Application of diphyllin in preparation of medicine for inhibiting poxvirus and preventing or treating poxvirus infection

Abstract

The invention discloses an application of diphyllin in preparation of drugs for inhibiting poxvirus and preventing or treating poxvirus infection, the poxvirus is common orthopoxvirus poxvirus, the poxvirus comprises vaccinia virus (VACV), vaccinia virus (CPXV) and rabbit poxvirus mucinoma virus (MYXV), the content of diphyllin is in a range of 1.25-2.5 [mu] M / mL, and the content of diphyllin is in a range of 1.25-2.5 [mu] M / mL. The compound has a remarkable inhibiting effect on infection of poxvirus family members such as CPXV, is expected to be used as a medicine for preventing and treating infection of CPXV and other poxviruses, and has important significance on prevention and control of diseases related to poxvirus infection.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and biotechnology, and specifically relates to the application of senna leaf extract in the preparation of drugs for inhibiting poxvirus and preventing or treating poxvirus infection. Background Technology

[0002] Monkeypox virus (MPXV) belongs to the family Poxviridae, subfamily Choropovagininae, and genus Orthopoxvirus. Its antigenic characteristics are similar to those of vaccinia virus, vaccinia virus, and smallpox virus, and cross-immunity exists between different viruses. MPXV is a large double-stranded DNA virus that replicates in the cytoplasm of vertebrate or invertebrate cells. Similar to vaccinia virus (CPXV), MPXV can enter host cells through fusion with the host cell membrane or endocytosis, and at least 16 proteins are involved in the entry process. Once inside the host cell, the virus initiates early gene transcription events, and DNA replication and synthesis occur at perinuclear sites called "viral factories."

[0003] For most patients infected with monkeypox, treatment is primarily symptomatic. Although there is no specific treatment for monkeypox, smallpox antiviral drugs such as Brincidofovir, Tecovirimat, and Cidofovir have shown some effectiveness due to their genetic similarity. However, there are still few specific antiviral drugs for monkeypox, and existing drugs lack sufficient validation of their effectiveness in large-scale clinical trials, with long-term use carrying the risk of viral immune escape. Regarding monkeypox vaccines: there are currently no vaccines specifically designed to prevent monkeypox virus infection. Due to cross-immunity, the use of smallpox vaccines (based on vaccinia virus) is recommended in the current monkeypox outbreak, but their supply is limited. Therefore, the importance of developing new therapies targeting host factors or virus-host interactions for treating orthopoxvirus infection cannot be underestimated. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide the application of senna leaf extract in the preparation of drugs for inhibiting poxvirus and preventing or treating poxvirus infection, so as to improve the current singularity and limitation of poxvirus treatment.

[0005] Currently, the prevention and treatment of monkeypox virus mainly relies on vaccination, and there is a lack of effective targeted therapeutic agents in clinical practice. This invention aims to screen effective antiviral drugs by evaluating the efficacy of VATPase inhibitors against CPXV and other viruses, and to determine their potential effects and antiviral mechanisms, in order to find a suitable therapeutic drug for poxvirus infection and provide a theoretical basis and technical direction for preventing poxvirus-related diseases. A VATPase inhibitor compound with preventive and therapeutic effects against poxvirus family members such as CPXV is provided.

[0006] The purpose of this invention is to improve the limited and singular nature of current poxvirus treatments and to provide a VATPase inhibitor that inhibits the replication of multiple poxvirus family members, including vaccinia virus (CPXV), vaccinia virus (VACV), and myxoma virus (MYXV). Such drugs have the potential to prevent and treat poxvirus-related infections and provide a potential drug direction for development to stop the occurrence and spread of poxvirus infections.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a drug for inhibiting poxviruses, which contains senna leaf extract. This drug can be used to inhibit poxvirus proliferation and / or poxvirus toxicity.

[0009] The present invention also provides a medicament for the prevention or treatment of poxvirus infection, the medicament containing senna leaf extract.

[0010] In the experimental study of this invention, the drug was in the form of a drug dissolved in DMSO, and then diluted with culture medium to different concentrations. That is, the drug of this invention can be in liquid form.

[0011] The poxviruses described in this invention are common poxviruses belonging to the genus Orthopoxvirus, including vaccinia virus (VACV), cowpox virus (CPXV), and myxoma virus (MYXV) belonging to the genus Leptopoxvirus.

[0012] Preferably, the content of kaempferol in the drug is 1.25–2.5 μM / mL. More preferably, in the application of medicaments for the prevention of poxvirus infection, the effective concentration of kaempferol is 1.25 μM / mL; and in the application of medicaments for the treatment of poxvirus infection, the effective concentration of kaempferol is 2.5 μM / mL.

[0013] Preferably, in the application of purpuric acid leaf extract for the prevention or treatment of CPXV infection, the effective concentration for cell treatment is 1.25–2.5 μM / mL, more preferably 1.25 or 2.5 μM / mL, wherein the cells are CPXV-infected host cells, preferably Vero cells. The preferred effect is inhibitory activity against poxvirus in vitro. Purpuric acid leaf extract can significantly inhibit the in vitro proliferation of vaccinia virus (CPXV) in the concentration range of 1.25–2.5 μM.

[0014] Preferably, in the application of kaempferol for the prevention or treatment of VCV infection, the effective concentration for cell treatment is 1.25–2.5 μM / mL, more preferably 1.25 or 2.5 μM / mL, wherein the cells are VCV-infected host cells, preferably BSC-1. Kaempferol can significantly inhibit the in vitro proliferation of VCV in the concentration range of 1.25–2.5 μM.

[0015] Preferably, the method of administering the drug is as follows: after viral infection, the drug is administered; including the following steps:

[0016] Methods: After 2 hours of viral infection (viral adsorption process), the liquid was discarded, the drug was added, and after 48 hours of treatment, the virus was fixed with 4% PFA, stained with crystal violet, and the number of viral pores was counted. The results were compared with the group infected with the virus but without the drug to determine the drug concentration range for inhibiting CPXV and VAV replication.

[0017] This invention also provides the use of kaempferol in the preparation of drugs for the prevention or treatment of poxvirus infection or for the preparation of drugs that inhibit poxvirus. The anti-poxvirus effects of kaempferol include inhibiting poxvirus proliferation and / or poxvirus toxicity.

[0018] The poxviruses mentioned in the application of the senna leaf extract of the present invention include vaccinia virus (VACV), vaccinia virus (CPXV) and myxoma virus (MYXV) of the rabbitpoxvirus genus, but are not limited to the above-mentioned viruses. In fact, the senna leaf extract is applicable to the prevention and treatment of various poxvirus infections and is applicable to the inhibition of the proliferation and / or toxicity of various poxviruses.

[0019] In a preferred embodiment of the present invention, the effects and potential uses of a VTPase compound called kaempferol, which has an inhibitory effect on poxviruses such as CPXV, are provided. The specific technical solution adopted is as follows:

[0020] The use of kaempferol, a VATPase compound that inhibits poxviruses such as CPXV, was investigated. Antiviral experiments were conducted using kaempferol, and by evaluating the anti-CPXV effect of kaempferol, a VATPase inhibitor, effective antiviral drugs were screened to clarify its potential uses.

[0021] The screening method for the senna leaf extract VATPase drug is based on antiviral experiments.

[0022] (1) Pre-treatment with viral infection: First, add lycopene to Vero cells and incubate for 1 hour, then inoculate with poxvirus (e.g., CPXV) for 1 hour, discard the liquid, add maintenance solution, collect samples after 48 hours to measure the viral inhibition effect, and evaluate the antiviral effect of the prevention mode.

[0023] (2) Simultaneous drug administration to infected virus: Poxvirus (e.g., CPXV) was added to Vero cells and then adsorbed at 4 degrees for 1 hour. The liquid was discarded and drug maintenance solution was added. After 48 hours, the sample was collected to determine the virus inhibition effect.

[0024] (3) Method of administering medication after viral infection: Vero cells are infected with poxvirus (e.g., CPXV) for 1 hour, the liquid is discarded, and lysimachia christinae is added. After 48 hours of treatment, samples are collected to measure the viral inhibition effect and evaluate the antiviral effect of the treatment modality.

[0025] Further, this experiment employed the previously described protocol of administering the drug after viral infection, and measured the RNA levels of CPXV infection (CPXV001, CPXV003, CPXV004, CPXV005, CPXV007, CPXV009, CPXV010, and CPXV012) at 2h, 4h, and 12h after drug administration. This further evaluated the inhibitory effect on the virus. The expression of all the aforementioned genes is related to viral adsorption and invasion; therefore, it was confirmed that senna leaf extract prevents CPXV from entering cells.

[0026] In another preferred embodiment of the present invention, the inhibitory effect of senna leaf extract on VACV was experimentally verified, demonstrating the antiviral effect of senna leaf extract on VACV under pre-administration, simultaneous administration, and post-infection administration modes. This indicates that senna leaf extract has a broad inhibitory effect on various poxviruses.

[0027] Furthermore, this experiment adopted the previously described protocol of administering medication after viral infection, and further evaluated the inhibitory effect on the virus by measuring the levels of viral proteins D8L, A27L, L1R, E3L, and WR117 expressed by VACV virus at 4h, 8h, 12h, and 24h after medication in both untreated and treated samples. This further clarified that senna leaf extract prevents the replication and assembly of viral proteins by VACV-associated poxvirus.

[0028] In another preferred embodiment of the present invention, the inhibitory effect of senna leaf extract on MYXV was experimentally verified, demonstrating the antiviral effect of senna leaf extract on MYXV under post-infection dosing mode. This indicates that senna leaf extract has a broad inhibitory effect on various poxviruses.

[0029] The above experiments have demonstrated that senna leaf extract can inhibit poxvirus replication and proliferation in vitro. Further, this experiment involved in vivo animal experiments on C57 mice. The procedure was as follows: Age-matched male C57 mice were randomly divided into two groups of eight mice each. The control group received an intraperitoneal injection of 6*10g of CPXV virus. 6 / ani; Treatment group: Intraperitoneal injection of CPXV virus 6*10 6Each mouse was given 5 mg / kg of senna leaf extract. After 3.5 days of normal feeding, the mice were euthanized, and the liver, fat, and spleen were collected to determine the viral titer in different organs. This was to assess the antiviral effect in vivo.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: through the evaluation of the anti-CPXV effect of senna leaf extract, it was discovered for the first time that senna leaf extract has a significant inhibitory effect on CPXV and other poxvirus family members. It is expected to be used as a drug for the prevention and treatment of CPXV and other poxvirus infections, which is of great significance for the prevention and control of poxvirus infection-related diseases. Attached Figure Description

[0031] Figure 1 The results of the toxicity assay of different concentrations of senna leaf extract on Vero cells in Example 1 of this invention are shown.

[0032] Figure 2 The results of the effect of different concentrations of senna leaf extract on Vero infection with CPXV in Example 2 of the present invention.

[0033] Figure 3 The results of the effects of kaempferol on Vero infection with CPXV at different time points in Example 3 of the present invention are shown.

[0034] Figure 4 This is the result of the effect of different treatment times after treatment with senna leaf extract on the expression level of CPXV RNA in Example 4 of the present invention.

[0035] Figure 5 This is the result of the effect of different treatment times on the expression level of VCV protein after VCV virus infection and treatment with senna leaf extract in Example 5 of the present invention.

[0036] Figure 6 This describes the antiviral effect of lotus leaf extract in vivo in Example 6 of the present invention.

[0037] Figure 7 This is a diagram illustrating the effect of senna leaf extract on inhibiting the proliferation of myxoma virus in host cells under the post-infection drug administration mode in Example 7 of the present invention. Detailed Implementation

[0038] The present invention will be described more clearly and completely through the following embodiments, but the described examples are only a part of the embodiments of the present invention, and not all of them. The embodiments are provided to help understand the present invention and should not be construed as limiting the scope of protection of the present invention.

[0039] This invention aims to screen effective antiviral drugs by evaluating the anti-poxvirus effects of VATPase inhibitors and verifying the effectiveness of several similar drugs in inhibiting poxviruses through in vitro and in vivo experiments. More importantly, it explores a new strategy for combating poxviruses, hoping to provide assistance for the active treatment and response to monkeypox outbreaks and offer important technical means for targeted prevention and control in clinical practice.

[0040] Arylnaphthalene lignan isolated from *Acanthopanax* has been proven to be an effective HIV-1 inhibitor, inhibiting lysosomal acidification in human osteoclasts and exhibiting anticancer and anti-inflammatory activities. Poxviruses pose a potential threat to public health, necessitating the discovery of effective antiviral drugs. Therefore, screening for inhibitory monomers of arylnaphthalene and exploring their target mechanisms will be an important direction for future anti-poxvirus drug development. This invention, through screening and antiviral experiments of arylnaphthalene, for the first time discovered that arylnaphthalene can significantly inhibit the in vitro proliferation of vaccinia virus (CPXV), a representative member of the orthopoxvirus genus, at a concentration range of 1.25–2.5 μM / mL. Furthermore, arylnaphthalene was also found to significantly inhibit the in vitro proliferation of various poxviruses such as VAVV and MYXV, showing promise as a preventative and therapeutic drug against poxviruses and providing potential research directions for subsequent poxvirus-related drug development. In summary, the above results evaluated the inhibitory activity of senna leaf extract against CPXV, indicating that senna leaf extract has certain potential value in the prevention and control of CPXV and other poxvirus infections.

[0041] The sources of materials used in the embodiments of this invention are:

[0042] Viruses and cells: CPXV-GFP (Brighton Red strain) (NCBI: 10243) strain, which is a publicly disclosed strain in the prior art and can be accessed by the public through literature resource sharing; VCV-GFP (Western Reserve strain) (NCBI: 10254) and MYXV-GFP (Lausanne strain) (NCBI: 31530) strains, which are publicly disclosed strains in the prior art and can be accessed by the public through literature resource sharing;

[0043] CPXV (Brighton Red strain) replicates in CV-1 cells. A CPXV derivative (vCpx-gfp) is produced by inserting an enhanced green fluorescent protein (EGFP) cassette between the open reading frames CPXV-107 and CPXV-108 of the CPXV genome. Following the manufacturer's instructions, the transfer plasmid was transfected into CPXV-infected CV-1 cells using Lipofectamine 2000 transfection reagent (Invitrogen). The resulting EGFP-labeled CPXV virus (CPXV-gfp) exhibits fluorescent green color development. In the VACV-GFP (Western Reserve strain) (NCBI: 10254) viral genome, the EGFP cassette is driven by the previously described synthetic VACV early / late promoter (vv sE / L). The MYXV-GFP (Lausanne strain) (NCBI: 31530) strain was also EGFP-labeled using the same method to obtain green fluorescent virions for subsequent experimental observation. Vero cells were purchased from ATCC (NA:Cat#CRL-1586); BSC-1 cells were purchased from ATCC (Cat#CCL-26).

[0044] Reagents and antibodies: The CCK-8 assay kit (Cell Counting Kit-8) was purchased from Beyotime Biotechnology Co., Ltd.; antibodies VACWR117 Antibody, L1R Antibody, A27L Antibody, E3L Antibody, and D8LA Antibody were prepared in this experiment by Huamei Biotechnology Co., Ltd.; HRP-labeled goat anti-mouse IgG was purchased from Cell Siganaling; fetal bovine serum was purchased from Gibco; and the BCA protein quantification kit was purchased from Beyotime Biotechnology Co., Ltd. All other reagents not mentioned in this invention are commercially available products.

[0045] Example 1

[0046] Evaluation of the cytotoxicity of senna leaf extract on Vero cells

[0047] Different concentrations of senna extract were prepared by dissolving it in dimethyl sulfoxide (DMSO). First, different concentrations of senna extract were added to approximately a monolayer of Vero cells and incubated for 48 h. Then, CCK-8 solution (10 μL / well) was added, and the cells were incubated at 37°C for 3 h. The absorbance at 450 nm was measured to evaluate the cytotoxic effect of senna extract. The CCK-8 kit used in this experiment judges cell viability based on the absorbance at 450 nm. The results are as follows: Figure 1The results show the toxicity assay of different concentrations of kaempferol on Vero cells. The toxicity of different concentrations of kaempferol on Vero cells was determined using the CCK-8 kit, and the half-maximal toxicity concentration (CC50) was calculated. The CC50 of kaempferol on cells for 48 h was 3.308 μM.

[0048] Example 2

[0049] Evaluation of the inhibitory effect of different concentration gradients of kaempferol on poxvirus

[0050] This experiment further evaluated the effect of different concentrations of kaempferol on Vero infection with CPXV. CPXV-GFP was inoculated into Vero cells at an MOI of 0.05. After 2 h of infection, kaempferol at concentrations of 1.25 μM / mL and 2.5 μM / mL was added for treatment. Samples were collected after 48 h, fixed in 4% PFA for 20 min, and then stained with 0.1% crystal violet. Figure 2 After rinsing with water, the bacterial plaques were counted visually, and the results were plotted using Graphpad Prism (version 8.3, Graphpad Software Diego, CA, USA) software. The effects of different concentrations of kaempferol on bsc-1 infection with VCV were further evaluated using the same method as before.

[0051] Example 3

[0052] Evaluation of the inhibitory effect of medication at different time points on poxvirus (i.e., preventive and therapeutic effects).

[0053] This experiment further evaluated the effect of adding kaempferol at different time points on Vero infection with CPXV. Three methods were used: (1) Pre-infection with the virus: Kaempferol (2.5 μm / ml) was added to Vero cells and incubated for 1 hour, followed by CPXV inoculation for 1 hour. The liquid was discarded, and kaempferol maintenance solution of the same concentration was added. After 48 hours, Vero cells were fixed for 20 minutes, stained with crystal violet, and the number of cell pores was counted visually to evaluate the antiviral effect of the prevention mode. (2) Simultaneous drug administration to the virus: CPXV was added to Vero cells first, followed by adsorption at 4 degrees Celsius for 1 hour. The liquid was discarded, and kaempferol maintenance solution (2.5 μm / ml) was added. After 48 hours, the virus inhibition effect was measured as described above. (3) Post-infection drug administration: Vero cells were infected with CPXV for 1 hour, the liquid was discarded, and senna leaf extract (2.5 μm / ml) was added. After 48 hours of treatment, the viral inhibition effect was measured as described above to evaluate the antiviral effect of the treatment modality. The protocol and results are as follows. Figure 3 .

[0054] Example 4

[0055] To evaluate the effect of different treatment times after CPXV virus infection and senna leaf extract treatment on CPXV RNA expression levels in Vero cells.

[0056] Furthermore, this experiment adopted the previously described protocol of administering medication after viral infection, and further evaluated the inhibitory effect on the virus by measuring the RNA levels of CPXV001, CPXV003, CPXV004, CPXV005, CPXV007, CPXV009, CPXV010, and CPXV012 at 2h, 4h, and 12h after drug administration and without drug administration. The expression of all the aforementioned genes is related to viral adsorption and invasion, thus clarifying that senna leaf extract prevents CPXV from entering cells.

[0057] Meanwhile, this embodiment also evaluated the effect of different treatment times after VCV virus infection and senna leaf extract treatment on CPXV RNA expression levels in Bsc-1 cells, such as... Figure 4 .

[0058] As described above, the RNA levels of E3 (early-stage expression gene), D13L (mid-stage expression gene), and A3L (late-stage expression gene) in VACV virus infection were measured at 4h, 8h, 12h, and 24h after drug administration, both in the absence of drug administration and after drug administration. The senna leaf extract inhibited the expression of genes throughout the entire VACV virus infection process. Further evaluation of the antiviral effect was conducted. Figure 4 .

[0059] Example 5

[0060] Evaluation of the effect of different treatment durations after kaempferol treatment on VCV protein expression levels in VCV virus infection.

[0061] Furthermore, this experiment adopted the previously described protocol of administering medication after viral infection, and further evaluated the inhibitory effect by measuring the levels of viral proteins A27L, L1R, E3L, and WR117 expressed by VACV virus at 4h, 8h, 12h, and 24h after medication in both untreated and treated samples. This further clarified that senna leaf extract inhibits the replication and assembly of viral proteins by VACV-associated poxvirus. Figure 5 .

[0062] Example 6

[0063] In vivo experiments have verified that physalis leaf extract can indeed inhibit the replication of CPXV in vivo, thereby achieving a therapeutic effect on the infection.

[0064] The above experiments have demonstrated that senna leaf extract can inhibit poxvirus replication and proliferation in vitro. Further, this experiment involved in vivo animal experiments on C57 mice. The procedure was as follows: Age-matched male C57 mice were randomly divided into two groups of eight mice each. The control group received an intraperitoneal injection of 6*10g of CPXV virus. 6 / ani; Treatment group: Intraperitoneal injection of CPXV virus 6*10 6 / mouse +5mg / kg of senna leaf extract. After 3.5 days of normal feeding, the mice were euthanized, and the spleen, visceral fat pad, and liver were harvested using DMEM containing 2.5% fetal bovine serum. The tissues were ground retrogradely using an Omni tissue homogenizer. The tissue extracts were serially diluted 10-fold, and the viral titer was determined by plaque assay on Vero cells. 48 hours after infection, Vero cells were fixed in 4% PFA for 20 minutes and then stained with 0.1% crystal violet. After rinsing with water, plaques were counted visually. The viral titer in different organs was determined to assess the in vivo antiviral effect. The results are as follows: Figure 6 The plant extract has the effect of inhibiting the replication of poxvirus in vivo.

[0065] Example 7

[0066] In the post-infection drug administration mode, lycopoietin (2.5 μm / ml) also showed antiviral effects against myxoma virus (MYXV) of the rabbitpoxvirus genus, as observed by GFP fluorescence after 48 hours, significantly inhibiting viral proliferation in host cells. Figure 7 .

[0067] The above results indicate that senna leaf extract has a significant inhibitory effect on infection with CPXV and other members of the poxvirus family.

Claims

1. Application of senna leaf extract in the preparation of drugs for inhibiting poxvirus and preventing or treating poxvirus infection.

2. The application according to claim 1, characterized in that, The poxvirus in question belongs to the genus *Orthopoxvirus*.

3. The application according to claim 1, characterized in that, The poxvirus is vaccinia virus (VACV), vaccinia virus (CPXV), or myxoma virus (MYXV) belonging to the genus rabbitpoxvirus.

4. The application according to claim 1, characterized in that, The content of the senna leaf extract in the drug is 1.25–2.5 μM / mL.

5. The application according to claim 1, characterized in that, In the preparation of drugs for the prevention of poxvirus infection, the effective concentration of kaempferol is 1.25 μM / mL; in the preparation of drugs for the treatment of poxvirus infection, the effective concentration of kaempferol is 2.5 μM / mL.

6. The application according to claim 1, characterized in that, The physalis leaf extract was screened using one of the following methods: (1) Pre-treatment with drug to infect the virus: first, add lycopene to Vero cells and incubate for 1 hour, then inoculate with poxvirus and incubate for 1 hour. Discard the liquid, add maintenance solution, collect samples after 48 hours to measure the virus inhibition effect and evaluate the antiviral effect of the prevention mode. (2) Simultaneous drug administration to infected virus: poxvirus was first added to Vero cells and then adsorbed at 4 degrees for 1 hour. The liquid was discarded, and drug maintenance solution was added. After 48 hours, the sample was collected to determine the virus inhibition effect. (3) Method of administering medication after viral infection: Vero cells were infected with poxvirus for 1 hour, the liquid was discarded, and senna leaf extract was added. After 48 hours of treatment, samples were collected to measure the viral inhibition effect and evaluate the antiviral effect of the treatment mode.

7. A drug for inhibiting poxvirus, characterized in that, This medicine contains senna leaf extract.

8. The drug for inhibiting poxvirus according to claim 7, characterized in that, The drug is in liquid form.

9. The drug for inhibiting poxvirus according to claim 7, characterized in that, The drugs that inhibit poxvirus include those that inhibit poxvirus proliferation and / or poxvirus toxicity.

10. A drug for the prevention or treatment of poxvirus infection, characterized in that, This medicine contains senna leaf extract.

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