Application of apigenin in preparation of medicine for preventing and / or treating monkey pox virus infection

Abstract

The invention relates to application of apigenin in preparation of a medicine for preventing and / or treating monkey pox virus infection, and belongs to the technical field of biological medicine. A natural product apigenin is selected, the anti-monkey pox virus activity of the apigenin is researched, and the result shows that the apigenin can effectively inhibit expression and replication of monkey pox viruses in host cells in vitro and shows remarkable antiviral activity. In addition, the apigenin is wide in source and low in cost, a new choice is provided for preparation of the anti-monkey pox virus medicine, and the apigenin has good popularization and application prospects.

Description

Technical Field

[0001] This invention relates to a novel use of apigenin, specifically its application in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, belonging to the field of biomedical technology. Background Technology

[0002] Monkeypox is a zoonotic disease caused by monkeypox virus (MPXV), a linear double-stranded DNA orthopoxvirus with a genome of approximately 197 kb containing nearly 190 open reading frames. MPXV shares high genetic homology with smallpox virus and has become a significant pathogen threatening human health. Since 2022, the global monkeypox epidemic has continued to spread. As of October 2025, more than 100 countries have reported over 120,000 confirmed cases and 283 deaths, posing a significant public health risk. Although most patients experience mild symptoms, infection can lead to serious complications such as pneumonia, encephalitis, and multi-organ damage in immunocompromised individuals, pregnant women, children, and the elderly, making clinical prevention and control a challenging task.

[0003] Currently, specific treatments for monkeypox virus are extremely limited. The only approved drug, tecovirex, exerts its medicinal value by inhibiting the function of the viral protein VP37, but its clinical application is constrained by insufficient animal experimental data and the emergence of drug-resistant strains. Another drug, cidofovir, while possessing some antiviral activity, exhibits significant nephrotoxicity, limiting its applicability. Regarding vaccines, JYNNEOS, although providing cross-immune protection, suffers from low global coverage due to limitations in production capacity, cost, and cold chain conditions. Therefore, developing natural drugs with broad-spectrum antiviral activity, low toxicity, and easy accessibility has become an urgent research need.

[0004] Natural products, due to their diverse structures, multiple mechanisms of action, and relatively high safety, have become an important source for antiviral drug development. Flavonoids, as a widely distributed class of plant secondary metabolites, possess various pharmacological activities, including anti-inflammatory, antioxidant, and immunomodulatory activities. Studies have shown that they exert antiviral effects by interfering with viral invasion, inhibiting viral enzyme activity, and regulating host immune responses. For example, quercetin and baicalin have shown inhibitory effects against coronaviruses and dengue viruses, providing a theoretical basis for the use of flavonoids in anti-poxvirus research.

[0005] Apigenin is a natural flavonoid compound widely found in plants such as celery and chamomile, possessing significant antioxidant, anti-inflammatory, antitumor, and antiviral effects. The apigenin molecule contains multiple hydroxyl and methoxy groups, which can form stable hydrogen bonds and hydrophobic interactions with biomolecules. Previous studies have shown that apigenin has inhibitory effects on various viruses, including influenza virus, herpes simplex virus, and human papillomavirus, through mechanisms including blocking viral adsorption and invasion, interfering with viral RNA replication, and regulating host antiviral signaling pathways. These findings suggest that apigenin has potential broad-spectrum antiviral properties. However, to date, there is no systematic research on the antiviral effect of apigenin in monkeypox virus infection, and its specific mechanism of action and application prospects require further exploration. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, the present invention provides the application of apigenin in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection.

[0007] The experimental data provided by this invention demonstrate that apigenin can effectively inhibit the replication and expression of monkeypox virus in host cells in vitro, and can be used to prepare drugs that have preventive and / or therapeutic effects against monkeypox virus infection.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The use of apigenin in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection.

[0009] Furthermore, the monkeypox virus strains are monkeypox virus type I and monkeypox virus type II.

[0010] Furthermore, the drug for preventing and / or treating monkeypox virus infection includes, but is not limited to, at least one of: plants containing apigenin, effective parts of plants containing apigenin, plant extracts containing apigenin, purified apigenin, and chemically synthesized apigenin.

[0011] Furthermore, the drug for the prevention and / or treatment of monkeypox virus infection comprises, but is not limited to, an active ingredient and a pharmaceutically acceptable carrier; the active ingredient is a plant extract of apigenin and / or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the drugs for preventing and / or treating monkeypox virus infection are pharmaceutically acceptable oral, injectable, inhaled, or topical formulations.

[0013] Furthermore, the drug for preventing and / or treating monkeypox virus infection may contain apigenin and / or a pharmaceutically acceptable salt thereof.

[0014] Furthermore, the apigenin and / or its pharmaceutically acceptable salts are one or both of apigenin glycosides and flavonols.

[0015] Furthermore, the plants containing apigenin are Chinese celery, Scrophulariaceae plant Agrostis chinensis, Polygonum cuspidatum (Polygonum cuspidatum), and Liliaceae plant Veratrum nigrum.

[0016] Furthermore, in the drug for preventing and / or treating monkeypox virus infection, the pharmaceutically acceptable carrier is one of liposomes or apigenin nanosuspensions.

[0017] Furthermore, the drug for preventing and / or treating monkeypox virus infection works by inhibiting the replication and proliferation of monkeypox virus.

[0018] Furthermore, the drug for preventing and / or treating monkeypox virus infection works by inhibiting the replication and expression of monkeypox virus, and the replication and expression of monkeypox virus in host cells gradually weakens as the concentration of apigenin in the drug for preventing and / or treating monkeypox virus infection increases.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1) This invention demonstrates through in vitro cell experiments that exogenous addition of apigenin can effectively inhibit monkeypox virus type I and monkeypox virus type II. The results show that apigenin can significantly inhibit the replication and gene expression of monkeypox virus type I and monkeypox virus type II, exhibiting significant antiviral activity. This proves that apigenin has a significant inhibitory effect on monkeypox virus type I and monkeypox virus type II, providing an effective new approach for the prevention and / or treatment of monkeypox virus infection. 2) The experimental data provided by this invention confirms that apigenin has anti-monkeypox virus activity at the cellular level, thereby establishing the feasibility of apigenin in the preparation of drugs for the prevention and / or treatment of monkeypox virus type I and monkeypox virus type II, and providing a new potential drug for the prevention and / or treatment of monkeypox virus type I and monkeypox virus type II infection. Attached Figure Description

[0020] Figure 1 Vero cells were pretreated with different concentrations of apigenin (12 μM, 6 μM, 3 μM, 1.5 μM, 0.75 μM, 0.375 μM, 0.18 μM, 0.09 μM, 0.045 μM, 0.0225 μM, and 0.011 μM) and then inoculated with monkeypox virus type I and type II at an MOI of 0.05. After 1 hour of adsorption, the virus-drug mixture was discarded, and the cells were cultured for 48 hours with the corresponding concentration of drug in maintenance medium. The cytopathic effect (CPE) was observed and recorded. The results showed that apigenin significantly inhibited the cytopathic effect induced by monkeypox virus type I / II, exhibiting certain antiviral activity.

[0021] Figure 2 Vero cells were treated with different concentrations (200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM) of apigenin for 48 hours, and cell viability was detected by the MTS assay. The results showed that the half-maximal cytotoxic concentration (CC50) of apigenin on Vero cells was... 50 Value > 200 μM.

[0022] Figure 3 Vero cells were pretreated with different concentrations of apigenin (12 μM, 6 μM, 3 μM, 1.5 μM, 0.75 μM, 0.375 μM, 0.18 μM, 0.09 μM, 0.045 μM, 0.0225 μM, 0.011 μM) for 1 h. Monkeypox virus type I and monkeypox virus type II were then inoculated at an MOI of 0.05. After 1 h of adsorption, the virus-drug mixture was discarded, and the cells were cultured for 48 h with the corresponding concentration of drug in maintenance medium. The viral genome copy number in the supernatant was detected by real-time quantitative PCR (qPCR). The results confirmed that apigenin significantly inhibited monkeypox virus type I replication in a dose-dependent manner, with an effective concentration (EC50) of [missing value]. 50 The EC50 of monkeypox virus type II was 13.77 nM, with a selection index (SI) > 14524. 50 The value is 6.9 nM, and the selection index SI > 28985.

[0023] Figure 4 To evaluate the inhibitory effect of apigenin on monkeypox virus infection at different treatment time points through cytopathic effect (CPE), all groups were inoculated with monkeypox virus type I and monkeypox virus type II (MOI=0.05) and treated with 25 μM apigenin at different treatment time points (full treatment group, pre-entry treatment group, and post-entry treatment group) to inhibit cytopathic effect (CPE). The results showed that the full treatment group had the highest CPE inhibition rate, which was significantly better than the pre-entry and post-entry treatment groups. Figure 5 To verify the inhibitory effect of apigenin on monkeypox virus type I / II protein expression at different treatment time points using Western blot experiments, Vero cells were seeded in 12-well plates and cultured until 80% confluence. Monkeypox virus was infected with 25 μM apigenin at three treatment rates: full treatment, pre-invasion treatment, and post-invasion treatment (MOI=0.05). Total protein was collected after 48 h for Western blot analysis, and viral protein expression was detected using anti-A42R antibody. Results showed that the A42R protein band was not detected in the full treatment group and the post-invasion treatment group, indicating a significantly better inhibitory effect than the pre-invasion treatment group.

[0024] Figure 6To analyze the inhibitory effect of apigenin on monkeypox virus (monkeypoxvirus type I and monkeypox virus type II) replication at different treatment time points using qPCR, Vero cells were treated with apigenin at a final concentration of 25 μM using three treatment methods: full-cycle treatment, pre-infection treatment, and post-infection treatment, and then infected with monkeypox virus (MOI=0.05). Viral genome copy number was measured 48 hours later. The results showed that the full-cycle treatment method had the most significant inhibitory effect on viral replication. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the embodiments.

[0026] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase.

[0027] The complete culture medium formulations used in the embodiments of this invention are as follows: DMEM complete medium: Add 1% (v%) of antibiotics and 8% (v%) of fetal bovine serum to commercial DMEM (Viva cell) medium. 2% DMEM maintenance solution: Add 1% (v%) of antibiotics and 2% (v%) of fetal bovine serum to commercial DMEM (Viva cell) culture medium. Serum-free culture medium: commercially available DMEM (Viva cell) medium.

[0028] The virus involved in this invention is the strain of monkeypox virus MPXV: monkeypox virus type I and monkeypox virus type II, provided by the National Kunming High-Level Biosafety Primate Experiment Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences.

[0029] The antibodies involved in this invention are shown in Table 1.

[0030] Table 1

[0031] The reagents involved in this invention are shown in Table 2.

[0032] Table 2

[0033] The preparation of the corresponding reagents in this invention is as follows: (1) The components of the transfer buffer are shown in Table 3.

[0034] Table 3

[0035] Add 600 mL of deionized water to the above ingredients, stir to dissolve, and then bring the volume up to 1 L with deionized water.

[0036] (2) The composition of 10×TBS is shown in Table 4.

[0037] Table 4

[0038] Add 400 mL of ultrapure water to the sodium chloride and Tris components mentioned above, stir to dissolve, adjust the pH to 7.5 with concentrated hydrochloric acid or NaOH, and then bring the volume to 500 mL with ultrapure water.

[0039] (3) The composition of 1×TBST is shown in Table 5.

[0040] Table 5

[0041] Mix thoroughly and use immediately after preparation.

[0042] (4) The composition of the sealing liquid is shown in Table 6.

[0043] Table 6

[0044] Mix thoroughly and use immediately after preparation.

[0045] (5) The composition of 0.25% pancreatic enzyme is shown in Table 7.

[0046] Table 7

[0047] After mixing, dispense and store in a -40°C refrigerator.

[0048] The English abbreviations involved in this invention and their corresponding Chinese and English names are shown in Table 8.

[0049] Table 8

[0050] Example 1 Vero cells were treated with apigenin and then infected with monkeypox virus type I and type II. The cytopathic effect (CPE) was observed to determine the antiviral activity of the drug. The specific steps are as follows.

[0051] Cell preparation: Well-grown Vero cells were passaged and seeded in 96-well plates with 100 μl of complete culture medium per well. The initial cell volume was 2 × 10⁶ cells / well. 4Cells / well were incubated overnight in a cell culture incubator at 37°C and 5% CO2 concentration. 2. Drug treatment: A 100 mM stock solution of apigenin was prepared using DMSO as the solvent. Apigenin solutions with final concentrations of 12 μM, 6 μM, 3 μM, 1.5 μM, 0.75 μM, 0.375 μM, 0.18 μM, 0.09 μM, 0.045 μM, 0.0225 μM, and 0.011 μM were added to 96-well plates, respectively. DMSO served as the negative control, and tecovirex served as the positive control. Each group had three replicates. After gentle mixing, the plates were pretreated at 37°C in a 5% CO2 incubator for 1 hour. 3. Virus infection and culture: Add 100 μl of monkeypox virus solution diluted in DMEM to each well (MOI = 0.05). Incubate at 37°C and 5% CO2 for 1 hour. Remove the drug and virus solution mixture, add maintenance medium containing the appropriate concentration of apigenin, and continue culturing for 48 hours. 4. Cytopathic Effect (CPE) Observation: After culture, the morphology of cells in each well was observed under a light microscope, and the degree of cytopathic effect (CPE) was photographed and recorded. Results showed that apigenin significantly inhibited Vero cell pathogenesis induced by monkeypox virus type I / II, exhibiting certain antiviral activity, such as... Figure 1 As shown.

[0052] Example 2 Evaluation of apigenin's cytotoxicity to Vero cells and CC 50 The specific steps for the determination are as follows: 1. Cell preparation: Take Vero cells in good growth condition, digest them and count the cells. Seed the cell suspension at 100 μL per well in a 96-well plate, with 3 replicates per group. Incubate overnight at 37℃ in a 5% CO2 incubator until the cells are fully attached. 2. Drug treatment: Apigenin was added to the wells of Vero cells at final concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM, and cultured in an incubator at 37°C and 5% CO2 for 48 hours. 3. Cell viability assay: After the culture in step 2 is completed, add 20 μL of MTS solution to each well and continue to incubate in an incubator for 1-4 h. Use a multi-functional microplate reader to detect the absorbance value of each well at a wavelength of 490 nm to reflect cell viability. 4. Data Analysis: GraphPad Prism software was used to process absorbance data, plot cell viability curves, and nonlinear regression analysis was used to calculate the half-maximal cytotoxic concentration (CMC) of apigenin in Vero cells. 50 ); 5. Through dose-toxicity curve analysis, the CC of apigenin on Vero cells was calculated. 50 Values ​​greater than 200 μM, experimental results as follows Figure 2 As shown.

[0053] Example 3 The half-maximal effective concentration (EC50) of apigenin against monkeypox virus type I and monkeypox virus type II was evaluated using qPCR. 50 The specific steps are as follows: Cell preparation: Well-grown Vero cells were passaged and seeded in 96-well plates with 100 μl of complete culture medium per well. The initial cell volume was 2 × 10⁶ cells / well. 4 Cells / well were incubated overnight in a cell culture incubator at 37°C and 5% CO2 concentration. 2. Drug treatment: A 100mM stock solution of apigenin was prepared using DMSO as the solvent. Apigenin solutions with final concentrations of 12μM, 6μM, 3μM, 1.5μM, 0.75μM, 0.375μM, 0.18μM, 0.09μM, 0.045μM, 0.0225μM, and 0.011μM were added sequentially to the wells. The solutions were incubated for 1 hour. DMSO served as a negative control, and tecovirex served as a positive control. Each group had three replicates. After gentle mixing, the cells were incubated at 37℃ in a 5% CO2 incubator for 1 hour. 3. Viral infection: Add 100 μL of monkeypox virus solution diluted with DMEM culture medium to each well, MOI=0.05. Incubate in a cell culture incubator with 5% CO2 concentration for 1 h, then remove the virus solution and drug mixture, add the corresponding concentration of apigenin, and continue to incubate in a cell culture incubator with 37℃ and 5% CO2 for 48 h. 4. qPCR detection: Collect cell supernatant, extract total DNA using a DNA extraction kit, and perform real-time quantitative PCR (qPCR) detection using monkeypox virus-specific primers (as shown in Table 1) to analyze the viral genome copy number; Table 1. Primer sequences for monkeypox virus

[0054] 5. Process the data and plot it using GraphPad software; 6. The results showed that apigenin inhibited monkeypox virus replication in a dose-dependent manner within the concentration range of 0.011-12 μM. Calculations showed that apigenin inhibited the EC50 of monkeypox virus type I. 50 The value was 13.77 nM, and the selection index SI > 14524; for EC of monkeypox virus type II 50 The value is 6.9 nM, the selection index SI > 28985, and the experimental results are as follows: Figure 3 As shown.

[0055] Example 4 Vero cells were treated with apigenin via three administration methods: full-cycle administration, pre-infection administration, and post-infection administration, followed by infection with monkeypox virus (monkeypox virus type I and monkeypox virus type II). Cell CPE results were observed after 48 hours. The specific steps are as follows: Cell preparation: Take Vero cells in good growth condition, at a density of 0.2 × 10⁶ cells per well. 6 The cells were seeded at a density of 100 μL of complete culture medium in each well of a 12-well plate and incubated overnight at 37°C in a 5% CO2 incubator. Different drug administration methods and viral infection: A 100 mM stock solution of apigenin was prepared using DMSO as the solvent. Full-process drug administration: After treating with apigenin at a final concentration of 25 μM for 1 h, monkeypox virus diluted in DMEM (MOI=0.05) was added to the wells and incubated for 1 h, followed by replacement with apigenin at a final concentration of 25 μM for 48 h of maintenance culture. Pre-cell invasion drug administration: Monkeypox virus diluted in DMEM (MOI=0.05) and apigenin at a final concentration of 50 μM were added to the wells and incubated together for 1 h, followed by replacement with maintenance medium for 48 h of maintenance culture. Post-cell invasion drug administration: Monkeypox virus diluted in DMEM (MOI=0.05) was added to the wells and incubated for 1 h, followed by replacement with apigenin at a final concentration of 25 μM for 48 h of maintenance culture. DMSO was added as a negative control for each drug administration method, and three replicates were set up. After gentle mixing, the cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 48 h. 3. Observation of cytopathic effect (CPE): After culture, the morphology of cells in each well was observed under an optical microscope, and the degree of CPE was photographed and recorded. 4. The results showed that apigenin treatment of Vero cells, with continuous dosing, had the most significant inhibitory effect on the monkeypox virus-induced Vero cell pathogenesis (CPE). The experimental results are as follows: Figure 4 As shown.

[0056] Example 5 Apigenin was administered to Vero cells via three methods: full-cycle administration, pre-infection administration, and post-infection administration, followed by infection with monkeypox virus (monkeypoxvirus type I and monkeypoxvirus type II). Western blotting was used to detect the expression of the monkeypox virus protein A42R. The specific steps are as follows: 1. Cell preparation. Passage well-grown Vero cells into 12-well plates, adding 1 ml of complete culture medium to each well. The initial cell volume is 1 × 10⁶ cells / well. 6 Cells per well were incubated overnight in a cell culture incubator at 37°C and 5% CO2 concentration.

[0057] 2. Different drug administration methods and viral infection: A 100mM stock solution of apigenin was prepared using DMSO as the solvent. Full-process drug administration: After treating the wells with a final concentration of 25μM apigenin for 1 h, monkeypox virus diluted in DMEM (MOI=0.05) was added and incubated for 1 h, then replaced with 25μM apigenin for 48 h of maintenance culture; Pre-cell incubation: Monkeypox virus diluted in DMEM (MOI=0.05) and apigenin at a final concentration of 50μM were added to the wells and incubated together for 1 h, then replaced with maintenance medium for 48 h of maintenance culture; Post-cell incubation: Monkeypox virus diluted in DMEM (MOI=0.05) was added to the wells and incubated for 1 h, then replaced with 25μM apigenin for 48 h of maintenance culture; DMSO was added as a negative control for each drug administration method, and three replicates were set up. After gently mixing, incubate in a cell culture incubator at 37°C and 5% CO2 concentration for 48 hours; 3. Protein Sample Harvesting: Remove the 12-well plate after 48 hours of culture, discard the cell culture supernatant, wash twice with colorless PBS, discard the PBS, and place the 12-well plate on ice. Add 15 μl of PMSF to 1.5 ml of RIPA lysis buffer on ice, mix well to obtain a lysis buffer containing PMSF. Add 50 μl of RIPA lysis buffer containing PMSF to each well and lyse on ice for 30 min. To ensure complete cell lysis, the culture flask should be shaken frequently. After lysis, transfer the cell debris and lysis buffer to a 1.5 ml centrifuge tube using a pipette. Centrifuge at 12500 rpm for 5 min at 4°C. Finally, aliquot the centrifuged protein supernatant into 1.5 ml centrifuge tubes and store at -80°C. 4. Western blot analysis of monkeypox virus protein A42R expression, as detailed below: 1) Sample Preparation: For BCA method to detect protein concentration, take the protein-containing supernatant obtained in step 3 and calculate the volume of solution containing 70 μg of protein as the loading volume (the loading volume of each well should be consistent; the sample concentration can be adjusted in advance to ensure consistent volume and mass; 5 μL for protein marker loading; the loading volume of a 15-well plate should not exceed 20 μL). Add 5×SDS loading buffer to a final concentration of 1×. Mix repeatedly by pipetting and incubate in boiling water for 10 min to denature the protein. Centrifuge at 10000g for at least 5 min, and take the supernatant as the sample, placing it on ice for later use. 2) SDS-polyacrylamide gel electrophoresis: Assemble the electrophoresis tank, add MOPS electrophoresis buffer, ensuring the inner wells are submerged and the outer wells reach the 4-Gels mark. Carefully remove the comb from the precast gel. Slowly add the sample, preferably to the middle wells. Wash the sample three times in electrophoresis buffer before adding the next sample to avoid contamination. Finally, add the protein marker. After loading, close the cap, connect the electrophoresis apparatus, and turn it on. Run at 80V for 30 minutes, then increase the voltage to 120V for a total electrophoresis time of 100 minutes. Stop electrophoresis when the bromophenol blue indicator migrates to a point 1-2 cm from the leading edge. 3) Transfer: After electrophoresis, gently pry open the two glass plates of the gel plate using a thin-plate tool. Using the protein marker as a guide, cut out the gel containing the target protein and carefully lay it flat on filter paper. Place the pre-cut, marked PVDF membrane over the corresponding gel, ensuring there are no air bubbles between them. Assemble the sandwich plate, with the black (gel) facing the black negative electrode and the transparent (membrane) facing the red positive electrode. Pour in the transfer buffer (wet transfer buffer), filling the entire transfer tank, and add ice for an ice bath transfer. Set the transfer current to a constant current of 300 mA. The transfer time depends on the protein size; generally, 1 kDa requires 1 minute. 4) Blocking: After the transfer is complete, remove the membrane with the protein side facing up, rinse once with 1×TBST, and then place it in blocking solution and block on a horizontal shaker at room temperature for 2 hours or more. 5) Primary antibody incubation: Dilute the primary antibody (A42R and GAPDH) with Beyotime primary antibody dilution buffer according to the dilution ratio recommended in the antibody instructions. After aspirating the blocking buffer, immediately add the diluted primary antibody and incubate on a horizontal shaker at room temperature for 2 hours, then place in a 4°C refrigerator overnight. Antibody dilution ratios: A42R (1:1000); GAPDH (1:20000). 6) Washing the membrane: After incubation, the primary antibody is collected and recovered (it can be reused 3-4 times within one or two weeks at 4℃). Add 1×TBST to a square food storage box, put the membrane in it, and make sure that 1×TBST covers the PVDF membrane. Shake at 70rpm for 10min on a shaker. Repeat the washing 4 times. 7) Incubation with secondary antibody: Dilute the secondary antibody with blocking buffer to the concentration specified in the antibody instructions, immerse the PVDF membrane with protein transfer into a square food storage box containing the antibody, and incubate at a low speed of 70 rpm for 2 hours on a shaker; 8) Washing the membrane: After incubation, take the secondary antibody, add 1×TBST to a square food storage box, put the membrane in it, so that 1×TBST covers the PVDF membrane, shake it at 70 rpm for 10 min on a shaker, and repeat the washing 4 times. 9) Development: Prepare the developing solution (prepare the developing solution A and B from the Beyotime Ultrasensitive ECL Chemiluminescence Kit in a 1:1 ratio and use immediately). During development, drop the developing solution onto the membrane until it is completely covered. After reacting in the dark for 1 minute, place the membrane in a UVP instrument to expose the protein bands and take pictures for recording. 5. The results showed that Vero cells were treated with apigenin throughout the entire treatment process, before cell entry, and after cell entry, and then infected with monkeypox virus. The A42R protein band was not detected in the groups treated throughout the entire treatment process and after cell entry, indicating a significantly better inhibitory effect than the group treated before cell entry. The experimental results are as follows: Figure 5 As shown.

[0058] Example 6 After Vero cells were treated with apigenin via three administration methods—full-process administration, pre-infection administration, and post-infection administration—they were infected with monkeypox virus (monkeypoxvirus type I and monkeypoxvirus type II). Viral nucleic acid levels were measured using qPCR, and the inhibition rate was calculated. The specific steps are as follows: 1. Cell preparation: Passage well-grown Vero cells into 12-well plates, adding 1 ml of complete culture medium to each well. The initial cell volume is 1 × 10⁶ cells / well. 6 Cells / well were incubated overnight in a cell culture incubator at 37°C and 5% CO2 concentration. 2. Different drug administration methods and viral infection: A 100mM stock solution of apigenin was prepared using DMSO as the solvent. Full-process drug administration: After treating with apigenin at a final concentration of 25μM for 1 h, monkeypox virus diluted in DMEM (MOI=0.05) was added to the wells and incubated for 1 h, followed by replacement with apigenin at a final concentration of 25μM for 48 h of maintenance culture. Pre-cell invasion drug administration: Monkeypox virus diluted in DMEM (MOI=0.05) and apigenin at a final concentration of 50μM were added to the wells and incubated together for 1 h, followed by replacement with maintenance medium for 48 h of maintenance culture. Post-cell invasion drug administration: Monkeypox virus diluted in DMEM (MOI=0.05) was added to the wells and incubated for 1 h, followed by replacement with apigenin at a final concentration of 25μM for 48 h of maintenance culture. DMSO was added as a negative control for each drug administration method, and three replicates were set up. After gentle mixing, the cells were incubated in a cell culture incubator at 37℃ and 5% CO2 for 48 h. 3. Take 100 μL of the supernatant and add it to 300 μL of Trizol for qPCR detection; 4. The results showed that the full-course dosing method had the most significant inhibitory effect. The experimental results are as follows: Figure 6 As shown.

[0059] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. The use of apigenin in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection.

2. The use of apigenin according to claim 1 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that, The monkeypox virus strains mentioned are monkeypox virus type I and monkeypox virus type II.

3. The use of apigenin according to claim 1 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that, The drug for the prevention and / or treatment of monkeypox virus infection includes, but is not limited to, at least one of the following: plants containing apigenin, effective parts of plants containing apigenin, plant extracts containing apigenin, purified apigenin, and chemically synthesized apigenin.

4. The use of apigenin according to claim 1 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that, The aforementioned drug for the prevention and / or treatment of monkeypox virus infection comprises, but is not limited to, an active ingredient and a pharmaceutically acceptable carrier; the active ingredient is a plant extract of apigenin and / or a pharmaceutically acceptable salt thereof.

5. The use of apigenin according to claim 1 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that, The drugs mentioned for the prevention and / or treatment of monkeypox virus infection are oral preparations, injections, inhaled preparations, or topical preparations.

6. The use of apigenin according to claim 1 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that, The drug for the prevention and / or treatment of monkeypox virus infection may contain apigenin and / or its pharmaceutically acceptable salts.

7. The use of apigenin according to claim 4 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that, The apigenin and / or its pharmaceutically acceptable salts are one or both of apigenin glycosides and flavonols.

8. The use of apigenin according to claim 3 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that... The plants containing apigenin are Chinese celery, Scrophulariaceae plant Achyranthes bidentata, Polygonum cuspidatum (Polygonaceae family), and Veratrum nigrum (Liliaceae family).

9. The use of apigenin according to claim 4 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that... In the drug for the prevention and / or treatment of monkeypox virus infection, the pharmaceutically acceptable carrier is one of liposomes or apigenin nanosuspensions.

10. The use of apigenin according to claim 1 in the preparation of drugs for the prevention and / or treatment of monkeypox virus infection, characterized in that... The drugs mentioned for the prevention and / or treatment of monkeypox virus infection work by inhibiting the replication and proliferation of monkeypox virus.

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