Application of apigenin-7-O-glucuronide in preparation of medicine for treating coronavirus

Abstract

The invention discloses application of apigenin-7-O-glucuronide in preparation of a medicine for treating coronavirus, and belongs to the technical field of pharmacology of traditional Chinese medicines. The application comprises the application of the apigenin-7-O-glucuronide serving as the RIG-I protein agonist and the application of the apigenin-7-O-glucuronide in preparation of the medicine for treating the coronavirus. Experiments prove that the apigenin-7-O-glucuronide has a good inhibition effect on the coronavirus by being used as the RIG-I protein agonist, and an experimental basis is provided for the apigenin-7-O-glucuronide to prevent and treat the coronavirus infection.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine pharmacology technology, and relates to the use of apigenin-7-O-glucuronide as a RIG-I protein agonist, specifically the application of apigenin-7-O-glucuronide in the preparation of drugs for treating coronaviruses. Background Technology

[0002] Members of the β-coronavirus genus (including SARS-CoV, MERS-CoV, and SARS-CoV-2) pose a persistent threat to public health due to their high pathogenicity and cross-species transmission characteristics. SARS-CoV-2, as a single-stranded positive-sense RNA virus, utilizes structural proteins (such as the spike protein S), accessory proteins (such as ORF6), and non-structural proteins (such as nsp1) to synergistically mediate immune evasion. For example, ORF6 inhibits the type I interferon (IFN-I) signaling pathway, leading to early failure of antiviral defenses. Clinical studies have confirmed that severely ill COVID-19 patients exhibit a delayed IFN-I response, a deficiency directly related to elevated viral load, cytokine storm, and lymphopenia. Early IFN-β treatment can significantly reduce viral load and shorten viral shedding time by 3 days in moderate cases. Therefore, enhancing the type I interferon response and improving the host's innate immune response are crucial for the prevention and treatment of coronavirus infection.

[0003] The Xuanfei Baidu Formula is composed of 13 traditional Chinese medicines, including ephedra, bitter almond, and gypsum. It is derived from five classic formulas, such as Ma Xing Shi Gan Tang, and has the effects of clearing the lungs and resolving dampness, clearing heat and expelling pathogens, and purging lung toxins. Pharmacological studies have confirmed that it alleviates acute lung injury by inhibiting the NF-κB pathway and relieves pulmonary fibrosis by blocking IL-6 / STAT3 signaling. Its active ingredient, verbascoside, can inhibit the activity of the SARS-CoV-2 main protease (Mpro). Sixty-two components were identified and compared with standards in the serum and lung tissue of treated rats. Apigenin-7-O-glucuronide is one of them.

[0004] Apigenin-7-O-glucuronide (A7G) is a component of verbena in the formula for clearing the lungs and detoxifying. Verbenae Herba ) and patchouli ( Pogostemonis Herba The key active ingredient of ) has been confirmed by previous studies to inhibit α-glucosidase activity (IC50). 50 = 8.3 μM) and regulate oxidative stress (increasing SOD activity by 2.1 times) and have anti-inflammatory effects (inhibiting TNF-α secretion by 67%). However, the inhibitory ability and mechanism of action of this compound against coronaviruses such as SARS-CoV-2 have not yet been clarified. Summary of the Invention

[0005] The purpose of this invention is to provide a novel use of apigenin-7-O-glucuronide in the preparation of drugs for treating coronaviruses, in order to overcome the shortcomings of the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Firstly, the application of apigenin-7-O-glucuronide in the preparation of RIG-I agonists.

[0007] The molecular formula of the apigenin-7-O-glucuronide is C2. 21 H 18 O 11 The structural formula is shown below: ; Secondly, the present invention also provides the use of apigenin-7-O-glucuronide in the preparation of drugs for treating coronaviruses.

[0008] Furthermore, the coronavirus is SARS-CoV-2.

[0009] Furthermore, the mass percentage of apigenin-7-O-glucuronide in the drug is ≥98%.

[0010] Furthermore, the drug also includes medically acceptable drug excipients or carriers.

[0011] Furthermore, the pharmaceutical excipients include one or more of fillers, binders, disintegrants, lubricants, and surfactants, and the carrier includes one or more of sodium carboxymethyl cellulose, glycerin, gelatin, and Tween-80.

[0012] Furthermore, the fillers include, but are not limited to, starch, lactose, dextrin, mannitol, glucose, cellulose, and inorganic salts; the binders include, but are not limited to, starch paste and polyvinylpyrrolidone; the disintegrants include, but are not limited to, dry starch, effervescent disintegrants, anhydrous sodium carbonate, glycine, tartaric acid, magnesium aluminum silicate, microcrystalline wax, calcium carboxymethyl cellulose, and potassium dihydrogen phosphate; and the lubricants include, but are not limited to, talc, silica, magnesium stearate, and micronized silica gel.

[0013] Furthermore, the dosage form of the drug includes tablets, granules, pills, emulsions, capsules, injections, suspensions, or solutions.

[0014] Furthermore, the routes of administration for the above-mentioned drugs include intravenous, oral, sublingual, intramuscular or subcutaneous, and skin / mucous membrane administration.

[0015] Thirdly, RIG-I agonists drive an immune response by inducing the expression of type I interferon / pro-inflammatory cytokines, thereby inhibiting tumor growth. Based on this mechanism, apigenin-7-O-glucuronide, as a RIG-I agonist, can be used to prepare drugs that inhibit tumor growth, including but not limited to: melanoma, pancreatic cancer, colorectal cancer, and non-small cell lung cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses for the first time the use of apigenin-7-O-glucuronide as a RIG-I protein agonist. Severe coronavirus infection patients have delayed type I interferon response, leading to excessive viral load and excessive inflammatory response. Experiments have shown that apigenin-7-O-glucuronide has a significant agonistic effect on RIG-I, thereby having a good inhibitory effect on coronavirus. This provides experimental evidence for the clinical application of apigenin-7-O-glucuronide in the prevention and treatment of coronavirus. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments; Figure 1 This is a diagram showing the experimental results of the agonistic effect of apigenin-7-O-glucuronide on RIG-I protein. Figure 1 (A) in the figure is a statistical graph showing the mRNA levels of IFNα, IFNβ, and interferon-stimulated genes in each group. Figure 1 (B) in the figure is a statistical graph showing the activation level of IFNβ-luc in each group after treatment with RIG-I, MDA-5, cGAS, and STING agonists, respectively. Figure 1 (C) in the diagram represents the docking experiment between apigenin-7-O-glucuronide and RIG-I molecules; Figure 2 This is a graph showing the results of the RIG-I and SARS-CoV-2 ORF6 overexpression experiments. Figure 2 (A) in the figure is a statistical graph of the activation level of IFNβ-luc in each group. Figure 2 (B) in the figure is a statistical graph of interferon mRNA levels in each group; Figure 3 This is a graph showing the inhibitory effect of apigenin-7-O-glucuronide on coronavirus; Figure 4 This is a graph showing the results of a cytotoxicity test on apigenin-7-O-glucuronide. Detailed Implementation

[0018] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, specific embodiments are described below to further illustrate the invention. However, the following embodiments are merely preferred embodiments of the invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without prior creative effort are all within the protection scope of this invention.

[0019] The raw material, apigenin-7-O-glucuronide, was purchased from Chengdu Desite Biotechnology Co., Ltd., with a compound purity of ≥98%. Other raw materials were ordinary commercially available products.

[0020] Example 1: Agonizing effect of apigenin-7-O-glucuronide on RIG-I protein.

[0021] (1) Experiment on the induction effect of natural interferon.

[0022] With a density of 3 × 10 5 RAW264.7 cells / mL and RPMI 1640 medium suspension were seeded into 12-well sterile culture plates, 1 mL per well, and cultured at 37℃ and 5% CO2 for 24 hours. The supernatant was discarded, and the control group was treated with fresh medium. The treatment group was treated with apigenin-7-O-glucuronide (final concentration 100 μM) for 8 hours. The effect of apigenin-7-O-glucuronide on the mRNA levels of IFNα, IFNβ and interferon-stimulated genes (ISG15, IFIT1, Mx1, Mx2, OAS1) was detected by qPCR.

[0023] like Figure 1 As shown in (A), after intervention with apigenin-7-O-glucuronide, the expression of IFNα, IFNβ and interferon-stimulated genes (ISG15, IFIT1, Mx1, Mx2, OAS1) mRNA increased, indicating that apigenin-7-O-glucuronide can promote the expression of interferon and interferon-related genes.

[0024] (2) Exploration experiment on the target of apigenin-7-O-glucuronide.

[0025] With a density of 6 × 10 5IFNβ promoter-Luciferase-THP-1 cells (cells / mL) and a suspension of diluted PMA in RPMI 1640 medium were seeded into 96-well sterile white culture plates. The concentration of PMA was controlled at 100 ng / mL, with 100 μL added to each well. The plates were incubated at 37°C and 5% CO2 for 24 hours. The supernatant was discarded. Fresh culture medium was added to the control and model groups, while the drug treatment groups were pre-treated with apigenin-7-O-glucuronide (final concentrations of 10 μM and 50 μM) solution for 24 hours. After 24 hours, low molecular weight poly(I:C), high molecular weight poly(I:C), HT-DNA, and cGAMP were added to the model and drug treatment groups, respectively, for 24 hours to construct RIG-I, MDA-5, cGAS, and STING activating models. Luciferase activity was detected using a luciferase reporter gene assay kit. Relative activation level = OD treatment group / OD control group.

[0026] like Figure 1 As shown in (B), the IFNβ promoter-luciferase activity was significantly enhanced in the RIG-I agonist group, and luciferase activity was further significantly increased after administration of apigenin-7-O-glucuronide. No significant differences were observed in other models.

[0027] (3) Apigenin-7-O-glucuronide docking experiment with RIG-I molecules.

[0028] First, the protein structure was obtained from the RCSB PDB database. The chemical structure of A7G was downloaded from PubChem and converted to PDB format using OpenBabel 3.1.1. Then, the protein and ligand were preprocessed using AutoDockTools 1.5.7, including removing water molecules, adding hydrogen atoms and partial charges, defining rotatable bonds (roots), and saving as PDBQT format. A grid box covering the protein's active site was set, and a Vina configuration file (config.txt) was generated. After adding key parameters (exhaustiveness = 24, num_modes = 20), docking calculations were performed using QuickVina-W. Finally, the two-dimensional interaction and three-dimensional binding conformation of the ligand-protein were visualized and analyzed using LigPlus 2.2 and PyMol 2.5, respectively.

[0029] like Figure 1 As shown in (C), apigenin-7-O-glucuronide has the lowest binding energy with RIG-I and can form six hydrogen bonds and some hydrophobic interactions with the DECH-box domain of RIG-I, showing potential for binding.

[0030] (4) RIG-I and SARS-CoV-2 ORF6 overexpression test.

[0031] With a density of 6 × 10 5 Cells / mL of IFNβ promoter-Luciferase-THP-1 or THP1-1 cells and a suspension of diluted PMA in RPMI 1640 medium were seeded into sterile culture plates. The concentration of PMA was controlled at 100 ng / mL. 100 μL / 96-well plate or 1 mL / 12-well plate was added to each well, and the plates were cultured at 37℃ and 5% CO2 for 24 hours. The plasmids pLV3-CMV-RIG-I-3×FLAG-Puro and SARS-CoV-2 ORF6 pLVX-EF1α-ORF6-2×Strep-IRES-Puro (500 ng each) were co-transfected using liposome transfection reagent. 24 hours after transfection, apigenin-7-O-glucuronide (final concentrations of 10 μM and 50 μM) was added, and the plates were cultured for another 24 hours for efficacy analysis.

[0032] like Figure 2 As shown in (A) and (B), in RIG-I and SARS-CoV-2 ORF6 overexpressing macrophages, apigenin-7-O-glucuronide can further enhance interferon promoter activity and promote the expression of interferon and its downstream genes.

[0033] Example 2: Inhibitory effect of apigenin-7-O-glucuronide on coronavirus SARS-CoV-2 (XBB subtype).

[0034] (1) Anti-coronavirus test.

[0035] The experiment was conducted in a BSL-3 (Biosafety Level 3) laboratory.

[0036] With a density of 2 × 10 5Vero E6 cells / mL and DMEM medium suspension were seeded into 96-well sterile culture plates, 100 μL per well, and cultured at 37°C and 5% CO2 for 24 hours. Coronavirus solution (Genebank accession no. MT123290.1) was diluted with DMEM medium to 100 TCID50. Simultaneously, the drug was diluted with DMEM medium, and an equal volume (100 μL: 100 μL) of virus solution and diluted drug solution were mixed and incubated together for 1 to 2 hours. The supernatant from the 96-well cell culture plates was then discarded. For the experimental group, 100 μL of the mixed drug and virus solution was added per well; for the virus control group, 100 μL of the 100 TCID50 virus solution was added; and for the blank control group, 100 μL of DMEM medium was added. The plates were incubated at 37°C and 5% CO2 for 2 hours. Discard the culture supernatant, add 100 μL of DMEM medium to each well, set up 3 replicates, and incubate for 3 days as usual.

[0037] Cytopathic effect (CPE) was observed under an optical microscope. The degree of cytopathic effect was recorded according to the following 6-level standard: "-" indicates no cytopathic effect; "±" indicates less than 10% cytopathic effect; "+" indicates approximately 25% cytopathic effect; "++" indicates approximately 50% cytopathic effect; "+++" indicates approximately 75% cytopathic effect; and "++++" indicates more than 75% cytopathic effect.

[0038] The half-maximal inhibitory concentration (IC50) was calculated using the Reed-Muench method. 50 ).

[0039] Experimental results are as follows Figure 3 As shown, apigenin-7-O-glucuronide had a half-maximal inhibitory concentration (IC50) of 26.25 μM against coronavirus, indicating a good anti-coronavirus effect.

[0040] (2) Detection of drug toxicity of apigenin-7-O-glucuronide.

[0041] With a density of 1 × 10 4 Vero cell suspension at a concentration of 100 μL / mL was seeded into 96-well sterile culture plates and cultured at 37°C and 5% CO2 for 24 hours. The supernatant was discarded. For the experimental groups, 100 μL of serially diluted (50, 25, 12.5, 6.25, 3.125 μM) apigenin-7-O-glucuronide was added, while for the blank control group, 100 μL of DMEM medium was added. Incubation continued for 72 hours.

[0042] After incubation, the supernatant of the 96-well cell culture plate was discarded, and 100 μL of CCK-8 solution diluted in DMEM was added to each well. The plates were then incubated at 37°C and 5% CO2 for 1 hour. The absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (Sample A - Blank A) / (Blank A - Blank A) * 100.

[0043] Experimental results are as follows Figure 4 As shown, apigenin-7-O-glucuronide showed no significant cytotoxicity within 72 hours.

[0044] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Application of apigenin-7-O-glucuronide in the preparation of RIG-I protein agonists.

2. The application as described in claim 1, characterized in that, The RIG-I protein agonist is prepared for use in the treatment of coronavirus.

3. Application of apigenin-7-O-glucuronide in the preparation of drugs for treating coronaviruses.

4. The application as described in claim 3, characterized in that, The coronavirus in question is SARS-CoV-2.

5. The application as described in claim 3, characterized in that, The mass percentage of apigenin-7-O-glucuronide in the drug is ≥98%.

6. The application as described in claim 3, characterized in that, The drug also contains medically acceptable drug excipients or carriers.

7. The application as described in claim 6, characterized in that, The drug excipients include one or more of fillers, binders, disintegrants, lubricants, and surfactants, and the carriers include one or more of sodium carboxymethyl cellulose, glycerin, gelatin, and Tween-80.

8. The application as described in claim 3, characterized in that, The dosage forms of the drug include tablets, granules, pills, emulsions, capsules, injections, suspensions, or solutions.

9. The application of apigenin-7-O-glucuronide in the preparation of drugs that inhibit tumor growth, characterized in that, The apigenin-7-O-glucuronide is used as a RIG-I protein agonist.

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