Hexameric protein-based method for screening of natural product antiviral drugs and use thereof

Abstract

The application discloses a kind of natural product antiviral drug screening method based on hexagon protein and application, belong to active ingredient screening and antiviral drug development field.The method includes: preparation nitrogen-doped mesoporous carbon and silver nanosheet composite solution, drop coating on the surface of pretreated glassy carbon electrode, after drying by activation, hexagon protein incubation, blocking and enzyme label antibody reaction, construct electrochemical sensing electrode;With the electrode, the sample to be screened natural product is incubated, and the current response signal is detected;According to signal change combination inhibition rate formula evaluation antiviral activity.The application takes hexagon protein as recognition target, based on the principle of competitive immunoassay, realizes the specific, high sensitivity detection of antiviral active ingredient in natural product, with the advantages of fast operation, low cost, small sample consumption, high throughput, etc., provides effective technical support for the mining of antiviral active ingredient in natural product such as traditional Chinese medicine.

Description

Technical Field

[0001] This invention relates to the fields of active ingredient screening and novel antiviral drug development, specifically to a method and application for screening natural product antiviral drugs based on hexagonal proteins. Background Technology

[0002] Adenoviruses (AdVs) are a class of non-enveloped, double-stranded DNA viruses. More than 100 serotypes have been identified, with Ad5 being the most common pathogenic subtype in humans. In recent years, the global incidence of AdV infection has shown a significant upward trend. According to data from the World Health Organization (WHO), approximately 5%-10% of respiratory infections in children are caused by AdV; in immunocompromised populations, the mortality rate from disseminated infection can reach as high as 80%. Notably, in the outbreaks of unexplained pneumonia in children across multiple regions of my country in 2023, the detection rate of AdV exceeded 30%, further highlighting the serious threat this virus poses to public health.

[0003] Currently, clinical treatment for Advanced Virus Infection (AdV) infection mainly relies on broad-spectrum antiviral drugs such as cidofovir, whose mechanisms of action include blocking viral adsorption and invasion, inhibiting viral replication enzyme activity, and modulating host immunity. However, these traditional regimens suffer from significant nephrotoxicity and increasing drug resistance, necessitating the development of novel, highly effective, and low-toxicity drugs. The primary step in AdV infection of host cells depends on the hexon protein on its capsid surface. The HVR5 region in the hypervariable region of this protein can directly bind with high affinity to the Coxsackievirus receptor (CAR) on the host cell surface. The interaction between specific receptor-binding domains (such as the HI loop) on the Hexon protein and the CAR is a core step in mediating viral adsorption and initiating the internalization process. Based on this mechanism, if an active ingredient can specifically bind to the key region on the Hexon protein responsible for interacting with the CAR, it can directly block the binding of Hexon and CAR through steric hindrance, thereby effectively preventing infection.

[0004] Natural products are a vital source of new drug discovery. Their structures can serve as excellent lead compounds or be directly developed into drugs, exhibiting unique mechanisms and therapeutic effects distinct from traditional synthetic compounds. In particular, the active components of traditional Chinese medicine (TCM) have become a focus of antiviral drug development due to their multi-target effects and relatively low toxicity. Currently, large-scale primary screening and mechanism validation of active components in TCM mainly rely on chromatographic techniques, affinity screening, and bioactivity-guided screening. However, these conventional techniques have significant limitations: difficulty in detecting trace amounts of active ingredients, easy omission of components acting on non-traditional targets, insufficient throughput in large-scale primary screening, and weak ability to elucidate the mechanisms of multi-component synergistic effects. These factors collectively hinder the efficient discovery of antiviral drugs from TCM.

[0005] In recent years, with the innovation of analytical techniques, electrochemical methods have demonstrated strong competitiveness in the early screening of active components due to their advantages such as real-time performance, sensitivity, small sample volume, and low cost. They are particularly suitable for scenarios with scarce samples and complex matrices, and have been widely used in the evaluation of antioxidant, antibacterial, and antitumor activities. Among them, electrochemical immunosensors combine specific molecular recognition with highly sensitive electrochemical detection, providing a new platform for the screening of natural product activity. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the nephrotoxicity and drug resistance of existing anti-adenovirus drugs, as well as the limitations of screening methods for antiviral active ingredients in natural products. The purpose is to provide a screening method and application of antiviral drugs in natural products based on hexagonal proteins. This method is sensitive, rapid, and low in cost, and can achieve efficient screening of anti-adenovirus active ingredients in natural products.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a method for screening natural product antiviral drugs based on hexagonal proteins, comprising the following steps: (1) A nitrogen-doped mesoporous carbon (NMC) and silver nanosheets (Ag NSs) composite solution was prepared and drop-coated onto the surface of a pretreated glassy carbon electrode (GCE). After drying, a nitrogen-doped mesoporous carbon / silver nanosheet modified glassy carbon electrode (NMC / Ag NSs@GCE) was obtained. (2) The nitrogen-doped mesoporous carbon / silver nanosheet modified glassy carbon electrode (NMC / Ag NSs@GCE) was immersed in an activator solution for activation treatment, and then a hexon protein solution was added for incubation. After washing to remove unbound hexon protein, the unbound sites on the electrode surface were blocked with a blocking solution, and then an enzyme-labeled anti-hexon antibody was added for reaction to obtain an electrochemical sensing electrode (Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE). (3) The natural product sample to be screened is co-incubated with the electrochemical sensing electrode, and the current response signal of the electrochemical sensing electrode is detected by an electrochemical detection system. (4) Based on the change in the current response signal and the formula for calculating the inhibition rate, evaluate the antiviral activity of the sample to be screened and complete the screening of antiviral drugs.

[0008] Further, in step (1), the preparation method of the nitrogen-doped mesoporous carbon (NMC) and silver nanosheet (Ag NSs) composite solution is as follows: 1 mg of NMC is dispersed in 1 mL of ethanol solution and sonicated for 30 min to make it uniformly dispersed to obtain NMC ethanol solution; 0.5 mg of Ag NSs is dispersed in 0.1 wt% polyvinylpyrrolidone (PVP) solution and sonicated for 1 h to make it uniformly dispersed to obtain Ag NSs PVP solution; 1 mL of each of the above two solutions is mixed to obtain NMC / Ag NSs solution, which is then refrigerated at 4℃ for later use.

[0009] Further, in step (1), the glassy carbon electrode (GCE) pretreatment includes: first polishing the chamois with 0.30μm and 0.05μm alumina powders in sequence, then ultrasonically cleaning it with methanol and distilled water respectively, then drying it at room temperature, and then activating it in 0.5M H2SO4 solution by cyclic voltammetry until it is stable. The potential (CV) range of the cyclic voltammetry is -0.2V to 1.5V, and the scan rate is 100mV / s.

[0010] Further, in step (2), the activator solution is an EDC / NHS (1-ethyl-3,3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) solution with a concentration of 50mM / 20mM, and the buffer is PBS (phosphate-buffered saline) at pH 7.4. The activation reaction is carried out at room temperature for 1 hour.

[0011] Further, in step (2), the concentration of the hexon protein solution is 10 μg / mL, the buffer is HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer, and the incubation conditions are 4℃ for 12h; the blocking solution is 1wt% BSA (bovine serum albumin) solution, and the blocking reaction is carried out at room temperature for 1h; the concentration of the enzyme-labeled anti-hexon antibody is 10 μg / mL, the buffer is PBS, and the reaction is carried out at room temperature for 2h.

[0012] Further, in step (3), the electrochemical detection system employs a three-electrode system, with the electrochemical sensing electrode (Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE) as the working electrode, the saturated calomel electrode (SCE) as the reference electrode, and the platinum wire electrode as the counter electrode. Square wave voltammetry is used, and the test solution contains 2.0 mM [Fe(CN)6]. 3- / 4- A mixed solution of 0.1M KCl.

[0013] Further, in step (4), the inhibition rate is calculated as follows: inhibition rate % = [(ΔI0-ΔI1) / ΔI0]×100%, where ΔI0 is the current difference between the peak current measured without a sample and its background current, and ΔI1 is the current difference between the peak current measured with a sample and its background current.

[0014] Furthermore, in step (4), the parameters for electrochemical detection using the square wave voltammetry are as follows: sensitivity 10 -5 Potential range: 0 V to -0.5 V; rest time: 2 s; voltage amplitude: 0.025 V; frequency: 15 Hz.

[0015] The present invention also provides the application of the above screening method in the screening of anti-adenovirus active ingredients in natural products.

[0016] Furthermore, the natural products include the traditional Chinese medicine Scutellaria baicalensis and its extract baicalin.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. The nitrogen-doped mesoporous carbon / silver nanosheet-modified glassy carbon electrode constructed in this invention utilizes the highly ordered pores, large specific surface area, and excellent stability of nitrogen-doped mesoporous carbon, combined with the anisotropic structure and localized surface plasmon resonance characteristics of silver nanosheets, to significantly improve the electrode's conductivity, electron transport capability, and electrochemical active surface area. The electrochemical active surface area of ​​the nitrogen-doped mesoporous carbon / silver nanosheet-modified glassy carbon electrode reaches 0.0688 cm². 2 It surpasses bare glassy carbon electrodes, nitrogen-doped mesoporous carbon-modified glassy carbon electrodes, and silver nanosheet-modified glassy carbon electrodes, providing a foundation for high-sensitivity detection.

[0018] 2. This invention uses hexagonal proteins as molecular recognition targets and, based on the principle of competitive immunoassay, achieves specific recognition of anti-adenovirus active ingredients in natural products. It can effectively avoid interference from impurities and has high screening specificity. The detection limit for the positive control drug cidofovir is 0.025 ng / mL (S / N=3), and the quantitation limit is 0.083 ng / mL (S / N=10). It has high sensitivity and can detect trace amounts of active ingredients.

[0019] 3. The method of this invention is quick and inexpensive, requires only a small amount of sample, and does not require complex pretreatment. It can achieve high-throughput screening, solving the problems of insufficient throughput, cumbersome operation, and high cost of traditional screening methods. At the same time, the method has good stability and repeatability, providing a new analytical means for screening antiviral active components of natural products.

[0020] 4. This invention experimentally verifies that baicalin, an extract of Scutellaria baicalensis, has a significant inhibitory effect on adenovirus in the concentration range of 1–200 μg / mL, with a maximum inhibition rate of 73.3% and an IC50 concentration of [missing value]. 50The concentration was 20 μg / mL, confirming the reliability of the method and providing technical support for the discovery of antiviral active ingredients in traditional Chinese medicine and other natural products. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of a natural product antiviral drug screening method based on hexagonal proteins according to the present invention. Figure 2 Characterization images of NMC / Ag NSs composite material and modified electrode are shown below; (A) FT-IR image; (B) Raman spectrogram; (C) SEM image of Ag NSs@GCE; (D) SEM image of NMC@GCE; (E) SEM image of NMC / Ag NSs@GCE.

[0022] Figure 3 Electrochemical characterization of the NMC / Ag NSs@GCE modified electrode in 2.0 mM potassium ferricyanide / potassium ferrocyanide substrate; (A) CV curves, (a) GCE, (b) Ag NSs@GCE, (c) NMC@GCE, (d) NMC / Ag NSs@GCE; (B) EIS curves; (C) CV curves of NMC / Ag NSs@GCE at scan rates of 10–200 mV / s; (D) Relationship between the logarithm of peak current and the logarithm of scan rate; (E) Chronocoulometric curves; (F) Qt 1 / 2 curve.

[0023] Figure 4 The graph shows the detection performance of the Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE electrochemical sensing electrode for Cidofovir in a 2.0 mM potassium ferricyanide / potassium ferrocyanide substrate. (A) DPV response at different concentrations of Cidofovir (0.1–100 ng / mL); (B) Relationship between different concentrations of Cidofovir and its DPV value; (C) Linear relationship between the logarithm of different concentrations of Cidofovir and its DPV value.

[0024] Figure 5 The graph shows the performance verification of the Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE electrochemical sensing electrode; where (A) selectivity; (B) repeatability; (C) reproducibility; and (D) stability.

[0025] Figure 6The inhibitory activity of Cidofovir on Hexon protein was determined for Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE; (A) Current response of Cidofovir to Hexon protein at different concentrations (0.1–100 ng / mL); (B) Dose-response curves of Cidofovir at different logarithmic concentrations.

[0026] Figure 7 The inhibitory activity of Baicalin was determined for Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE; (A) Standard curve of absorbance measured at 278 nm wavelength; (B) Current response of Baicalin to Hexon; (C) Dose-response curve of Baicalin at different concentrations. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] like Figure 1 As shown, the present invention provides a method for screening natural product antiviral drugs based on hexagonal proteins, comprising the following steps: (1) Prepare a composite solution of NMC and Ag NSs, drop it onto the pretreated GCE surface, and dry it to obtain NMC / Ag NSs@GCE; (2) The NMC / Ag NSs@GCE was immersed in an activator solution for activation treatment, then Hexon protein solution was added dropwise for incubation, unbound Hexon protein was washed away, the unbound sites on the electrode surface were blocked with a blocking solution, and then enzyme-labeled Anti-Hexon was added dropwise for reaction to obtain Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE; (3) The natural product sample to be screened is co-incubated with the electrochemical sensing electrode, and the current response signal of the electrochemical sensing electrode is detected by an electrochemical detection system. (4) Based on the change in the current response signal and the formula for calculating the inhibition rate, evaluate the antiviral activity of the sample to be screened and complete the screening of antiviral drugs.

[0029] Example A. Preparation and characterization of NMC / Ag NSs@GCE 1 mg NMC was dispersed in 1 mL of ethanol solution and sonicated for 30 min to obtain a uniform NMC ethanol solution. 0.5 mg Ag NSs was dispersed in 0.1 wt% polyvinylpyrrolidone (PVP) solution and sonicated for 1 h to obtain an AgNSs PVP solution. 1 mL of each solution was mixed to obtain an NMC / Ag NSs solution, which was then refrigerated at 4°C for later use. GCE was first polished on chamois leather using 0.30 μm and 0.05 μm alumina powders, then ultrasonically cleaned with methanol and distilled water respectively, and dried at room temperature. Next, it was activated to stability in 0.5 M H₂SO₄ solution using cyclic voltammetry. The cyclic voltammetry potential (CV) range was -0.2 V to 1.5 V, and the scan rate was 100 mV / s. 15 μL of NMC / Ag NSs solution was drop-coated onto the treated GCE surface to obtain NMC / Ag NSs@GCE. The solution was dried under an infrared lamp and stored in a refrigerator at 4°C.

[0030] The prepared NMC / Ag NSs@GCE was characterized by infrared spectroscopy, Raman spectroscopy, SEM electron microscopy, and elemental analysis. The results are as follows: Figure 2 As shown, Figure 2 The results showed that NMC and Ag NSs were successfully combined, and the NMC / Ag NSs@GCE modified electrode was successfully prepared.

[0031] Electrochemical characterization was then performed to characterize the electrode's fabrication and performance using electrochemical testing methods. Electrochemical measurements included cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), chrono-coulometric voltammetry (CC), differential pulse voltammetry (DPV), and square wave voltammetry (SWV).

[0032] Among them, the CV method has a measurement sensitivity of 10. -4 The potential range is -0.4 V to 0.8 V, and the scan rate is 0.1 V / s; EIS method: the measurement frequency parameter is 1 Hz to 1 × 10⁻⁶. -6 Hz, rest time 2 s, voltage amplitude 0.005 V. CC method: measurement sensitivity 10 -4 The potential range is 0 V to 0.8 V, the step count is 1, the pulse width is 0.25, the sampling interval is 0.00025 s, and the rest time is 2 s. DPV method: sensitivity is 10. -4 The potential range is 0.1 V to -0.3 V, the rest time is 2 s, the voltage amplitude is 0.05, the pulse period is 0.5 s, and the sampling width is 0.0167 s. The sensitivity of SWV is 10. -5The potential range was 0 V to -0.5 V, the settling time was 2 s, the voltage amplitude was 0.025 V, and the frequency was 15 Hz. The test solution contained 2.0 mM [Fe(CN)6]. 3- / 4- A mixed solution of 0.1 M KCl.

[0033] The results are as follows Figure 3 As shown, Figure 3 The results show that NMC and Ag NSs were successfully combined, and the NMC / Ag NSs@GCE modified electrode was successfully prepared; furthermore, we calculated the electrochemical active surface area of ​​NMC / Ag NSs@GCE ( Figure 3 A) is 0.0688cm 2 It is higher than the naked GCE (0.0084 cm). 2 ), NMC@GCE (0.0679cm) 2 ), Ag NSs@GCE (0.0186 cm) 2 ) and N-Gr / MWCNTs@GCE (0.245cm) 2 The electrochemically active surface area of ​​NMC / Ag NSs is shown in the figure. This data confirms that NMC / Ag NSs possess excellent electron mobility and can achieve a good current response.

[0034] B. Fabrication and performance evaluation of Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE electrochemical sensing electrode The NMC / Ag NSs@GCE electrode was immersed in EDC / NHS solution and reacted at room temperature for 1 h. Then, Hexon protein solution was added dropwise and incubated at 4 °C for 12 h. Unbound protein was washed away, and the electrode was then immersed in 1 wt% BSA solution and blocked at room temperature for 1 h. Finally, enzyme-labeled Anti-Hexon was added dropwise and reacted at room temperature for 2 h to obtain the electrochemical sensing electrode Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE, which was stored at 4 °C for later use.

[0035] The concentration of EDC / NHS solution was 50 mM / 20 mM, and the buffer was PBS at pH 7.4; the concentration of Hexon protein solution was 10 μg / mL, and the buffer was HEPES buffer; the washing was performed 3 times with PBS buffer; the concentration of enzyme-labeled Anti-Hexon was 10 μg / mL, and the buffer was PBS.

[0036] Under optimal experimental conditions, the current response of the electrochemical sensing electrode Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE to Cidovir (0.1–100 ng / mL) was detected using differential pulse voltammetry. The results are as follows: Figure 4As shown. Figure 4 A shows the relationship between different concentrations of Cidofovir (0.1–100 ng / mL) and the corresponding peak DPV current (the potential corresponding to the peak is 0.18 V). Figure 4 Figure B shows that the peak current of the DPV gradually decreases as the concentration of Cidofovir increases. Figure 4 C shows that, within the concentration range of 0.1–100 ng / mL, the peak current (I0.05) is... p The correlation coefficient (R²) showed a good linear relationship with the logarithm of Cidofovir concentration. 2 The value is 0.9894, and the linear regression equation is: I p (μA) = -37.1151LogC Cidofovir The limit of detection (LOD) was +119.5591 (S / N=3), and the limit of quantitation (LOQ) was 0.083 (S / N=10). These results indicate that the electrochemical sensing electrode Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE can accurately detect trace levels of Cidofovir within the therapeutic concentration range, thus ensuring its reliability in drug monitoring or efficacy studies.

[0037] Next, the selectivity, repeatability, reproducibility, and stability of the electrochemical sensing electrode Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE were verified and tested under optimized conditions. The results are as follows: Figure 5 As shown. Figure 5 The results show that the electrochemical sensing electrode Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE has good selectivity, repeatability, reproducibility and stability.

[0038] To verify the reliability of the Hexon / Anti-Hexon / BSA / NMC / Ag NSs@GCE assay for Hexon inhibitory activity under optimized conditions, Cidofovir was used as the positive test drug. The SWV method was employed to determine the inhibitory activity of different concentrations (0.1–100 ng / mL) of Cidofovir against Hexon. The results are as follows: Figure 6 As shown in Table 1, the inhibition rate of cidofovir ranged from 22.1% to 84.1%, and different concentrations of cidofovir exhibited varying degrees of inhibitory effect on hexon. Within a certain concentration range, drug concentration and inhibition rate showed a positive correlation. The equations for the cidofovir inhibition rate versus hexon concentration, obtained by the SWV method, are: y = -6.3787x2 +25.3570x+53.6911 (R) 2 =0.9724), its IC 50 The value was 0.56 ng / mL (2.0 nM). The experimental results indicate that Cidofovir has strong inhibitory activity against Hexon, and it can be further used as a positive control to evaluate the inhibitory effect of baicalin on AdV.

[0039] Table 1. Inhibitory activity of different concentrations of Cidofovir on Hexon protein

[0040] C. Extraction, purification, and inhibitory activity determination of baicalin (1) Baicalin extraction and separation: Aqueous extraction and acid precipitation method was used. 50.0 g of Scutellaria baicalensis powder was weighed into a beaker, 500 mL of water was added, and the mixture was boiled for 30 min. After cooling and filtration, the residue was boiled again with 10 times the amount of water for 30 min. The extracts were combined, and the filtrate was kept at a constant temperature of 70-80℃. 6 M concentrated HCl was added to adjust the pH to 1-2, and the mixture was allowed to stand for 1 day to allow the precipitate to fully form. The precipitate was then filtered, and the yellow precipitate was retained. The filtrate was discarded. The yellow precipitate was added to water and stirred to form a suspension. Then, 20.0% NaOH aqueous solution was added to adjust the pH to 7-7.5. The solution became clear, and then an equal volume of 95% ethanol was added. After stirring, the mixture was allowed to stand for 12 h. Impurities were removed by filtration, and the filtrate was retained. The filtrate was kept at a constant temperature of 80℃, and concentrated HCl was added to adjust the pH to 1-2. Baicalin precipitated. The precipitate was obtained by filtration, washed with 20.0 mL of 95% ethanol, placed in a petri dish, and dried in an oven at 60 °C to constant weight to obtain a relatively pure Baicalin extract.

[0041] (2) Qualitative identification of Baicalin: Chemical colorimetric identification and thin-layer chromatography were used.

[0042] Chemical colorimetric identification: Weigh 0.01g of Baicalin standard and extract and dissolve them in 1.0 mL of ethanol. Add 0.1g of zinc powder and then add two drops of concentrated HCl. Observe the color change. Then, spot the product solution onto filter paper, dry it, and spray it with 1.0% magnesium acetate methanol solution. Observe the control under ultraviolet light at 365nm.

[0043] Thin-layer chromatography identification: Weigh 0.01 g of Baicalin standard and extract separately, add 1.0 mL of methanol (HPLC grade), and sonicate to dissolve, preparing methanol solutions of appropriate concentrations. Spot the solutions onto the same silica gel plate using a capillary tube. Use ethyl acetate, methanol, formic acid, and water (volume ratio 7:2:0.5:0.5) as the developing solvent. After development, allow to dry. Finally, spray with 1.0% ferric chloride aqueous solution and lead acetate solution respectively; the spots appear green and yellowish-brown, respectively. Compare the position and color of the spots.

[0044] (3) Baicalin content determination: Using ultraviolet-visible spectrophotometry, accurately weigh 9.30 mg of Baicalin standard, dissolve it in 50.0% ethanol aqueous solution, and dilute to a 25.0 mL volumetric flask to prepare a Baicalin standard solution with a mass concentration of 0.372 g / L. Pipette 0.75, 1.00, 1.25, 1.50, 1.75, and 2.00 mL of the standard solution into 10.0 mL volumetric flasks, dilute to a volume with 50.0% ethanol aqueous solution, and measure the absorbance at 278 nm using an ultraviolet-visible spectrophotometer to plot a standard curve. Accurately weigh 10.0 mg of Baicalin extract, dissolve it in 50.0% ethanol aqueous solution, and dilute to a 25.0 mL volumetric flask; filter through filter paper, discard the initial filtrate, and pipette 5.0 mL of the subsequent filtrate into a 25.0 mL volumetric flask. A 50.0% ethanol aqueous solution was used as a blank control, and the absorbance was measured at a wavelength of 278 nm. The corresponding concentration was calculated according to the regression equation. The content of baicalin was then calculated from the corresponding concentration. Results calculation: ω (%) = 5ρx × 25 / m × 100 In the formula, ω represents the content of Baicalin (mass fraction %), ρx is the mass concentration of the sample calculated according to the regression equation (g / L), 25 is the volume V (mL) of the Baicalin ethanol solution, and m is the mass of the Baicalin extract (mg).

[0045] (4) Determination of the inhibitory activity of baicalin against hexon protein: Take 6 test tubes and label them A, B, C, D1, D2, D3, D4, and D5 respectively. A is the blank group, without hexon protein, without anti-Hexon (using test base solution instead), and without inhibitor; B is the negative control group, with hexon protein and anti-Hexon added, without inhibitor; C is the positive control group, with hexon protein, anti-Hexon added, and inhibitor added; D1, D2, D3, D4, and D5 are the sample groups, with hexon protein, anti-Hexon added, and different concentrations (1-200 μg / mL) of baicalin solution added. Cidofovir was used as the positive control for the inhibitors in all the above groups.

[0046] Square wave voltammetry was used to determine the inhibitory activity of different concentrations (1–200 μg / mL) of baicalin against hexamethylenetetramine protein. The results are as follows: Figure 7 As shown in Table 2.

[0047] Table 2. Inhibitory activity of different concentrations of baicalin against hexon protein

[0048] The inhibition rate of baicalin against hexon protein can be calculated using the formula for the inhibition rate. Therefore, the IC50 can be determined by plotting the inhibition rate-concentration curve. 50 ,like Figure 7 C. That is: Inhibition rate % = [(ΔI0 - ΔI1) / ΔI0] × 100% Wherein, ΔI0 is the difference between the peak current measured without a sample (blank group A) and its background current, and ΔI1 is the difference between the peak current measured with a sample (sample group D) and its background current.

[0049] Table 2 shows the inhibition rates of hexon protein by the baicalin groups (1–200 μg / mL). The IC50 of baicalin against hexon can be calculated based on the baicalin concentration and inhibition rate. 50 = 20 μg / mL (44.8 μM). Based on experimental data, the IC50 of baicalin is... 50 (44.8 μM) is lower than the IC50 of the positive control drug Cidofovir. 50 The concentration was 0.56 ng / mL (2.0 nM). Although the inhibitory efficacy of baicalin was much lower than that of cidofovir, this result still indicates that baicalin has a significant inhibitory effect on AdV at higher concentrations. Its inhibition rate increased with increasing concentration, reaching a maximum of 73.3% (20 μg / mL).

Claims

1. A method for screening natural product antiviral drugs based on hexagonal proteins, characterized in that, Includes the following steps: (1) Prepare a nitrogen-doped mesoporous carbon and silver nanosheet composite solution, drop-coat it onto the surface of a pretreated glassy carbon electrode, and dry it to obtain a nitrogen-doped mesoporous carbon / silver nanosheet modified glassy carbon electrode. (2) The nitrogen-doped mesoporous carbon / silver nanosheet modified glassy carbon electrode is immersed in an activator solution for activation treatment, then hexagonal protein solution is added dropwise for incubation, unbound hexagonal protein is washed away, the unbound sites on the electrode surface are blocked with a blocking solution, and then enzyme-labeled anti-hexagonal antibody is added dropwise for reaction to obtain an electrochemical sensing electrode. (3) The natural product sample to be screened is co-incubated with the electrochemical sensing electrode, and the current response signal of the electrochemical sensing electrode is detected by an electrochemical detection system. (4) Based on the change in the current response signal and the inhibition rate calculation formula, evaluate the antiviral activity of the sample to be screened and complete the antiviral drug screening.

2. The method for screening natural product antiviral drugs based on hexamethylenetetramine proteins according to claim 1, characterized in that: In step (1), the preparation method of the nitrogen-doped mesoporous carbon and silver nanosheet composite solution is as follows: 1 mg NMC is dispersed in 1 mL ethanol solution and sonicated for 30 min to make it uniformly dispersed, so as to obtain NMC ethanol solution; 0.5 mg Ag NSs was dispersed in 0.1 wt% PVP solution and sonicated for 1 h to obtain Ag NSs PVP solution. 1 mL of each of the two solutions was mixed to obtain NMC / Ag NSs solution, which was then refrigerated at 4 °C for later use.

3. The method for screening natural product antiviral drugs based on hexamethylenetetramine proteins according to claim 1, characterized in that: In step (1), the glassy carbon electrode pretreatment includes: first polishing the chamois with 0.30μm and 0.05μm alumina powders in sequence, then ultrasonically cleaning it with methanol and distilled water respectively, then drying it at room temperature, and then activating it in 0.5M H2SO4 solution by cyclic voltammetry until it is stable. The potential (CV) range of the cyclic voltammetry is -0.2V to 1.5V, and the scan rate is 100mV / s.

4. The method for screening natural product antiviral drugs based on hexamethylenetetramine proteins according to claim 1, characterized in that: In step (2), the activator solution is an EDC / NHS solution with a concentration of 50mM / 20mM, the buffer is PBS with pH 7.4, and the activation reaction is carried out at room temperature for 1 hour.

5. The method for screening natural product antiviral drugs based on hexamethylenetetramine proteins according to claim 1, characterized in that: In step (2), the concentration of the hexagonal protein solution is 10 μg / mL, the buffer is HEPES buffer, and the incubation conditions are 4℃ for 12h; the blocking solution is 1wt% BSA solution, and the blocking reaction is carried out at room temperature for 1h; the concentration of the enzyme-labeled anti-hexagonal antibody is 10 μg / mL, the buffer is PBS, and the reaction is carried out at room temperature for 2h.

6. The method for screening natural product antiviral drugs based on hexamethylenetetramine proteins according to claim 1, characterized in that: In step (3), the electrochemical detection system employs a three-electrode system, with the electrochemical sensing electrode as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum wire electrode as the counter electrode. Detection is performed using square wave voltammetry, and the test solution contains 2.0 mM [Fe(CN)6]. 3- / 4- A mixed solution of 0.1M KCl.

7. The method for screening natural product antiviral drugs based on hexamethylenetetramine proteins according to claim 1, characterized in that: In step (4), the inhibition rate is calculated as follows: inhibition rate % = [(ΔI0-ΔI1) / ΔI0]×100%, where ΔI0 is the current difference between the peak current measured without a sample and its background current, and ΔI1 is the current difference between the peak current measured with a sample and its background current.

8. The method for screening natural product antiviral drugs based on hexamethylenetetramine proteins according to claim 1, characterized in that: In step (4), the parameters for electrochemical detection using square wave voltammetry are as follows: sensitivity 10 -5 Potential range: 0 V to -0.5 V; rest time: 2 s; voltage amplitude: 0.025 V; frequency: 15 Hz.

9. The application of the natural product antiviral drug screening method based on hexagonal protein as described in any one of claims 1-8 in the screening of anti-adenovirus active ingredients in natural products.

10. The application according to claim 9, characterized in that: The natural products include the traditional Chinese medicine Scutellaria baicalensis and its extract baicalin.

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