Application of morusin M in preparation of anti-enterovirus drugs
By using the method of combining Sancin M and/or its salt with a vector, drugs with anti-enterviral activity were prepared, especially with a significant inhibitory effect on enterovirus types 71 and CA16, which solved the problem of the lack of effective anti-enterviral drugs in the prior art, and achieved effective prevention and treatment of these viruses.
Patent Information
- Application Number
- CN202510328166.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-19
- Publication Date
- 2025-05-09
AI Technical Summary
No effective anti-enterviral drugs have been found in the prior art, especially for enterovirus type 71 and enterovirus type CA16.
Anti-enterviral drugs are prepared by adding a pharmaceutically acceptable carrier, especially at the adsorption stage of the enterovirus to inhibit the adsorption of the virus to cells by the virus.
Sancin M significantly inhibits the viral titers of enterovirus type 71 and enterovirus type CA16, has excellent antiviral activity, is low toxic to cells, and has good biosafety.
Smart Images
Figure CN119950478A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to application of sansinol M in the preparation of anti-enteroviral drugs. Background Art
[0002] Enterovirus is a type of virus with small particles, icosahedral, 24~30nm in diameter, no lipids, single-stranded RNA in the core, resistance to ether and other lipid solvents, acid resistance, and resistance to various antibiotics, antiviral drugs, and detergents. Most enteroviruses produce cytopathic effects in cell culture. Enterovirus belongs to the Picornaviridae family and is a naked virus. Different enteroviruses can cause the same symptoms, and the same virus can cause different clinical manifestations. Enteroviruses are often latent infections, which can cause symptoms such as mild upper respiratory tract infection, abdominal discomfort, and diarrhea. Occasionally, they invade the central nervous system and cause flaccid paralysis.
[0003] Enterovirus 71 is the main pathogen causing hand, foot and mouth disease in infants and young children in the Asia-Pacific region. Although EV71 vaccines are available, they have no cross-protection against other enteroviruses. Hand, foot and mouth disease is still a very serious public health problem, but there are no effective preventive measures or treatments so far, so the search for antiviral drugs is urgent. Summary of the invention
[0004] In view of the technical problems existing in the background technology, the present invention aims to explore drugs with broad-spectrum resistance to enteroviruses and provide new means for the prevention and treatment of enteroviruses.
[0005] In a first aspect, the present invention provides the use of sansinsu M and / or its pharmaceutically acceptable salt in the preparation of anti-enteroviral drugs.
[0006] Moracin M is a phenolic component extracted from mulberry bark. Its structural formula is shown below:
[0007] Moracin M is a phenolic component in the bark of Morus alba, a plant of the Moraceae family. It can be extracted from the bark of Morus alba or synthesized artificially. It is reported that Moracin M has anti-inflammatory activity, and as a phosphodiesterase-4 (PDE4) inhibitor, it can be used to treat pneumonia caused by the new coronavirus. However, there are currently no reports that Moracin M has anti-enteroviral efficacy. When the inventors were exploring anti-enteroviral drugs from existing small molecule compounds, they first discovered that Moracin M has anti-enteroviral activity, especially against enterovirus 71 and enterovirus CA16.
[0008] The present invention further explores the mechanism of action of Moracin M on enterovirus, and the experimental results show that Moracin M acts on the adsorption stage of enterovirus, so it is more conducive to the preparation of anti-enterovirus drugs.
[0009] In the above scheme, after adding mulberry extract M and / or its pharmaceutically acceptable salt to a pharmaceutically acceptable carrier, it can be prepared into any dosage form of tablets, sprays, granules, capsules, oral liquids, and injections.
[0010] In a second aspect, the present invention provides a use of mulberry extract M and / or a pharmaceutically acceptable salt thereof as a product for inhibiting virus adsorption to cells. In one embodiment of the present invention, mulberry extract M can inhibit the adsorption of enterovirus 71 to cells.
[0011] In a third aspect, the present invention provides an anti-enteroviral drug comprising sangsin M and / or a pharmaceutically acceptable salt thereof.
[0012] Preferably, in the above-mentioned medicine, the enterovirus is enterovirus type 71 and / or enterovirus type CA16.
[0013] Preferably, the above-mentioned medicine also contains a pharmaceutically acceptable carrier.
[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention proposes for the first time that sansinol M has enteroviral activity and explains its mechanism of action, providing a new approach for the prevention and treatment of enterovirus infection and a theoretical basis for the development of anti-enteroviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.
[0016] Figure 1 This is a diagram showing the cytotoxicity test results of Sansinsu M in an embodiment of the present invention; Figure 2 This is a fluorescence microscopy observation result of RD cells after being treated with EV71 and different concentrations of Sancin M in the embodiment of the present invention; Figure 3 This is a flow cytometric analysis result of RD cells treated with EV71 and different concentrations of Sancin M in the embodiment of the present invention; Figure 4 The expression of viral RNA after treatment with different concentrations of Sangxinsu M in the embodiment of the present invention is changed; Figure 5 The TCID50 changes of the virus after being treated with different concentrations of Sangsinsu M in the examples of the present invention; Figure 6 This is a graph showing the effect of sansinol M on the adsorption, entry and replication of enterovirus 71 in an embodiment of the present invention; Figure 7 This is a comparison chart of viral RNA expression under different treatment conditions in the embodiments of the present invention; Figure 8 This is a graph showing the effect of mulberry extract M on the replication of enterovirus CA16 in an example of the present invention. DETAILED DESCRIPTION
[0017] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.
[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" and any variations thereof used herein are intended to cover non-exclusive inclusions; the term "and / or" used herein is merely a description of the association relationship of associated objects, indicating that three relationships may exist, for example, A and / or B can represent: the existence of A alone, the existence of A and B at the same time, and the existence of B alone.
[0019] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.
[0020] Example 1 This example demonstrates the positive effects of Sansin M on enterovirus 71, including the following experiments and results: (1) Cytotoxicity test of sansin M.
[0021] RD cells were plated in 96-well plates and cultured in a 37°C, 5% CO2 cell culture incubator for about 12 hours. The cell culture medium was discarded and cell maintenance medium containing different concentrations of Sancin M was added to continue culturing. After 36 hours, the cell survival rate was determined by CCK-8 method. Graph Pad software was used to calculate the median cytotoxic concentration (CC 50 ). Cell survival rate = (mean OD of drug group 450 - Average OD of blank group 450 / Average OD of cell control group 450 - Average OD of blank group 450 )×100%, where the control group was the single cell group without drug addition, and the blank group was the PBS group.
[0022] Test results such as Figure 1 As shown, the effect of sansin M on CC 50 It is 90.33 μM. It can be seen that Sansinsu M has low toxicity to cells and has good biosafety.
[0023] (2) Trial on the effect of sansin M on enterovirus 71.
[0024] RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, EV71-GFP with MOI=1 was added to infect the RD cells. After 2 hours, the culture medium was discarded, and 80μM Ribavirin or different concentrations of Sancin M (12.5μM, 25μM, 50μM, 100μM, 125μM) were added to treat the RD cells. After 24 hours, the expression of viral green light was observed by fluorescence microscopy and detected by flow cytometry. The results were as follows: Figure 2 and 3 At the same time, RT-qPCR was used to detect the expression of viral RNA and the TCID50 of the virus. The results are shown in Figure 4 and Figure 5 shown.
[0025] from Figure 3 and 4 It can be seen that as the concentration of mulberry extract M increases, the detected green fluorescence signal becomes weaker, indicating that mulberry extract M can significantly inhibit the viral titer of enterovirus type 71 in a dose-dependent manner. Figure 4 and Figure 5 The results showed that the viral RNA and titer decreased with the increase of drug concentration.
[0026] Example 2 This case tested the effect of sansin M on the adsorption, entry and replication of enterovirus 71 to explore its antiviral mechanism, including the following experiments: (1) Adsorption stage experiment.
[0027] RD cells were plated in 12-well plates at a seeding density of 2 × 10 5 Cells / well, cultured overnight. Virus group: EV71-GFP virus (MOI=1) was co-incubated with cells at 4°C for 1 hour, the supernatant was discarded, the cells were washed once with 1×PBS, and the culture medium was added to continue culturing in the incubator. Drug group: 50μM Sancin M and EV71-GFP virus (MOI=1) were added to the cells together, incubated at 4°C for 1 hour, the supernatant was discarded, the cells were washed once with 1×PBS, and the culture medium was added to continue culturing in the incubator. Cells in each group were cultured for 12 hours, and the proportion of GFP-positive cells was detected by flow cytometry or cell protein samples were collected for Western blot detection.
[0028] (2) Entering the stage experiment.
[0029] Vero cells were plated in 12-well plates and the inoculation number was 2×10 5 Cells / well, continue to culture overnight. Virus group: Infect cells with EV71-GFP virus (MOI=1), adsorb at 4℃ for 1h, discard supernatant, wash once with 1×PBS; add culture medium, incubate at 37℃ for 1h, discard supernatant, wash once with 1×PBS, and continue to culture with culture medium. Drug group: Infect cells with EV71-GFP virus (MOI=1), adsorb at 4℃ for 1h, discard supernatant, wash once with 1×PBS; add culture medium containing 50μM sangsin M, incubate at 37℃ for 1h, discard supernatant, wash once with 1×PBS, and continue to culture with culture medium. After 12h, the proportion of GFP-positive cells was detected by flow cytometry or cell protein samples were collected for Western blot detection.
[0030] (3) Replication phase experiment.
[0031] Vero cells were plated in 12-well plates at a seeding density of 2 × 10 5 Cells / well, continue to culture overnight. Virus group: Infect cells with EV71-GFP virus (MOI=1), incubate at 37℃ for 2h, discard supernatant, wash once with 1×PBS; add culture medium and continue to culture. Drug group: Incubate EV71-GFP virus (MOI=1) with cells at 37℃ for 1h, discard supernatant, wash once with 1×PBS; add culture medium containing 50μM sansin M and continue to culture. After 12h, detect the proportion of GFP-positive cells by flow cytometry or collect cell protein samples for Western blot detection.
[0032] Test results such as Figure 6 As shown, sansin M can inhibit the adsorption stage of enterovirus 71 virus.
[0033] Example 3 In order to further determine whether Sancin M acts on target cells or viruses, the following experiments were performed in this case: After incubating the cells with Sancin M and RD at 4°C for 1 hour, Sancin M was removed and EV71-GFP with MOI=1 was added. The cells were cultured at 37°C for 12 hours and then collected. The same volume of DMSO was used as a control. The expression level of viral nucleic acid RNA was detected by fluorescence quantitative PCR. The test results are shown in Figure 7 As shown in A. After incubating Sancin M and EV71-GFP with MOI=1 at 4℃ for 4 hours, the mixture of Sancin M and virus was diluted 100 times and added to RD cells containing culture medium. After culturing for 12 hours, the cells were collected and the same volume of DMSO was used as a control. The proportion of GFP-positive cells was detected by flow cytometry. The test results are shown in Figure 7 As shown in B.
[0034] The above results show that Sangxinsu M has obvious antiviral effect.
[0035] Example 4 This example demonstrates the positive effects of Sansin M on enterovirus CA16, including the following experiments and results: Vero cells were seeded in 12-well plates. The next day, when the cells grew to 90%, CA16-WT with an MOI of 1 was added to infect the Vero cells. After 2 hours, the culture medium was discarded and different concentrations of Sancin M (12.5 μM, 25 μM, and 50 μM) were added to treat the Vero cells. After 24 hours, RT-qPCR was used to detect the expression of viral RNA. The results are as follows: Figure 8 As shown in A.
[0036] Vero cells were seeded in 12-well plates. The next day, when the cells grew to 90%, CA16-WT with MOI=1 was added to infect the Vero cells. After 2 hours, the culture medium was discarded, and 80μM Ribavirin or different concentrations of Sancin M (12.5μM, 25μM, 50μM, 100μM) were added to treat the Vero cells. After 24 hours, the expression of viral proteins was detected by Western blot. The results are shown in Figure 8 As shown in B.
[0037] From the above results, it can be seen that Sancin M has anti-enterovirus CA16 activity.
[0038] In summary, Sansinsu M has significant anti-enteroviral activity and good biosafety, and is expected to be developed into an anti-enteroviral drug, which has positive significance for the prevention and treatment of diseases caused by enterovirus.
[0039] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.
Claims
1. Use of sangxinsu M and / or its pharmaceutically acceptable salt in the preparation of anti-enteroviral drugs.
2. The use according to claim 1, characterized in that: The enterovirus includes enterovirus 71 and enterovirus CA16.
3. The use according to claim 1, characterized in that: The sansinsu M and / or its pharmaceutically acceptable salt is added to a pharmaceutically acceptable carrier to prepare any dosage form selected from tablets, sprays, granules, capsules, oral liquids, and injections.
4. Use of Sancinsu M and / or its pharmaceutically acceptable salt as a product for inhibiting the adsorption of enterovirus to cells.
5. The use according to claim 4, characterized in that: The enterovirus includes enterovirus 71 and enterovirus CA16.
6. An anti-enteroviral drug, characterized in that: The medicine comprises sangxinsu M and / or a pharmaceutically acceptable salt thereof.
7. The drug according to claim 6, characterized in that The enterovirus includes enterovirus 71 and enterovirus CA16.
8. The drug according to claim 6, characterized in that The medicament also includes other anti-enteroviral active substances.
9. The drug according to claim 6, characterized in that The medicine contains a pharmaceutically acceptable carrier.
10. The drug according to claim 6, characterized in that The dosage form of the drug is any one of tablets, sprays, granules, capsules, oral liquids or injections.