Application of monkshood extract in orthopoxvirus inhibitor

Abstract

The invention provides an application of a monkshood extract in an orthopoxvirus inhibitor. The invention discloses an antiviral mechanism that the radix aconiti carmichaeli extract activates innate immunity of host cells through a cGAS-STING pathway for the first time, and the radix aconiti carmichaeli extract has no side effect in treatment of orthopoxvirus, and is suitable for long-term use.

Description

Technical Field

[0001] The present invention relates to the field of new applications of drugs, and particularly to the application of aconite extract as an orthopoxvirus inhibitor. Background Art

[0002] The genus Orthopoxvirus is a genus with the largest number of members, the widest host range, and the most serious harm in the mammalian subfamily. All orthopoxviruses in this genus, such as Monkeypox virus (MPXV), Cowpox viruses (CPXV), Vaccinia virus (VACV), Variola Virus (VARV), Camelpox virus (CMPV), Ectromelia virus (ECTV), Taterapox virus (TATV), etc., have the ability to infect humans.

[0003] Orthopoxviruses pose a very serious threat to humans. Among them, the fatality rate of patients infected with Variola virus is as high as 30%, the fatality rate of patients infected with West African clade Monkeypox is about 3.6%, and the fatality rate of patients infected with Congo Basin clade Monkeypox can reach 10.6%. The clinical symptoms of Monkeypox virus-infected patients mainly include headache, fever, lymphadenopathy, and symptoms such as vesicular-like rashes. Among existing drugs, for example, cidofovir has a certain effect on the treatment of Monkeypox. However, some patients will experience adverse reactions such as nausea, vomiting, and diarrhea after using cidofovir, which greatly limits the application scope of this drug in the treatment of Monkeypox.

[0004] Natural drugs have become a hot spot in antiviral research due to their multi-target effects and low toxicity characteristics. Aconitum carmichaelii, as a traditional Chinese medicine, has a wide range of pharmacological activities, and the potential of aconite extract in antiviral aspects has been gradually revealed. However, the application of aconite extract in the field of anti-orthopoxvirus has not been reported yet. Summary of the Invention

[0005] In view of the problem that drugs for treating orthopoxviruses in the prior art can cause adverse reactions, the present invention provides an application of aconite extract as an orthopoxvirus inhibitor.

[0006] The technical method of the present invention is as follows:

[0007] An application of aconite extract as an orthopoxvirus inhibitor.

[0008] Orthopoxviruses, including monkeypox virus, vaccinia virus, cowpox virus, smallpox virus, camelpox virus, mousepox virus, and African gerbilpox virus, all have an envelope on their outermost layer. Moreover, after infecting cells, the proteins encoded by orthopoxviruses inhibit the activation of the host's innate immune pathway.

[0009] In the present invention, the aconite extract can directly and indirectly inhibit orthopoxviruses.

[0010] Direct inhibition: The aconite extract disrupts the envelope of orthopoxviruses and reduces the virus's adsorption ability.

[0011] Indirect inhibition: The aconite extract activates the cGAS-STING signaling pathway, thereby activating the host's innate immune pathway and significantly inhibiting the replication of orthopoxviruses.

[0012] Among them, as an important sensor of the innate immune system, cGAS exerts its biological function by specifically recognizing double-stranded DNA (dsDNA) in the cytoplasm. When dsDNA binds to cGAS, it induces a significant conformational change in this enzyme, thereby activating its catalytic domain. Activated cGAS, through its unique cyclase activity, converts the substrates ATP (adenosine triphosphate) and GTP (guanosine triphosphate) into the second messenger molecule 2',3'-cyclic GMP-AMP (cGAMP). This signaling molecule binds to the endoplasmic reticulum-localized STING (stimulator of interferon genes) protein through diffusion, triggering its conformational change and oligomerization. The activated STING complex activates the TANK-binding kinase 1 (TBK1) and nuclear factor κB (NF-κB) signaling pathways through a cascade of signal transduction. Specifically, TBK1 can specifically phosphorylate interferon regulatory factor 3 (IRF3), prompting IRF3 to form a homodimer and translocate into the nucleus. At the transcriptional level, the phosphorylated IRF3 dimer binds to the interferon-stimulated response element (ISRE) to initiate the transcription program of type I interferon (IFN-I) and interferon-stimulated genes (ISGs), ultimately mediating the activation of antiviral immune responses and inflammatory reactions.

[0013] Specifically, the aconite extract upregulates the expression of key proteins in the cGAS-STING signaling pathway (e.g., STING and / or IRF3 (interferon regulatory factor 3)), promotes the secretion of type I interferon (e.g., IFN-β), and enhances the host's antiviral immune response.

[0014] In the present invention, the aconite extract is prepared by the water extraction method.

[0015] Specifically, the steps of the water extraction method include:

[0016] S1. Wash, dry, and pulverize aconite to obtain aconite granules;

[0017] S2. Perform water extraction on the aconite granules to obtain a filtrate;

[0018] S3. Concentrate and dry the filtrate to obtain an aconite extract.

[0019] In step S1, the origin of the aconite is Sichuan, Shaanxi, Guizhou and other places. The retention rate of aconitum alkaloids in aconite is the highest (determined by HPLC ≥ 85%). Deionized water or pure water is used for cleaning. Low-temperature drying is used for drying, and the drying temperature is 50 - 60 °C. Preferably, it is 60 °C. Conventional comminution equipment in the art is used for comminution. The particle size of the comminuted aconite granules is less than or equal to 50 μm.

[0020] In step S2, the steps of water extraction include: putting the aconite granules into water and extracting by heating. Among them, the solid-liquid ratio is 1:10 - 30 (w / v), and the heating temperature is 80 - 100 °C. Preferably, the solid-liquid ratio is 1:10 (w / v). The heating temperature is 80 °C. Here, the filtrate can be collected at regular intervals, and all the collected filtrates are combined. Preferably, the time can be 2 hours.

[0021] In step S3, the density of the concentrated filtrate is 1.0 - 1.4 g / mL. Preferably, the density of the concentrated filtrate is 1.2 g / mL. Spray drying can be used for drying, with an inlet air temperature of 160 °C and an outlet air temperature of 80 °C.

[0022] In the present invention, the yield of the water extraction method is ≥ 18%, and the aconite extract is a light brown powder.

[0023] The present invention also provides an application of the aconite extract in a medicament for treating orthopoxvirus. Here, the preparation method of the orthopoxvirus medicament includes: taking the aforementioned orthopoxvirus inhibitor, adding pharmaceutically acceptable excipients or auxiliary components, and preparing into a dosage form.

[0024] Furthermore, the dosage form includes but is not limited to tablets, capsules, powders, soft capsules, dripping pills, honey pills, pills, granules, honey-refined ointments, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, injection preparations or external preparations, etc.

[0025] The beneficial effects of the present invention are:

[0026] First, no side effects have been found in the aconite extract of the present invention in the treatment of orthopoxvirus, and it is suitable for long-term use.

[0027] Second, the present invention for the first time reveals the antiviral mechanism of the aconite extract to activate the innate immunity of host cells through the cGAS-STING pathway.

[0028] III. The aconite extract of the present invention has broad-spectrum antiviral potential and is applicable to the prevention and treatment of various viruses. For example, it shows inhibitory effects on the Vaccinia virus WR strain (EC50 = 28.5 ± 3.2 μg / mL). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A It is the first effect experimental diagram of the aconite extract against the Vaccinia virus Western Reserve (VACV-WR) strain on Vero-E6 cells;

[0030] Figure 1B It is the second effect experimental diagram of the aconite extract against the Vaccinia virus Western Reserve (VACV-WR) strain on Vero-E6 cells;

[0031] Figure 2A It is the third effect experimental diagram of the aconite extract against the Vaccinia virus Western Reserve (VACV-WR) strain on Vero-E6 cells;

[0032] Figure 2B It is the fourth effect experimental diagram of the aconite extract against the Vaccinia virus Western Reserve (VACV-WR) strain on Vero-E6 cells;

[0033] Figure 3A It is the fifth effect experimental diagram of the aconite extract against the Vaccinia virus Western Reserve (VACV-WR) strain at the mouse level;

[0034] Figure 3B It is the sixth effect experimental diagram of the aconite extract against the Vaccinia virus Western Reserve (VACV-WR) strain at the mouse level;

[0035] Figure 4A It is the observation diagram of the Western blotting experimental effect of the aconite extract against the Vaccinia virus Western Reserve (VACV-WR) strain on Vero-E6 cells;

[0036] Figure 4B It is the data diagram of the Western blotting experiment of the aconite extract against the Vaccinia virus Western Reserve (VACV-WR) strain on Vero-E6 cells;

[0037] Figure 5A Observation diagram of the effect of aconite extract on activating the cGAS-STING pathway and IFNB in HeLa cells;

[0038] Figure 5B Observation diagram of the effect of aconite extract on activating the cGAS-STING pathway and ISG15 in HeLa cells;

[0039] Figure 5C Observation diagram of the effect of aconite extract on activating the cGAS-STING pathway and ISG20 in HeLa cells;

[0040] Figure 5D Observation diagram of the effect of aconite extract on activating the cGAS-STING pathway and STING in HeLa cells;

[0041] Figure 5E Observation diagram of the effect of aconite extract on activating the mRNA level of the cGAS-STING pathway and the protein level of STING in HeLa cells. Detailed implementation manners

[0042] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] In the present invention, unless otherwise specified, all preparation raw materials are commercially available products well-known to those skilled in the art.

[0044] Example 1 Preparation of aconite extract

[0045] The present invention uses aconite from Sichuan Province, and it is confirmed that the retention rate of aconitum alkaloids is the highest (determined by HPLC ≥ 85%), and the aconite extract is prepared according to the above ratio.

[0046] Specifically, the method for preparing the aconite extract includes:

[0047] (1) Raw material treatment: The aconite medicinal materials are washed, dried at 60°C at low temperature, and pulverized to obtain ultrafine pulverization (particle size ≤ 50 μm).

[0048] (2) Extraction process: The ultrafine pulverization is subjected to water extraction in water at 80°C, where the solid-liquid ratio is 1:10 (w / v), and extraction is carried out 2 times (2 hours each time), and the filtrates after extraction are combined.

[0049] (3) Concentration and drying: The filtrate was concentrated under reduced pressure to a density of 1.2 g / mL and then spray-dried (inlet air temperature 160 °C, outlet air temperature 80 °C) to obtain a light brown powder (yield ≥ 18%).

[0050] Example 2 Detection of in vitro antiviral activity

[0051] Experimental design: Vero-E6 cells were divided into a blank control group, a virus control group, and a drug group (10 - 100 μg / mL). Among them: Vero-E6 cells were used as the experimental model for cell culture. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum.

[0052] Virus infection: The vaccinia virus (VACV-WR) strain was inoculated into Vero-E6 cells at a multiplicity of infection (MOI) of 0.1.

[0053] Drug treatment: After virus infection, different concentrations of the compound aconite extract (10 μg / mL, 50 μg / mL, 100 μg / mL) were added respectively and cultured for 48 hours.

[0054] Results: Figure 1A This is the first effect experimental diagram of the aconite extract against the vaccinia virus Western Reserve (VACV-WR) strain on Vero-E6 cells. By detecting the RNA level of the E9L gene of the VACV-WR strain, the inhibitory effect of the aconite extract on the virus was observed. Figure 1B This is the second effect experimental diagram of the aconite extract against the vaccinia virus Western Reserve (VACV-WR) strain on Vero-E6 cells. By detecting the virus titer of the VACV-WR strain, the inhibitory effect of the aconite extract on the virus was observed. As Figure 1A and Figure 1B shown, the aconite extract (100 μg / mL) decreased the virus replication rate by 89.7% (qPCR was used to detect viral DNA, **p < 0.01), and the effect was better than that of cidofovir (decrease of 72.4%).

[0055] Figure 2A and Figure 2B are the observations of the inhibitory effect of the aconite extract on the virus by confocal fluorescence microscopy to observe the number of positive virus cells. Figure 3A This is the fifth effect experimental diagram of the aconite extract against the vaccinia virus Western Reserve (VACV-WR) strain at the mouse level; by detecting the RNA level of the E9L gene of the VACV-WR strain in mouse lung tissue, the inhibitory effect of the aconite extract on the virus was observed. Figure 3BThis is the sixth effect experiment diagram of the anti-Vaccinia virus Western Reserve (VACV-WR) strain of aconite extract at the mouse level. By detecting the virus titer of the VACV-WR strain in the lung tissue of mice, the inhibitory effect of aconite extract on the virus was observed. As Figure 2A , Figure 2B , Figure 3A and Figure 3B shown, by gPCR detection and fluorescence confocal microscopy observation of virus replication, the results showed that aconite extract significantly inhibited the replication of the Vaccinia virus (VACV-WR) strain.

[0056] Example 3 Mouse Model Verification

[0057] Animal grouping: BALB / c mice (n = 40) were randomly divided into 4 groups: normal group, infection group, extract group (100 mg / kg·d). Among them:

[0058] Animal model: BALB / c mice were used as experimental animals and inoculated with the Orthopoxvirus (VACV-WR) strain.

[0059] Drug treatment: After virus infection, compound aconite extract (100 mg / kg) was orally administered daily for 7 consecutive days.

[0060] Results: The survival rate of the extract group reached 90%, the viral load (lung tissue) decreased by 3.2 log10 (**p < 0.01), and there was no liver and kidney toxicity (serum ALT / AST normal).

[0061] Figure 4A This is the observation diagram of the Western bloting experiment of the anti-Vaccinia virus Western Reserve (VACV-WR) strain of aconite extract on Vero-E6 cells; Figure 4B This is the data diagram of the Western bloting experiment of the anti-Vaccinia virus Western Reserve (VACV-WR) strain of aconite extract on Vero-E6 cells. By Western blot detection of the viral load, the results showed that aconite extract significantly reduced the viral load on Vero-E6 cells.

[0062] Example 4 Molecular Mechanism Analysis

[0063] Signaling pathway: By Western blot and qPCR techniques, the activation of signaling pathways such as cGAS-STING in HeLa cells treated with aconite extract was detected.

[0064] The experimental results showed that the mRNA expressions of IFNB1, ISG15, ISG20, and STING in the extract treatment group were up-regulated by 31, 13, 12, and 13 times respectively (qPCR), and the protein expression level of STING was significantly increased (Western blot). Figure 5A Figure for observing the effect of aconite extract on activating the IFNB of the cGAS-STING pathway in HeLa cells; Figure 5B Figure for observing the effect of aconite extract on activating the ISG15 of the cGAS-STING pathway in HeLa cells; Figure 5C Figure for observing the effect of aconite extract on activating the ISG20 of the cGAS-STING pathway in HeLa cells; Figure 5D Figure for observing the effect of aconite extract on activating the STING of the cGAS-STING pathway in HeLa cells; Figure 5E Figure for observing the effect of aconite extract on the mRNA level of the cGAS-STING pathway and the protein level of STING in HeLa cells. As Figure 5A 、 Figure 5B 、 Figure 5C 、 Figure 5D and Figure 5E shown, after stimulating HeLa cells with aconite (10 ng) for 12 hours, the activation of the cGAS-STING pathway was detected by RT-PCR. The results showed that aconite could significantly up-regulate the mRNA levels of IFNB, ISG15, ISG20, STING and the protein level of STING. Aconite extract may enhance the innate immune response of host cells by activating the cGAS-STING signaling pathway, thereby inhibiting virus replication.

[0065] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of aconite extract in orthopoxvirus inhibitors.

2. The application according to claim 1, wherein The orthopoxviruses include monkeypox virus, vaccinia virus, cowpox virus, smallpox virus, camelpox virus, ectromelia virus, and African gerbilpox virus.

3. The application according to claim 1, wherein The aconite extract can damage the envelope of orthopoxviruses.

4. The application according to claim 1, characterized in that The aconite extract activates the cGAS-STING signaling pathway.

5. The application according to claim 1, wherein The aconite extract is prepared by a water extraction method.

6. The application according to claim 5, wherein The steps of the water extraction method include: S1. Wash, dry, and pulverize aconite to obtain aconite granules; S2. Perform water extraction on the aconite granules to obtain a filtrate; S3. Concentrate and dry the filtrate to obtain aconite extract.

7. The application according to claim 6, wherein The particle size of the aconite granules is less than or equal to 50 μm.

8. The application according to claim 6, wherein The steps of the water extraction include: Put the aconite granules into water and extract by heating; wherein, the solid-liquid ratio is 1:10 - 30 (w / v), and the heating temperature is 80 - 100 °C.

9. The application according to claim 6, characterized in that, The density of the concentrated filtrate is 1.0 - 1.4 g / mL.

10. Application of aconite extract in the medicament for treating orthopoxviruses in the application according to any one of claims 1 - 9.

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