Formulations for use against african swine fever virus and uses thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a preparation for resisting African swine fever virus and application thereof.The preparation is obtained by carbonizing kaempferol in an alkaline environment;the particle size of the kaempferol carbon dots is 1nm-20nm;and the solubility of the kaempferol carbon dots in water is 0.1mg / mL-0.5mg / mL.The kaempferol carbon dots obtained by a solvothermal method have significantly improved water solubility and biocompatibility, reduced cytotoxicity, and are more suitable for use in a drug delivery system.The kaempferol carbon dots have a significant inhibitory effect on African swine fever virus, can reduce virus titer and reduce virus protein expression.The kaempferol carbon dots can inhibit the TLR4 signaling pathway, reduce the production of inflammatory cytokines, and remove active oxygen through SOD-like activity, thereby playing an antiviral role.

Description

Preparations for combating African swine fever virus and their applications Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a preparation for combating African swine fever virus and its application. Background Technology

[0002] African swine fever (ASF) is a highly contagious and deadly disease of pigs caused by the African swine fever virus (ASFV), resulting in enormous economic losses to the global pig farming industry. Although vaccine development efforts are ongoing, no effective vaccine has yet been approved for use. Therefore, developing novel anti-ASFV drugs is of paramount importance.

[0003] Traditional Chinese medicine has garnered attention in the medical community due to its natural sources, diverse composition, and minimal side effects. Kaempferol, a tetrahydroxyflavonoid extracted from various vegetables and fruits, also known as kaempferol 3, kaempferol flavonol, or kaempferol flavonol, belongs to the dietary flavonoid class. It primarily originates from the rhizome of the ginger family plant *Kaempferia galanga* and is widely found in various fruits, vegetables, and natural plants, making it a very common compound in nature. Kaempferol acts on numerous signaling targets in vivo, possessing broad pharmacological activities such as antitumor, anti-inflammatory, antioxidant, cardioprotective, and hepatoprotective effects. However, its poor water solubility and high cytotoxicity limit its application in antiviral therapy. The development of nanotechnology has offered possibilities to improve these shortcomings of kaempferol. Carbon dots (CDs), due to their small particle size, high chemical stability, and good biocompatibility, have shown great potential in antiviral research. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a formulation for combating African swine fever virus and its application. The specific technical solution is as follows.

[0005] An antiviral agent for African swine fever virus, wherein the agent is obtained by carbonizing kaempferol in an alkaline environment;

[0006] The pH value of the alkaline environment is 9~10, the carbonization temperature is 180℃~185℃, and the time is 8h~9h;

[0007] The particle size of the formulation is 1 nm to 20 nm;

[0008] The solubility of the preparation in water is 0.1 mg / mL to 0.5 mg / mL.

[0009] In another preferred embodiment, the formulation further includes purification after carbonization; the specific purification process is as follows: after carbonization, the supernatant is collected by centrifugation, filtered, and dialyzed using a 1000 Da dialysis membrane.

[0010] A second aspect of the present invention provides the use of the aforementioned formulation in the preparation of antiviral inhibitors.

[0011] In another preferred embodiment, the antiviral inhibitor refers to an inhibitor against African swine fever virus.

[0012] In another preferred embodiment, the inhibitor is a lyophilized powder or solution.

[0013] In another preferred embodiment, the lyophilized powder is obtained by directly freeze-drying the kaempferol carbon dots.

[0014] In another preferred embodiment, the concentration of the kaempferol carbon dots in the solution is 0.1 mg / mL to 0.5 mg / mL.

[0015] In another preferred embodiment, the solvent of the solution is water.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention improves the solubility of kaempferol by carbonizing it under alkaline conditions, achieving a water solubility of 0.1 mg / mL to 0.5 mg / mL compared to regular kaempferol. This significantly enhances kaempferol's biocompatibility and reduces cytotoxicity, making it more suitable for drug delivery systems. The carbonized kaempferol formulation in this invention exhibits significant inhibitory effects against African swine fever virus, reducing viral titers and viral protein expression. Kaempferol carbon dots exert their antiviral effect by inhibiting the TLR4 signaling pathway, reducing the production of inflammatory cytokines, and scavenging reactive oxygen species through SOD-like activity. This invention combines traditional Chinese medicine with nanotechnology, providing new ideas and methods for research on combating African swine fever virus. Attached Figure Description

[0018] Figure 1 shows the synthesis diagram of kaempferol carbon dots.

[0019] Figure 2 shows the transmission electron microscope (TEM) image and size distribution of kaempferol carbon dots; (A) is the TEM image with a scale bar of 20 nm; (B) is the size distribution.

[0020] Figure 3 shows the infrared spectrum of kaempferol carbon dots.

[0021] Figure 4 shows the XPS spectra of carbon dots of kaempferol, where (A) is the full scan XPS spectrum and (B) is the high-resolution C1s XPS spectrum.

[0022] Figure 5 shows the effect of different concentrations of kaempferol carbon dots on the anti-ASFV activity of red blood cells adsorbed on the results; (A) shows the anti-ASFV activity of different concentrations of kaempferol carbon dots; (B) shows the effect of different concentrations of kaempferol carbon dots on the change of ASFV particle number.

[0023] Figure 6 shows the analysis results of kaempferol carbon dots on ASFV-infected cells; (A) shows the immunoblotting analysis results of ASFV-infected PAM cells, and (B) shows the RT-qPCR detection of different concentrations of kaempferol carbon dots on ASFV-infected PAM cells.

[0024] Figure 7 shows the results of the analysis of the regulatory effect of kaempferol carbon dots on the TLR4 signaling pathway; (A) shows the results of Western blotting detection of the expression levels of TLR4, MyD88 and NF-κB proteins after KPL-CDs treatment; (B) shows the results of IL-1β expression analysis; (C) shows the results of IL-6 expression analysis; (D) shows the results of IL-18 expression analysis; and (E) shows the results of TNF-α expression analysis.

[0025] Figure 8 shows the antioxidant capacity results of kaempferol carbon dots; (A) shows the total antioxidant capacity results of kaempferol carbon dots verified by the kit, and (B) shows the results of in vitro experiments verifying the ability of kaempferol carbon dots to scavenge reactive oxygen species. In the figure, b is a photograph of the in vitro experimental results, and MB is the mitochondrial superoxide probe. Detailed Implementation

[0026] The technical solutions of this invention will be clearly and completely described in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0027] Unless otherwise specified, the methods described in the various embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available.

[0028] Kaempferol was purchased from Amex Chemicals; alveolar macrophages were prepared from the lavage fluid of 6-week-old specific pathogen-free (SPF) piglets.

[0029] Example 1: Preparation of an anti-African swine fever virus formulation.

[0030] Dissolve 100 mg of kaempferol in 10 mL of deionized water, and add 0.2 mM NaOH aqueous solution dropwise to adjust the pH of the solution to 9.0.

[0031] The solution was sonicated at 40 kHz for 10 minutes, then transferred to a 25 mL polytetrafluoroethylene-lined high-pressure reactor. The reactor was heated to 180 °C and maintained for 8 hours. The mixture was then allowed to cool naturally to room temperature.

[0032] The mixture was centrifuged at 8000 rpm for 10 minutes to remove large-particle precipitates, and the supernatant was collected. The supernatant was filtered through a 0.22-micron filter membrane to remove smaller-particle precipitates. Deionized water was dialyzed through a dialysis membrane with a molecular weight cutoff of 1000 Daltons for 24 hours, with the deionized water replaced every 6 hours. The dialysate was collected and freeze-dried for 24 hours to obtain kaempferol carbon dots, denoted as KPL-CDs.

[0033] The prepared KPL-CDs were structurally characterized, and the transmission electron microscopy (TEM) image is shown in Figure 2. These carbon dots exhibit a nearly spherical morphology with an average diameter of 8.53 ± 1.61 nm. The infrared spectrum is shown in Figure 3, at 3318 nm. -1 1624m -1 1550m -1 1260m -1 and 1010m -1 There are five distinct absorption peaks at C1s, corresponding to OH, C=O, C=C, CO, and CC, respectively. The full-scan XPS spectrum of KPL-CDs is shown in Figure 4. In the composition of KPL-CDs, carbon accounts for 64.02% and oxygen accounts for 35.98%. Its high-resolution XPS spectrum in the C1s region can be decomposed into three peaks. The peak at 284.8 eV is attributed to the C−C / C=C bond, while the other two peaks are located at 286.3 eV and 288.2 eV, respectively, corresponding to the CO and C=O bonds.

[0034] Example 2: Antiviral activity test of KPL-CDs.

[0035] Alveolar macrophages (PAMs) were seeded in 24-well plates and incubated with ASFV (MOI=0.3) at 37°C for 48 hours. KPL-CDs were added to the culture medium at concentrations of 0.005 mg / mL, 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 0.5 mg / mL.

[0036] The supernatant was collected, and viral load was detected by qPCR, while viral titer was detected by HAD50 assay. Cells were collected and analyzed by Western blotting and RT-qPCR to detect the expression levels of p72 protein and B646L gene in ASFV.

[0037] As shown in Figure 5, within the concentration range of 0.005 mg / mL to 0.5 mg / mL, the viral titer decreased in a dose-dependent manner after treatment with KPL-CDs. At the highest concentration of 0.5 mg / mL, the viral titer decreased from 5.02 log... 10 HAD50 / mL decreased to 1.71 log 10 HAD50 / mL was reduced by approximately 66%.

[0038] Western blot analysis and immunofluorescence assays, as shown in Figure 6, revealed that KPL-CDs significantly reduced the expression level of ASFV p72 protein. At a concentration of 0.1 mg / mL, p72 protein expression was reduced by approximately 90% compared to the ASFV control group. RT-qPCR analysis showed that KPL-CDs significantly reduced the transcriptional level of the B646L gene in ASFV in a dose-dependent manner. At a concentration of 0.1 mg / mL, B646L gene expression was reduced by approximately 85% compared to the ASFV control group.

[0039] The results above show that KPL-CDs can significantly reduce viral titer and viral protein expression, and have good antiviral activity.

[0040] Example 3: The moderating effect of KPL-CDs on the TLR4 signal path.

[0041] Western blotting was used to detect the expression levels of TLR4, MyD88, and NF-κB proteins after KPL-CDs treatment. p-P65, a key transcription factor in the NF-κB signaling pathway, was chosen to characterize the expression levels of NF-κB proteins. RT-qPCR was used to detect the expression levels of inflammatory cytokines IL-1β, IL-6, and TNF-α.

[0042] Western blot analysis, as shown in Figure 7(A), revealed that KPL-CDs significantly downregulated the expression levels of TLR4 and MyD88. At a concentration of 0.5 mg / mL, the expression levels of TLR4 and MyD88 were reduced by approximately 70% and 60%, respectively, compared to the ASFV infection group. RT-qPCR analysis, as shown in Figures 7(B)–(E), indicated that KPL-CDs significantly inhibited the expression of inflammatory cytokines such as IL-1β, IL-6, TNF-α, and IL-18. At a concentration of 0.5 mg / mL, the expression levels of IL-1β, IL-6, and TNF-α were reduced by approximately 80%, 75%, and 70%, respectively, compared to the ASFV infection group. These results demonstrate that KPL-CDs can significantly downregulate the expression of proteins related to the TLR4 signaling pathway and reduce the production of inflammatory cytokines.

[0043] Example 4: Antioxidant capacity test of KPL-CDs.

[0044] Changes in intracellular reactive oxygen species (ROS) levels after KPL-CDs treatment were detected using the DCFH-DA fluorescent probe. The antioxidant capacity of KPL-CDs was assessed using a total antioxidant capacity assay kit. As shown in Figure 8, KPL-CDs significantly reduced intracellular ROS levels, demonstrating good antioxidant capacity.

[0045] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A preparation for treating African swine fever virus, characterized in that, The formulation is obtained by carbonizing kaempferol in an alkaline environment; the pH value of the alkaline environment is 9~10, the carbonization temperature is 180℃~185℃, and the time is 8h~9h; the particle size of the formulation is 1nm~20nm; the solubility of the formulation in water is 0.1mg / mL~0.5mg / mL.

2. The preparation for treating African swine fever virus according to claim 1, characterized in that, The formulation also includes purification after carbonization; the specific purification process is as follows: after carbonization, the supernatant is collected by centrifugation, filtered, and dialyzed with a 1000Da dialysis membrane.

3. The use of the formulation according to claim 2 in the preparation of antiviral inhibitors, characterized in that, The antiviral inhibitors mentioned refer to inhibitors against African swine fever virus.

4. The application according to claim 3, characterized in that, The inhibitor is a lyophilized powder or solution.

5. The application according to claim 4, characterized in that, The lyophilized powder is obtained by directly freeze-drying the preparation.

6. The application according to claim 5, characterized in that, The concentration of the preparation in the solution is 0.1 mg / mL to 0.5 mg / mL.

7. The application according to claim 6, characterized in that, The solvent for the solution is water.

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