Use of magnoline in preparation of antiviral drug

Abstract

The present invention provides use of magnoline in the preparation of an antiviral drug, which belongs to the technical field of antiviral drugs. The present invention demonstrates at the cellular level that magnoline exhibits an inhibitory effect on various common infectious disease viruses in domestic pigs, especially in vitro exhibits a significant inhibitory effect on the proliferation of African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV). Further disclosed in the present invention are an antiviral composition consisting of the active ingredient magnoline or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient, and use of the composition in the preparation of a viral infection inhibitor and / or in the preparation of a pharmaceutical formulation for preventing and / or treating viral infectious diseases, thereby providing a basis for the development of new broad-spectrum antiviral drugs.

Description

Application of magnoline in preparation of antiviral drugs TECHNICAL FIELD

[0001] The present application belongs to the technical field of antiviral drugs, and particularly relates to application of magnoline in preparation of antiviral drugs. BACKGROUND

[0002] African swine fever (ASF) is an acute and severe infectious disease caused by African swine fever virus (ASFV) infection, with a mortality rate of 100%. ASF was first discovered in Kenya in 1921, and then gradually spread to sub-Saharan Africa, Europe, the Americas, Asia and more than 60 countries and regions, and the epidemic area is increasing, which is the No. 1 threat to the global pig industry. In August 2018, China first appeared ASF epidemic, and then spread rapidly throughout the country, which seriously damaged the economic interests of China's pig industry. At present, there is no vaccine and treatment drug to provide effective prevention and treatment for ASF, and safe and effective drugs can improve the survival rate of infected pigs, reduce economic losses and prevent the spread of the virus.

[0003] Porcine reproductive and respiratory syndrome (PRRS) is one of the most common infectious diseases affecting the pig industry worldwide, caused by porcine reproductive and respiratory syndrome virus (PRRSV). The virus is transmitted by direct or indirect contact, and its characteristics are reproductive failure in sows and respiratory disease in pigs of all ages. PRRSV has characteristics such as antigen variation, macrophage tropism, persistent infection, antibody-dependent enhancement, secondary and mixed infection, and so far there is no effective prevention and treatment method. At present, the protective effect of PRRS vaccine on the disease is limited, therefore, it is of great significance to develop new, safe, efficient and broad-spectrum anti-PRRSV drugs.

[0004] Porcine circovirus (PCV) is a non-enveloped icosahedral virus containing a single-stranded negative sense circular DNA genome, belonging to the Circoviridae family, Circovirus genus, and is one of the smallest animal viruses ever discovered. There are currently four known PCV serotypes: PCV1, PCV2, PCV3 and PCV4. Since its discovery in 1998, PCV2 has been recognized as one of the most important pathogens in pig populations worldwide. PCV2 mainly attacks 5-12 week old weaned piglets and is the main pathogen of postweaning multisystemic wasting syndrome, porcine dermatitis and nephropathy syndrome, proliferative and necrotic pneumonia, reproductive disorders, peripartum myocarditis, congenital tremor in piglets and other diseases, which are collectively referred to as porcine circovirus-associated diseases, which seriously affect pork production.

[0005] Porcine epidemic diarrhea virus (PEDV) is the pathogen of porcine epidemic diarrhea, and PEDV belongs to the Coronaviridae family, Alphacoronavirus genus, and is a single-stranded positive-sense RNA virus with an envelope. PEDV has brought huge economic losses to the global pig industry and has become one of the most threatening viral diseases in pigs. The main means of prevention and control of PEDV is to rely on vaccines to produce mucosal immunity, however, the main victim group of PEDV is the mammalian piglets whose mucosal immune system has not yet developed completely, therefore, the current immune effect is not ideal.

[0006] The above viruses have high mutation frequency, fast pathogenic speed, high mortality, and unsatisfactory vaccine protection, and lack of safe and effective specific drugs in clinical practice, therefore, it is of great significance to develop new, safe, efficient and broad-spectrum antiviral drugs.

[0007] Magnoline, with a molecular formula of C 36 H 40 N2O6, is a double benzyl isoquinoline alkaloid, and Magnoline is derived from the dried flower buds of Magnolia kobus (DC.) Spach of Magnoliaceae Magnolia genus in Japan, and has good biological activity, such as antiarrhythmia, anti-inflammatory, antitumor, etc., but there is no related report on antiviral research at present. SUMMARY

[0008] The present application aims to solve the technical problems in the prior art and provide the application of Magnoline in the preparation of antiviral drugs.

[0009] To achieve the above purpose, the specific technical scheme of the present application is as follows:

[0010] In one aspect of the present application, the use of magnoflorine in the preparation of an antiviral drug is provided.

[0011] The chemical structure of the magnoflorine is shown in Formula I:

[0012] Preferably, the virus includes African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV).

[0013] In another aspect of the present application, an antiviral drug composition is provided, which includes the active ingredient magnoflorine or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and / or excipients.

[0014] Preferably, the active ingredient magnoflorine in the drug composition accounts for 0.1% to 99.9% of the total weight.

[0015] Preferably, the drug composition further contains one or more other antiviral drugs.

[0016] Preferably, the other antiviral drugs are compounds and / or traditional Chinese medicine extracts with antiviral activity, and / or traditional Chinese medicine compositions with antiviral effects.

[0017] In yet another aspect of the present application, the use of the above-mentioned antiviral drug composition in the preparation of a virus infection inhibitor and / or in the preparation of a pharmaceutical preparation for preventing and / or treating viral infectious diseases is provided.

[0018] The administration of the pharmaceutical preparation is oral or injection or external use.

[0019] The pharmaceutical preparation includes, but is not limited to, tablet, capsule, granule, gel, dispersion, oral liquid, injection, spray, drop, ointment, etc. Advantages:

[0020] The present application proves from the cellular level that magnoflorine can inhibit a variety of RNA viruses and DNA viruses, especially African swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 virus, and porcine epidemic diarrhea virus, providing a potential broad-spectrum antiviral drug for the prevention and treatment of a variety of viral infectious diseases. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the cell survival rate of magnoflorine acting on PAMs cells;

[0022] Figure 2 is the CC of magnoflorine on PAMs cells 50 Fitting results;

[0023] Figure 3 is a Western Blot method for determining the expression of African swine fever virus p72 protein in PAMs cells treated with magnoline;

[0024] Figure 4 is a software analysis of the gray value of the Western Blot protein band, the inhibition rate of the corresponding concentration is calculated, and the IC 50 value is fitted; Figure 5 is a Western Blot method for determining the expression of PRRSV virulence protein M protein in Vero cells treated with magnoline, and the ratio of M protein to internal reference protein β-actin is calculated according to the protein gray value;

[0025] Figure 6 is a qPCR method for determining the transcription level of PCV2 Cap gene in PK15 cells treated with magnoline and positive control ribavirin;

[0026] Figure 7 is the cell activity inhibition rate of magnoline on Vero cells. DETAILED DESCRIPTION

[0027] The above will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter to the following examples. Any technology realized based on the content of the present application is within the scope.

[0028] Example 1 In vitro inhibition of African swine fever virus (ASFV) by magnoline

[0029] 1. Determination of the half cytotoxic concentration (CC50) of magnoline on PAMs

[0030] PAMs were recovered and seeded in a 96-well plate at 2×10 4 After 6h of cell adhesion, the cell culture solution was removed and different concentrations of magnoline (1 μM, 3 μM, 10 μM, 30 μM, 60 μM, 100 μM) were added to each well, with three replicate wells for each concentration, and negative and blank controls were set. After 24h of incubation, 10 μL of CCK-8 (cell counting kit-8) was added to each well, and the OD value was measured at 450 nm using a microplate reader after 2h of incubation. The cell survival rate % was calculated according to the formula: cell survival rate % = (A sample - A blank) / (A negative - A blank) × 100%, and the results are shown in Figure 1. The CC50 value of magnoline was fitted, as shown in Figure 2. The CC50 of magnoline on PAMs was 37.08 μM, and magnoline had no obvious toxicity to PAMs cells (cell activity > 90%) when the concentration was less than 10 μM. Therefore, there was no significant difference between the treated cell group and the untreated group when the concentration of magnoline was less than 10 μM.

[0031] 2. Magnoliaine's inhibitory activity against African swine fever virus infection in PAMs cells.

[0032] PAMs cells at 2×10 6 Cells were seeded per well in 6-well plates. After complete cell adhesion, different concentration gradients of magnolamine (0.3 μM, 1 μM, 3 μM, 6 μM) were added to the cells. After 2 h of incubation, 0.1 MOI of virus adsorption solution was added to each well for 1 h. After washing twice with PBS, different concentration gradients of magnolamine (0.3 μM, 1 μM, 3 μM, 6 μM) were added. After culturing for 24 h, cells were harvested, and the expression of p72 protein was determined by immunoblotting. The results are shown in Figure 3. Image J was used to analyze the gray values ​​of the protein bands, obtaining the gray values ​​of p72 protein and internal control bands in each lane. The inhibition rate of magnolamine against ASFV at each concentration was calculated using the formula: 1 - [(gray value of target band in experimental group / gray value of internal control band in experimental group) / (gray value of target band in control group / gray value of internal control band in control group)]. The half-maximal inhibitory concentration (IC50) of magnolamine against ASFV was then fitted based on the inhibition rate. 50 As shown in Figure 4, the IC50 of magnolol is... 50 =1.621 μM. Selectivity index SI = CC 50 / IC 50 The higher the SI value, the larger the therapeutic window and safe concentration of the drug, and the stronger the inhibitory effect. The SI value of magnolol is 22.87.

[0033] Example 2: In vitro inhibitory effect of magnolol on porcine reproductive and respiratory syndrome virus (PRRSV)

[0034] Vero cells (African green monkey kidney cells) were seeded in 6-well plates and cultured for 6 to 8 hours. Vero cells were then infected with 0.1 MOI PRRSV. After 1 hour of adsorption, the washing solution was discarded. Different concentrations of magnolamine (0 μM, 0.3 μM, 1 μM, 3 μM) were added to the cells, with 0 μM serving as a negative control. Cells were collected 36 hours after viral infection, and the expression of viral virulence protein M was detected by immunoblotting (Figure 5). The results showed that magnolamine significantly inhibited the expression of PRRSV virulence protein M in a dose-dependent manner.

[0035] Example 3: In vitro inhibitory effect of magnolol on porcine circovirus type 2 (PCV2)

[0036] PK15 cells (porcine kidney cells) were inoculated in 24-well plates, and when the cells grew to about 80%, the PK15 cells were infected with PCV2 at a MOI of 0.1. After 2 hours of adsorption, the infection solution was removed and the cells were washed, and different concentrations of magnolia base (0 μM, 0.3 μM, 1 μM, 3 μM) and the positive control ribavirin (10 μM) were added to the cells. After 48 hours of viral infection, the PCV2 cap gene copy number was detected by qPCR.

[0037] The PCV2 cap primer sequence is:

[0038] PCV2 cap-F: 5'-TACATTTCCAGCAGTTTG-3';

[0039] PCV2 cap-R: 5'-CTCCCGCCATACCATAA-3';

[0040] The results (Figure 6) show that the Cap gene copy number of each group of magnolia base was significantly reduced in a dose-dependent manner compared with the infection group.

[0041] Example 4 In vitro inhibition of porcine epidemic diarrhea virus (PEDV) by magnolia base

[0042] Vero cells (African green monkey kidney cells) were inoculated in 96-well plates, and when the cells grew to about 80%, the Vero cells were infected with PEDV at a MOI of 0.1. After 2 hours, the infection solution was removed and the cells were washed, and different concentrations of magnolia base (0 μM, 0.3 μM, 1 μM, 3 μM) were added to the cells. After 24 hours of viral infection, the culture medium was removed, CCK-8 was added, and incubation was continued for 2 hours. The OD value was measured at 450 nm by a microplate reader, and the cell activity was calculated. The results (Figure 7) show that magnolia base can significantly inhibit the proliferation of PEDV in a dose-dependent manner.

Claims

1. Use of magnolol in the preparation of an antiviral drug.

2. Use according to claim 1, characterized in that, The chemical structural formula of the magnolia base is shown as formula I:

3. Use according to claim 1, characterized in that, The virus includes African swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine epidemic diarrhea virus.

4. An antiviral drug composition, which is composed of an active ingredient magnolol or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable carriers and / or excipients.

5. The pharmaceutical composition according to claim 4, characterized by The mass percentage of magnolol or its pharmaceutically acceptable salt in the drug composition is 0.1%-99.9%.

6. The pharmaceutical composition according to claim 4, wherein The drug composition further contains one or more other antiviral drugs.

7. The pharmaceutical composition according to claim 6, characterized in that, The other antiviral drugs are compounds and / or traditional Chinese medicine extracts with antiviral activity, and / or traditional Chinese medicine compositions with antiviral effect.

8. Use of an antiviral drug composition according to any one of claims 4-7 in the preparation of a virus infection inhibitor and / or in the preparation of a drug preparation for preventing and / or treating viral infectious diseases.

9. The pharmaceutical composition according to claim 8, characterized by The drug preparation is administered orally or by injection or externally.

10. The pharmaceutical composition according to claim 9, characterized by The drug preparation includes tablets, capsules, granules, gels, dispersions, oral liquids, injections, sprays, drops, and ointments.

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