 37 ℃ and 5% CO2. When the cell density just reached the confluence of monolayers (100%), SVV was inoculated at a dose of MOI=5. After 1 h of infection, the medium was discarded, the cells were washed twice with PBS, and DMEM maintenance medium (containing 2% FBS) containing different concentrations (1 μM, 10 μM, 20 μM) of salivarius was added and the cells were cultured for another 12 h. At the same time, a virus infection control group (containing only DMSO solvent) and a cell blank group (containing no virus and drug) were set up. Cell samples were then collected and gene expression analysis was performed by Western blot. The results showed that, as shown in Figure 3, different concentrations of quercetin could inhibit the expression of viral protein VP3 (VP3 is used to indicate viral protein expression, and β-actin is used as an internal reference protein), and this inhibitory effect was dose-dependent.

[0070] (3) TCID50 assay for virus titer: PK-15 cells and BHK-21 cells were seeded in 24-well plates with DMEM medium containing 10% fetal bovine serum and cultured at 37 ℃ and 5% CO2. When the cell density reached 100% confluence, SVV was inoculated at an MOI of 5. After 1 h of infection, the medium was discarded, and the cells were washed twice with PBS. Then, DMEM maintenance medium (containing 2% FBS) containing different concentrations (1 μM, 10 μM, 20 μM) of quercetin was added and the cells were cultured for another 12 h. A drug-free virus infection control group (containing only DMSO solvent) was set up. The cell supernatant was then collected and the SVV titer was determined by TCID50 assay. The results, as shown in Figure 4, indicated that compared with the control group, the SVV titers in the 1 μM, 10 μM, and 20 μM hydroquinone treatment groups were reduced by approximately 10-fold, 1000-fold, and 10000-fold, respectively, and similar results were obtained in both cell types, suggesting that hydroquinone can dose-dependently inhibit SVV replication.

[0071] (4) Timing analysis of the inhibitory effect of hydroquinone on SVV replication: PK-15 cells and BHK-21 cells were seeded in 24-well plates with DMEM medium containing 10% fetal bovine serum and cultured at 37 ℃ and 5% CO2. When the cell density just reached the confluence of monolayers (100%), SVV was inoculated at a dose of MOI=5. After 1 h of infection, the medium was discarded, the cells were washed with PBS several times, and DMEM maintenance medium (containing 2% FBS) containing 10 μM hydroquinone was added. Subsequently, cell samples were collected at 3 h, 6 h, 9 h, and 12 h post-infection, and the expression level and viral titer of VP3 protein were analyzed by Western blot and TCID50. The results showed that hydroquinone treatment significantly inhibited VP3 protein expression 9 h post-infection compared with the control group (Figure 5). In addition, TCID50 results showed that hydroquinone had a significant inhibitory effect on SVV replication in the early stage (6 h) (Figure 6).

[0072] In summary, hydroquinone can effectively inhibit SVV replication and has good anti-SVV activity, and can be used to prepare anti-SVV drugs.

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

Claims

1. Application of hydroquinone in the preparation of anti-Seneca virus drugs.

2. The application according to claim 1, characterized in that, The hydroquinone is isolated from the seeds of hydroquinone, and its purity is over 99%.

3. The application according to claim 1 or 2, characterized in that, The anti-Seneca virus drug includes an active ingredient, which includes hydroquinone.

4. The application according to claim 3, characterized in that, The effective concentration range of the hydroflavin in the anti-Seneca virus drug is 1-20 μM.

5. The application according to claim 4, characterized in that, The anti-Seneca virus drug includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that, The dosage forms of the anti-Seneca virus drugs include: injections, oral liquids, pills, powders, ointments, tablets, granules, powders, or capsules.

7. The application according to claim 6, characterized in that, The routes of administration for the anti-Seneca virus drugs include: intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, or transdermal administration.

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