Application of sophocarpidine in preparation of medicine for resisting foot and mouth disease virus type O and continuous infection of foot and mouth disease virus type O

Abstract

The invention relates to a novel application of matrine, in particular to an application of matrine in preparation of a medicine for resisting O-type foot and mouth disease virus and continuous infection thereof. It is proved for the first time that matrine has anti-foot-and-mouth disease virus activity, especially anti-infection ability to O-type foot-and-mouth disease persistently infected virus strains; the invention provides a new application of matrine in preparation of medicines for resisting foot-and-mouth disease and continuous infection thereof for the first time. Matrine belongs to a traditional Chinese medicine extract, is low in cytotoxicity and high in safety, and has an application prospect in the aspects of prevention and treatment of the foot and mouth disease virus and continuous infection thereof. The invention also provides a pharmaceutical composition taking sophocarpidine as a main active component, and a theoretical basis and a practical basis are provided for development of medicines for preventing and treating foot-and-mouth disease and sustained infection thereof.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of drug application, and particularly relates to application of a traditional Chinese medicine extract, matrine, in preparation of a medicine for resisting O-type foot-and-mouth disease virus and persistent infection. BACKGROUND

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease caused by foot-and-mouth disease virus (FMDV), which mainly infects domestic and wild cloven-hoofed animals such as cattle, buffaloes, pigs, sheep and goats. As one of the highly contagious diseases of animals widely prevalent in the Asian continent, FMDV can be transmitted through direct contact with the secretions and excretions of infected animals, or indirectly through contaminated feed, utensils and aerosols. Infected animals often develop fever, and blisters form on the mucosa of the oral cavity, the skin of the hooves and the udder, and the blisters rupture to form ulcers, resulting in lameness, difficulty in lactation and other problems, and young animals may also suddenly die of myocarditis. This disease not only causes growth and development of animals to be hindered, and milk and meat production to be greatly reduced, but also causes international trade restrictions on animal products due to the risk of cross-border transmission, resulting in serious economic losses to the livestock industry of each country. Foot-and-mouth disease is listed as the first animal epidemic by the World Organization for Animal Health (OIE).

[0003] In addition to acute clinical symptoms, infected animals also develop a long-term virus-carrying and virus-excreting state after infection with FMDV, which is characterized by no obvious clinical symptoms but the continuous isolation of live virus in the nasopharynx and oropharynx fluid. This long-term virus-carrying state is related to the persistent replication of the virus in the upper respiratory tract and upper digestive tract of the host, and the immune response of the host is difficult to effectively eliminate the virus, and the continuous excretion of the carrier significantly increases the risk of hidden transmission of the virus, resulting in a substantial increase in the difficulty of epidemic prevention and control. Studies have shown that during the period of foot-and-mouth disease virus persistent infection, co-evolution occurs between the virus and the host cells, and under the persistent selection pressure, the nucleic acid sequence of the virus mutates accordingly, so that the persistently infected virus exhibits stronger viral virulence and greater ability to form persistent infection, increasing the difficulty of epidemic prevention and control. At the same time, the host cells gradually evolve the ability to limit virus replication and lysis in order to maintain FMDV persistent infection, and wild-type FMDV cannot effectively replicate in these evolved host cells, but the virus harvested from the persistent infection cell line can successfully replicate by activating different host response modes, and can quickly re-establish persistent infection. Therefore, when FMDV persistent infection is cured, the "self-healing" or "cured" animals still have a high risk of re-forming persistent infection, and it is particularly important to eliminate FMDV persistent infection virus in a timely manner rather than to enhance the immune response of the host.

[0004] After the outbreak of FMD, in order to eradicate FMDV, all infected animals are usually killed; on the other hand, in order to prevent the introduction of FMDV into areas without epidemic, the livestock trade in epidemic areas will be restricted. The existing prevention and control and treatment means of FMD mainly depend on vaccination. So far, there is no commercialized specific drug for FMDV and persistent infection virus, and no related drug treatment research for FMDV persistent infection has been seen.

[0005] Matrine is a natural quinoline alkaloid rich in Sophora alopecuroides Linn, Sophora tonkinensis, and is the main effective monomer of Sophora alopecuroides Linn, which has anti-tumor, anti-inflammatory, analgesic, anti-fibrosis, anti-viral, anti-arrhythmia, anti-bacterial and insecticidal and other biological effects.

[0006] At present, there is no related research report on the treatment effect of matrine on FMDV and persistent infection. Through the study of the mechanism of matrine in directly inhibiting O-type FMDV, blocking the virus replication cycle and regulating the host immunity, a new target and theoretical basis can be provided for the natural medicine antiviral research, and the blank of the mechanism research of matrine against FMDV and persistent infection is filled. SUMMARY

[0007] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide the application of matrine, a traditional Chinese medicine extract, in the preparation of an anti-FMDV and persistent infection drug. Matrine is a traditional Chinese medicine extract, which has low cytotoxicity and high safety, and has application prospects in the prevention and treatment of FMDV and persistent infection.

[0008] The matrine involved in the present application is an alkaloid extracted from Sophora plants, which inhibits the infection of O-FMDV and persistent infection virus strains in host cells; the molecular weight thereof is 248.36, the molecular formula is C 15 H 24 N2O, and the structural formula is:

[0009] One object of the present application is to provide the application of matrine, which is (a) and / or (b) and / or (c) and / or (d): (a) the application of matrine in the preparation of an anti-FMDV infection drug; (b) the application of matrine in the preparation of an anti-FMDV persistent infection drug; (c) the application of matrine in the preparation of a product for preventing FMD, wherein the product is a drug or a feed additive; (d) the application of matrine in the preparation of a drug for preventing or treating FMD or FMDV persistent infection.

[0010] The matrine is capable of resisting virus by blocking expression and replication of the virus.

[0011] The FMDV virus is O FMDV and its persistent infection virus strain.

[0012] The foot-and-mouth disease is a disease caused by O FMDV and its persistent infection virus strain or a disease caused by a virus variant strain.

[0013] Another object of the present application is to provide a medicine or a pharmaceutical composition for resisting FMDV persistent infection or treating FMDV persistent infection, which contains a matrine compound.

[0014] Another object of the present application is to provide a medicine or a pharmaceutical composition for resisting FMDV infection or preventing / treating foot-and-mouth disease, which contains a matrine compound.

[0015] The concentration of matrine in the medicine or the pharmaceutical composition is 1-625 ng / mL.

[0016] The medicine or the pharmaceutical composition can be prepared into different required dosage forms, such as injection preparations or oral preparations, such as capsules, tablets, etc.

[0017] In summary, the above technical solutions and solved technical problems have the following advantages: First, the present application proves for the first time that matrine has the activity of resisting foot-and-mouth disease virus, especially the anti-infection ability to O foot-and-mouth disease persistent infection virus strain. The present application provides a new use of matrine, provides a basis for the development of an antiviral drug for foot-and-mouth disease persistent infection and foot-and-mouth disease virus, and provides more methods and means for preventing and treating foot-and-mouth disease and persistent infection.

[0018] Second, the prior art lacks a specific drug for foot-and-mouth disease virus persistent infection. The existing prevention and treatment means for foot-and-mouth disease mainly rely on vaccination, but when the vaccinated animals are exposed to persistent infection virus, there is still a risk of infection. Timely blocking and preventing virus infection can effectively prevent and control the epidemic and avoid repeated infection of animals. The present application provides a new drug for treating foot-and-mouth disease, provides a more reliable prevention and control means for livestock breeding, reduces economic losses, and promotes the healthy development of the industry. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Detection results of the toxicity of matrine to BHK21 cells.

[0020] Figure 2 : Western-blot detection of the expression level of FMDV-3D protein after BHK21 cells were infected with FMDV mixed with different concentrations of matrine for 24 h.

[0021] Figure 3 Figure 6: Western-blot analysis of FMDV-3D protein expression level after BHK21 cells were infected with FMDV persistent strain mixed with different concentrations of matrine for 24h.

[0022] Figure 4 Figure 7: Western-blot analysis of FMDV-3D protein expression level after BHK21 cells were infected with FMDV mixed with matrine for 24h.

[0023] Figure 5 Figure 8: Western-blot analysis of FMDV-3D protein expression level after BHK21 cells were infected with FMDV, and then added with matrine for 24h.

[0024] Figure 6 Figure 9: Analysis of potential host gene targets of matrine against FMDV infection. DETAILED DESCRIPTION

[0025] The present application is further described in the following Examples. It should be understood that these Examples are included merely to illustrate aspects of the present application and should not be considered in any way limiting to the scope of the application.

[0026] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by a person of ordinary skill in the art.

[0027] Experimental materials used in the following: BHK21 cells were obtained from China Center for Type Culture Collection (CCTCC), O type foot-and-mouth disease virus (FMDV) was Akesu / 58 / 2002, and FMDV persistent strain was FMDV-Op. CCK-8 was purchased from EnoGene company, and matrine was purchased from Selleck company.

[0028] Example 1: Cytotoxicity experiment of matrine Matrine powder was dissolved with DMSO and then was aliquoted. BHK21 cells were seeded in 96-well plates at a density of 2×104 cells / well, and then were treated with different concentrations of matrine for 24h. Cell viability was detected by CCK-8 assay. 4BHK21 cells were plated in 96-well plates at a concentration of 1 cell / well, incubated at 37°C in CO2for 24 h, and then grown to 70-80% confluence. The culture medium was discarded, the cells were washed twice with PBS, and the PBS was discarded. Different concentrations of matrine (0, 59.31, 79.8, 105.4, 140.6, 187.5, 250, and 500 ng / mL) were added to 2% FBS MEM medium, and the cells were incubated for 24 h. The concentration of 0 was replaced with DMSO. Blank control groups without DMSO and drug were also designed. After 24 h of incubation, 10 μL / well of CCK-8 solution was added, and the cells were incubated in the dark for 3 h. The OD value was then determined by an enzyme marker at A450, and the cell viability was calculated. Each group was repeated 8 times, and the average value and standard deviation were calculated. The results are shown in the graph of Figure 1 .

[0029] Cell activity calculation formula:

[0030] As shown in the graph of Figure 1 , the cell activity of the cells treated with different concentrations of matrine was not significantly different from that of the untreated cells, which proved that matrine in the concentration range did not have significant cytotoxicity.

[0031] Example 2: Inhibition of foot-and-mouth disease virus by different concentrations of matrine BHK21 cells with a confluence of 90% were plated in 12-well plates, incubated at 37°C in CO2for 24 h, and then grown to 80% confluence. The culture medium was discarded, the cells were washed three times with serum-free MEM, and the MEM was discarded. Different dilution ratios of matrine were mixed with 10 2.25 TCID50 / mL titer of FMDV to obtain different concentrations of matrine virus mixtures (0, 250, 625, and 1250 ng / mL). The concentration of 0 was replaced with an equal volume of DMSO as a negative control. 500 μL of each concentration of matrine virus mixture was used to infect BHK21 cells, which were incubated at 37°C for 1 h. The virus supernatant was then discarded, the cells were washed three times with serum-free MEM, and the washing solution was discarded. 2% FBS-containing MEM was then added, and the corresponding concentration of matrine was added to maintain the culture. The cells were incubated at 37°C in CO2for 24 h until CPE appeared. The culture supernatant was discarded, and cell lysis solution and SDS were added for protein sampling. The expression level of FMDV-3D protein was detected by Western blot, and the housekeeping gene GAPDH was used as an internal control.

[0032] The results are shown in the graph of Figure 2, matrine significantly inhibited the expression of FMDV-3D protein, and with the increase of matrine concentration, the inhibition effect was more obvious, showing dose-dependent. The above experimental results showed that matrine could significantly inhibit the expression of FMDV replication related gene 3D protein, and had strong antiviral effect on FMDV.

[0033] Example 3 Inhibition effect of different concentrations of matrine on foot-and-mouth disease persistent virus strain FMDV persistent virus strain (FMDV-OP) was the virus liquid collected from the previous FMDV persistent cell line, which was stored after plaque purification and titer determination, and the titer was 10 5.25 TCID50 / mL.

[0034] BHK21 with a confluence of 90% was plated in a 12-well plate and incubated at 37°C CO2 for 24h. When the cells grew to 80% confluence, the culture medium was discarded, and the serum-free MEM was washed three times, and the MEM culture medium was discarded. Different dilution ratios of matrine were mixed with 10 2.25 TCID50 / mL dilution of FMDV persistent virus strain (FMDV-OP) to obtain different concentrations of matrine virus mixture (0, 250, 625, 1250 ng / mL), and the concentration of 0 group was added with equal volume of DMSO as negative control. 500 μL of the above matrine virus mixture with different concentrations was used to infect BHK21, and adsorbed at 37°C for 1h. Then the virus supernatant was discarded, and the serum-free MEM was washed three times, and the washing liquid was discarded. Then 2% FBS-containing MEM was added, and the corresponding concentration of matrine was added for culture; and then incubated at 37°C CO2 for 24h until the cells appeared obvious CPE, and the culture supernatant was discarded, and the cell lysis solution and SDS were added for protein sampling. Western blot was used to detect the expression level of FMDV-3D protein, and the housekeeping gene GAPDH was used as an internal control.

[0035] The results are as follows Figure 3 , matrine significantly inhibited the expression of FMDV-OP 3D protein, and with the increase of matrine concentration, the inhibition effect was more obvious, showing dose-dependent. The above experimental results showed that matrine could limit the inhibition of FMDV persistent virus strain 3D protein expression, and had strong antiviral effect on FMDV persistent virus strain.

[0036] Example 4 Evaluation of the direct killing effect of matrine on foot-and-mouth disease virus BHK21 with a confluence of 90% was plated in a 12-well plate and incubated at 37°C CO2 for 24h. When the cells grew to 70-80% confluence. A certain concentration of matrine was mixed with 10 2.25FMDV mixed at TCID50 / mL dilution was incubated at 4°C for 1 h to allow the drug to mix well with the virus. DMSO and FMDV were incubated as a negative control group. The BHK21 cells at 70-80% confluence were discarded, washed with serum-free MEM three times, and then the drug-virus mixture was added. The cells were adsorbed at 37°C for 1 h, washed with serum-free MEM three times, and then the washing solution was discarded. The cells were cultured in MEM containing 2% FBS until the CPE was observed at 37°C in CO2for 24 h. The culture supernatant was discarded, and the cell lysate and SDS were added for protein sampling. At the same time, the matrine-treated virus infection process was used as a positive control group, and the DMSO group was used as a negative control. The FMDV-3D protein expression level was detected by Western blot, and the housekeeping gene GAPDH was used as an internal control.

[0037] The results are shown in Table 1. Figure 4 The matrine-treated FMDV infection cycle significantly inhibited the expression of FMDV 3D protein, but the matrine directly incubated with FMDV did not significantly affect the expression of FMDV 3D protein, indicating that the matrine inhibited the FMDV infection, and was not due to the direct killing of the virus or the inhibition of the virus adsorption to the host cells.

[0038] Example 5: Evaluation of the antiviral effect of matrine on foot-and-mouth disease virus infection BHK21 cells at 90% confluence were plated in a 12-well plate and cultured at 37°C in CO2for 24 h. The cells were grown to 70-80% confluence, the cell culture medium was discarded, and the cells were washed with serum-free MEM three times. 500 μL of 10 2.25 FMDV at a TCID50 / mL titer was added to each cell well, and the cells were incubated at 37°C for 1 h. The virus infection solution was discarded, and the cells were washed with serum-free MEM three times. Then, the cells were cultured in MEM containing a certain concentration of matrine and 2% FBS at 37°C in CO2. The DMSO mixed with 2% FBS was used as a negative control group. At the same time, the matrine-treated virus infection process was used as a positive control group, and the DMSO group was used as a negative control. The cells were cultured for 24 h until the CPE was observed, the culture supernatant was discarded, and the cell lysate and SDS were added for protein sampling. The FMDV-3D protein expression level was detected by Western blot, and the housekeeping gene GAPDH was used as an internal control.

[0039] The results are shown in Table 1. Figure 5, Matrine treatment FMDV infection cycle significantly inhibited FMDV 3D protein expression, at the same time, after the cell infected FMDV, given Matrine treatment can significantly inhibit FMDV 3D protein expression. It is illustrated that Matrine mainly blocks the expression and replication of FMDV to resist virus; timely giving Matrine treatment after FMDV infection can significantly inhibit virus proliferation.

[0040] Example 6 Matrine potential target against foot and mouth disease virus infection Login Pubchem database search "Matrine" to obtain Matrine chemical formula and save in SDF format to PharmMapper, by selecting Targets target number 300, click to get the top 300 targets, and convert the protein name to gene name by UniProt.

[0041] In CTD (http: / / ctd.mdibl.org) input "foot and mouth disease" to query FMDV related target genes, and intersect with the above Matrine potential target, and use the Venn diagram to show, get Matrine potential related host key target genes against FMDV. Results are as follows Figure 6 , Mitogen-activated protein kinase 8 (Mapk8), Lysozyme (Lyz), Deoxycytidine kinase (Dck), Heme oxygenase 1 (Hmox1), Retinoic acid receptor beta (Rarb) are potential target genes.

[0042] The above examples are only illustrative of the present application and do not constitute a limitation on the scope of protection of the present application. It should be noted that after reading the above content of the present application, those skilled in the art can supplement and modify the present application, and these equivalent forms of modification also belong to the scope defined in the present application patent.

Claims

1. Use of matrine in the preparation of a medicine for resisting foot-and-mouth disease virus infection.

2. Use of matrine in the preparation of a medicine for resisting foot-and-mouth disease virus persistent infection.

3. Use of matrine in the preparation of a product for preventing foot-and-mouth disease.

4. Use according to claim 3, characterized in that: The product is a medicine or a feed additive.

5. Use of matrine in the preparation of a medicine for preventing or treating foot-and-mouth disease or foot-and-mouth disease virus persistent infection.

6. Use according to any one of claims 1 to 5, characterized in that, Matrine resists virus by blocking expression and replication of the virus.

7. A medicament or pharmaceutical composition for preventing / treating foot-and-mouth disease or for resisting foot-and-mouth virus infection or persistent infection, characterized by, The medicine or pharmaceutical composition contains matrine as an effective component.

8. The medicament or pharmaceutical composition according to claim 7, characterized by, It also includes a pharmaceutically acceptable carrier.

9. The medicament or pharmaceutical composition according to claim 7, characterized by, The dosage form of the medicine or pharmaceutical composition includes injection preparation, oral preparation, such as capsules, tablets.

10. The use according to any one of claims 1 to 5 or the medicament according to any one of claims 7 to 9, characterized in that, The foot-and-mouth disease virus includes O-type foot-and-mouth disease virus, O-type foot-and-mouth disease virus persistent infection strain, and O-type foot-and-mouth disease virus variant strain.

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