New application of 5-O-methylvisammioside in prevention and treatment of HCMV infection

Abstract

The invention provides an application of 5-O-methylvisammioside in preparation of a medicine for inhibiting HCMV (human cytomegalovirus) infection. According to the present invention, the 5-O-methylvisammioside can significantly reduce the cytopathic effect caused by HCMV infection, and the DNA copy numbers of the immediate early protein IE1 / 2, the early protein p52, the immediate early gene UL123, the early gene UL44 and the late gene UL32 of the HCMV are selected as the detection indexes to discover that the 5-O-methylvisammioside can provide the significant inhibition effect, and finally, a medicine addition experiment finds that the 5-O-methylvisammioside can play an antiviral role in multiple stages of a virus life cycle. The 5-O-methylvisammioside has small toxic and side effects, plays an antiviral role in multiple stages, has good application prospects and development advantages, and is expected to be developed into an ideal medicine for treating and / or preventing HCMV infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a new use of 5-O-methylvisamidoside in preventing and treating HCMV infection. Background Art

[0002] Human cytomegalovirus (HCMV) has the largest genome in the herpesvirus family, encoding over 200 proteins. HCMV infection is widespread worldwide. HCMV infection typically causes asymptomatic infection in healthy individuals but can lead to severe disease in neonates, the elderly, and immunocompromised individuals, particularly those with AIDS. HCMV can infect a variety of cell types and organs, with inflammation as the primary pathological manifestation, including gastrointestinal ulcers, hepatitis, pneumonia, and retinitis, as well as autoimmune diseases, transplant rejection, cardiovascular disease, and cancer. Currently, there is no vaccine to prevent HCMV infection, and medication is the only treatment option. Ganciclovir, foscarnet, and cidofovir are clinically approved anti-HCMV drugs that inhibit viral replication by interfering with viral DNA synthesis. However, long-term use of these drugs has demonstrated significant toxicity and drug resistance, necessitating the urgent need for new drug development.

[0003] Windproof ( Saposhnikovia divaricata ) is a high-quality Chinese medicine in China. It is a perennial herb of the Apiaceae family. It has a pungent and sweet taste. Its medicinal function was first recorded in the "Shennong Bencao Jing". 5-O-Methylvisamminol glycoside (4'-O-beta-Glucopyranosyl-5-O-Methylvisamminol) is a phenolic compound derived from the dried roots of the Chinese herbal medicine Saposhnikovia divaricata. Its molecular formula is C 22 H 28 O 10 5-O-Methylvisaminol glycoside is readily soluble in organic solvents such as methanol and ethanol. It is a key component of Saposhnikovia divaricata. Together with the total content of cimicifuga glycosides, it is a chemical indicator for evaluating Saposhnikovia divaricata quality as specified in the Chinese Pharmacopoeia. Studies have shown that it exhibits significant anti-tumor, anti-inflammatory, and antidepressant effects, particularly anti-inflammatory activity, with significant therapeutic effects on inflammatory diseases such as ulcerative colitis and arthritis. However, to date, 5-O-methylvisaminol glycoside has not been used in the preparation of drugs for the prevention and / or treatment of HCMV. Summary of the Invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a drug that has little toxicity and side effects and is safe and effective in inhibiting HCMV replication.

[0005] The purpose of the present invention is achieved through the following technical solutions: The present invention provides application of 5-O-methylvisamidoside in preparing medicine for inhibiting HCMV infection.

[0006] The inhibition of HCMV refers to the inhibitory effect of 5-O-methylvisamidoside on HCMV immediate early protein IE1 / 2, early protein p52 and immediate early gene UL123 , early genes UL44 and late genes UL32 The inhibitory effect on DNA copy number.

[0007] In the application, 5-O-methylvisamidoside can exert an anti-HCMV effect both before and after the virus enters the cell.

[0008] In the application, 5-O-methylvisaminol glycoside can exert an antiviral effect in the stage before the virus enters the cell - pre-treatment of host cells and pre-treatment of viruses. 5-O-methylvisaminol glycoside can inactivate virus particles in vitro and block receptors on the surface of host cells, preventing the virus from entering the host cell.

[0009] In the application, 5-O-methylvisamidoside can exert an antiviral effect through a certain mechanism after the virus enters the cell.

[0010] In the application, the medicine further includes a pharmaceutically acceptable carrier, diluent, and excipient.

[0011] In the application, the drug preparation is a liquid preparation, granules, tablets, granules, capsules, sustained-release preparations or injections.

[0012] The present invention uses commercially available pure 5-O-methylvisaminol glycoside (CAS No. 84272-85- 5 ), conducted a series of biological experiments, and provided a theoretical basis for the new use of 5-O-methylvisamidoside in preventing and / or treating HCMV infection.

[0013] The 5-O-methylvisaminol glycoside described in the present invention can be used to prepare anti-HCMV drugs. Experiments were conducted using human diploid fibroblast WI-38 cells as host cells to evaluate the anti-HCMV activity of 5-O-methylvisaminol glycoside. The results showed that 5-O-methylvisaminol glycoside, at non-cytotoxic concentrations, significantly alleviated the cytopathic effects (CPE) caused by HCMV infection; significantly inhibited the expression of HCMV immediate early proteins IE1 / 2 and early protein p52; and effectively inhibited the expression of HCMV genes, including the immediate early gene UL123 , early genes UL44 and late genes UL32 At the same time, drug addition experiments showed that 5-O-methylvisamidoside can significantly reduce the number of DNA copies of immediate early genes in pre-treatment of cells, pre-treatment of viruses and after the virus enters the cells. UL123 , early genes UL44 and late genes UL32 The results indicate that 5-O-methylvisamidoside can play an antiviral role at different stages of the viral life cycle.

[0014] The results of the biological experiments of the present invention show that: 1. 5-O-Methylvisamidoside treated HCMV host cell WI-38 alone, and had no obvious cytotoxicity to WI-38 at the concentration gradient of 2.5 μM, 5 μM, 10 μM, and 210 μM ( Figure 1 B).

[0015] 2. 5-O-Methylvisamidoside at concentrations of 2.5 μM and 10 μM can significantly alleviate the WI-38 cell pathological effect caused by HCMV infection, as reflected by a significant reduction in the range of cell pathological changes. Figure 2 Middle circle logo.

[0016] 3. 5-O-Methylvisamidoside has an inhibitory effect on the immediate early protein IE1 / 2 protein at a low concentration (2.5 μM), but there is no statistically significant difference; however, there is a significant difference in the inhibitory effect of 5 μM and 10 μM on the immediate early protein IE1 / 2 protein ( * P <0.05). The three concentrations tested (2.5 μM, 5 μM, 10 μM) all had significant inhibitory effects on the HCMV early protein p52 ( ** P <0.01, *** P <0.001), the results are as follows Figure 3 As shown, PFA (phosphocarboxylic acid) is a positive control drug.

[0017] 4. 5-O-Methylvisamidoside showed no significant effect on the immediate early genes at the three tested concentrations (2.5 μM, 5 μM, and 10 μM), except for the low concentration of 2.5 μM. UL123 Except for the DNA copy number inhibition effect of α-catenin, which had no statistically significant effect, the others could significantly reduce the expression of α-catenin UL123 , early genes UL44 and late genes UL32 The DNA copy number is as follows Figure 4 As shown, this result is also consistent with Figure 3The viral protein detection results were consistent with those of

[0018] 5. Drug addition experiments were performed with 5-O-methylvisamidoside at a concentration of 10 μM ( Figure 5 A), by detecting immediate early genes UL123 The results showed that 5-O-methylvisaminidine could significantly reduce the DNA copy number of immediate early genes in all five treatments. UL123 The DNA copy number of HCMV infection was significantly decreased, indicating that 5-O-methylvisamidoside can play a certain antiviral role in multiple stages of the HCMV infection host cell life cycle ( Figure 5 B).

[0019] The medicaments of the present invention also include pharmaceutically acceptable carriers, diluents, and excipients. Pharmaceutically acceptable carriers include any and all substances compatible with pharmaceutical administration, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-retarding agents, and other substances and compounds compatible with pharmaceutical administration. Unless a conventional medium or agent is incompatible with the active compound, its use in the compositions of the present invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.

[0020] Useful pharmaceutical carriers for preparing compositions thereof can be solid, liquid, or gaseous; thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enteric-coated or otherwise protected formulations (e.g., bound to ion exchange resins or packaged in lipoprotein vesicles), sustained-release formulations, solutions, suspensions, elixirs, aerosols, and the like. The carrier can be selected from a variety of oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water, saline, aqueous dextrose, and ethylene glycol are preferred liquid carriers, particularly (when isotonic with blood) for injectable solutions. For example, formulations for intravenous administration include sterile aqueous solutions of the active ingredient, which are prepared by dissolving the solid active ingredient in water to produce an aqueous solution and rendering the solution sterile. Suitable pharmaceutical excipients include starch, cellulose, talc, talc, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, anhydrous skim milk, glycerol, propylene glycol, water, ethanol, etc. The composition may contain conventional pharmaceutical additives such as preservatives, stabilizers, wetting agents or emulsifiers, salts for adjusting osmotic pressure, buffers, etc. In any case, such a composition will contain an effective amount of the active compound together with a suitable carrier to prepare a suitable dosage form for appropriate administration to the recipient.

[0021] The medicament of the present invention can be administered in any convenient administration form, for example, tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, gels, emulsions, gels, patches, etc. Such compositions may contain conventional ingredients in pharmaceutical preparations, for example, diluents, carriers, pH adjusters, sweeteners, fillers and other active agents.

[0022] In order to administer the 5-O-methylvisaminol glycoside of the present invention other than by parenteral administration, it may be necessary to coat the 5-O-methylvisaminol glycoside with a material that prevents its inactivation or to administer it together with the 5-O-methylvisaminol glycoside. Supplementary active compounds may also be added to the composition. In specific implementations, the 5-O-methylvisaminol glycoside of the present invention is co-formulated and / or co-administered with one or more other therapeutic drugs that can be used to treat a disease. This combined use can advantageously utilize lower doses of the therapeutic drug, thereby avoiding possible toxicity or complications associated with various monotherapies.

[0023] The preparation of the present invention can be prepared into one or more liquid preparations such as aqueous solutions, oil suspensions or other liquid preparations, such as one or more syrups or elixirs; when used for parenteral administration, it can be prepared into one or more solutions, aqueous solutions or oil suspensions for injection.

[0024] Among the above-mentioned forms of use, preferred forms are one or more of tablets, coated tablets, capsules, suppositories or injections, and more preferred forms are one or more of tablets, capsules or injections.

[0025] As an alternative, the dosage form can be a powder injection. Powder injections are generally prepared using a conventional freeze-drying method with water as the solvent. The steps are: taking 5-O-methylvisaminol glycoside, adding excipients, dissolving in water, adding activated carbon, filtering and sterilizing, filling, semi-plugging, freeze-drying, and plugging and capping. The excipients used are selected from one or more of mannitol, hydrolyzed gelatin, glucose, lactose, dextran, and the like.

[0026] As an optional method, the powder injection of the present invention can also be prepared by a spray drying method with water as the solvent, and the steps are: taking 5-O-methyl visamidoside, with or without excipients, dissolving it in water, adding activated carbon, filtering and sterilizing, spray drying, aseptically packaging, and plugging and capping.

[0027] As an optional mode of the present invention, the dosage form can be a small injection, which can be prepared using water for injection as a solvent, and can also be prepared with an appropriate amount of excipients, which are selected from one or more of ethanol, propylene glycol, glycerol, polyethylene glycol, benzyl benzoate, and dimethylacetamide.

[0028] Pharmaceutical compositions of 5-O-methylvisaminol must generally be sterile and stable under manufacturing and storage conditions. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions are prepared by adding the desired amount of 5-O-methylvisaminol along with one or a combination of the aforementioned ingredients to an appropriate solvent, followed by sterile filtration. Generally, dispersions are prepared by adding 5-O-methylvisaminol to a sterile solvent containing a basic dispersion medium and the other ingredients described above. In the case of sterile powders for the preparation of sterile injectable solutions, vacuum drying and freeze drying are recommended methods of preparation. For example, proper fluidity of the solution can be maintained by coating with a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, such as monostearate or gelatin.

[0029] The advantages and beneficial effects of the present invention are as follows: The present invention provides a new use of 5-O-methylvisaminol glycoside in the preparation of drugs for treating and / or preventing HCMV, 5-O-methylvisaminol glycoside can significantly reduce the cytopathic effect caused by HCMV infection, selects HCMV immediate early protein IE1 / 2, early protein p52 and immediate early gene UL123 , early genes UL44 and late genes UL32 Using DNA copy number as a detection indicator, 5-O-methylvisaminol glycoside was found to exert a significant inhibitory effect. Finally, through drug addition experiments, it was found that 5-O-methylvisaminol glycoside can exert antiviral effects at multiple stages of the viral life cycle. 5-O-methylvisaminol glycoside has minimal toxicity and side effects, and exerts antiviral effects at multiple stages. It has promising application prospects and development advantages, and is expected to be developed as an ideal drug for the treatment and / or prevention of HCMV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A is the structural formula of 5-O-methylvisamidoside. Figure 1 B is the CCK8 assay to determine the cytotoxicity of 5-O-methylvisaminol on HCMV host cells—human embryonic lung fibroblasts (WI-38). Compared with the control group, which did not contain 5-O-methylvisaminol and was treated with solvent (DMSO), treatment with 5-O-methylvisaminol at four concentrations (2.5 μM, 5 μM, 10 μM, and 20 μM) showed no significant cytotoxicity to the host cells.

[0031] Figure 2The cytopathic effect of 5-O-methylvisamidoside on HCMV infection of human embryonic lung fibroblasts WI-38 was investigated. Control group: control group cells, not infected with HCMV, without the addition of solvent DMSO and 5-O-methylvisamidoside; The HCMV infection treatment group was infected with HCMV alone without the addition of 5-O-methylvisamidoside; HCMV+DMSO treatment group: cells were pre-treated with solvent DMSO for 2 h and then inoculated with HCMV; HCMV+PFA treatment group: cells were pre-treated with phosphocarboxylic acid (PFA), a positive control, for 2 h before inoculation with HCMV, and the concentration of PFA was 200 μg / ml; HCMV+5-O-methylvisamidoside treatment group: cells were pretreated with 5-O-methylvisamidoside at two concentrations (2.5 μM and 10 μM) for 2 h before inoculation with HCMV; All HCMV-infected groups were inoculated at an MOI of 0.5 and observed and photographed 5 days after HCMV infection (dpi). The cytopathic area is indicated by a red circle.

[0032] Figure 3 Western blotting was used to detect the effects of pretreatment of WI-38 cells with 5-O-methylvisaside at three different concentrations (2.5 μM, 5 μM, 10 μM) on HCMV immediate early protein IE1 / 2 and early protein p52 (GAPDH was used as an internal reference); Figure A shows the expression levels of HCMV immediate early protein IE1 / 2 and early protein p52 in cells treated with 5-O-methylvisaside (2.5 μM, 5 μM, 10 μM) (representative images of the three groups of experiments); Figure B is a quantitative analysis of the proteins in Figure A.

[0033] (+) group: HCMV infection alone, without adding solvent DMSO or 5-O-methylvisamidoside treatment, this group served as the experimental control group; (+) DMSO group: cells were pre-treated with solvent DMSO for 2 hours before virus infection, serving as the drug solvent control group; (+) PFA group: cells were pre-treated with the positive drug phosphodiesterone (PFA) for 2 hours before virus infection, serving as the positive drug control group; the PFA concentration was 200 μg / ml.

[0034] Drug treatment groups: cells were pretreated with 5-O-methylvisamidoside at three drug concentrations (2.5 μM, 5 μM, and 10 μM) for 2 h before virus infection, serving as experimental groups; All HCMV-infected groups were inoculated at an MOI of 0.5, and samples were collected five days after infection (5 dpi). Proteins were extracted using Biyuntian's Western lysis buffer (P1003) for detection. All drug-treated groups, including the solvent DMSO-treated group, were statistically compared with the HCMV-infected group (+). * P <0.05, ** P <0.01, *** P <0.001.

[0035] Figure 4 The qPCR method was used to determine the effect of different concentrations (2.5 μM, 5 μM, 10 μM) of 5-O-methylvisamidoside on the immediate early gene of HCMV. UL123 , early genes UL44 and late genes UL32 Effects of DNA copy number.

[0036] Control group: HCMV infection group, without drug or solvent DMSO, serving as the control group of the experiment; PFA group: PFA pretreatment was followed by virus infection, serving as the positive drug control group of the experiment. The PFA concentration was 200 μg / ml.

[0037] All groups infected with HCMV were inoculated at an MOI of 0.5, and samples were collected three days after infection (3 dpi) to extract viral DNA for detection. The treatment groups with different concentrations of 5-O-methylvisamin (2.5 μM, 5 μM, 10 μM) were compared with the control group. * P <0.05, ** P <0.01, *** P <0.001.

[0038] Figure 5 This is the stage where drug addition experiments confirm that 5-O-methylvisamidoside exerts an anti-HCMV effect. Figure 5 A is a schematic diagram of the drug addition experiment; Figure 5 B is the qPCR assay for the expression of HCMV immediate early genes by different addition methods UL123 , early genes UL44 and late genes UL32The effect of DNA copy number. The experimental results show that 5-O-methylvisamin can inactivate HCMV in vitro and block cell surface receptors before the virus enters the cell, thereby preventing the virus from entering the cell; after the virus enters the cell, 5-O-methylvisamin can also exert an antiviral effect through a certain mechanism. All groups infected with HCMV were inoculated at a dose of MOI = 0.5, and samples were collected three days after infection (3 dpi) to extract viral DNA for detection. The concentration of 5-O-methylvisamin was 10 μM, and the treatment group was compared with the HCMV single infection treatment group (+), ** P <0.01, *** P <0.001. DETAILED DESCRIPTION

[0039] The present invention is further described below with reference to specific examples. The examples of the present invention are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] The human embryonic lung fibroblast cell line WI-38 and the human cytomegalovirus (HCMV) Towne strain involved in the present invention are both from the American type culture collection (ATCC).

[0043] Example 1 Effect of 5-O-methylvisamidoside treatment alone on WI-38 cell cytotoxicity The CCK-8 assay was used to detect the cytotoxicity of 5-O-methylvisaminol. WI-38 cells were trypsinized and cultured using conventional methods in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS).

[0044] The cytotoxicity determination method was slightly modified according to the instructions of the CCK8 kit: WI-38 cells in the logarithmic growth phase were trypsinized and counted using a hemocytometer. Then, the cells were seeded at a density of 5000 cells / well in a 96-well cell culture plate and cultured in a CO2 incubator. The next day, the cells were observed to grow into a monolayer, and different concentrations (0 μM, 2.5 μM, 5 μM, and 10 μM) of 5-O-methylvisamidoside working solutions were prepared in DMEM medium containing 0.2% FBS (20 mM 5-O-methylvisamidoside stock solution was dissolved in DMSO and sterilized by filtration with a 0.22 μm filter). The original culture medium was discarded, and fresh culture medium containing different concentrations of 5-O-methylvisamidoside was added. Six parallel wells were set for each concentration, and a negative control group without 5-O-methylvisamidoside and a blank control group without cells were also set up. After 5 days of continuous culture, 10 μl of 5-O-methylvisamidoside was added to each well. CCK-8 solution; place the 96-well plate in a CO2 incubator and continue incubation for 1-2 hours; then remove the plate and measure the absorbance at 450nm. Finally, calculate the relative cell viability based on the absorbance of each well. The cell viability of the blank group without any solvent DMSO or drug is set as 100%. The experimental results are analyzed as follows: cell viability = [OD (drug added) - OD (blank)] / [OD (0 drug added) - OD (blank)]. The results of the effect of 5-O-methylvisamidoglycoside on cytotoxicity are shown in Figure 1 B. The results showed that compared with the control group without 5-O-methylvisaminol glycoside and solvent (DMSO) treatment, 5-O-methylvisaminol glycoside treatment at four concentrations (2.5 μM, 5 μM, 10 μM, 20 μM) had no obvious toxicity to the host cells.

[0045] Example 2 HCMV inoculation and drug treatment WI-38 cells were subcultured according to conventional methods, and the number of cells was 2×10 4 / cm 2Cells were plated at a density of 1 / 400 μg / ml in a 12-well plate and cultured in a 37°C, 5% CO2 incubator. After the cells reached monolayer growth, the culture medium was replaced with DMEM supplemented with 0.2% FBS and cultured for an additional 2 days. This procedure improves the efficiency of subsequent HCMV infection. HCMV was inoculated at a multiplicity of infection (MOI) of 0.5 and incubated in a 37°C, 5% CO2 incubator for the designated times. Cytopathic morphology was observed, and protein and DNA were extracted for Western blotting and DNA copy number analysis. For treatments with 5-O-methylvisaminidine, the solvent DMSO, or the positive agent phosphodiesterone (PFA), the drug, DMSO, or PFA was added to the culture medium 2 hours before HCMV infection, followed by inoculation with HCMV (MOI = 0.5). The PFA concentration was 200 μg / ml.

[0046] Example 3 Effect of 5-O-methylvisamidoside Pretreatment on HCMV Cytopathic Effects in WI-38 Cells WI-38 cells were cultured at 2×10 4 / cm 2 Cells were plated at a density of 100 μg / mL in a 12-well cell culture plate and treated as in Example 2 above. HCMV infection alone served as a control, the DMSO-treated group served as a solvent control, and the positive drug phosphodiesterase (PFA)-treated group served as a positive drug control. Cells were pretreated with different concentrations of 5-O-methylvisaminol (2.5 μM, 10 μM) 2 hours in advance and then inoculated with HCMV (MOI = 0.5). Three days later, the HCMV infection-induced cytopathic effect (CPE) in the HCMV infection alone group was observed under a microscope. The main characteristic was that the virus-infected cells became larger and rounder, and the intercellular spaces increased. After five days, the cytopathic effect became more obvious. Figure 2 The red circle indicates the cytopathic effect (CPE) in the DMSO-treated group, similar to that in the HCMV-only infection group, was observed, with enlarged intercellular spaces and rounded and swollen cells. Treatment with a low concentration (2.5 μM) of 5-O-methylvisaminidine significantly reduced the CPE, and treatment with a high concentration (10 μM) further reduced the CPE. Treatment with the positive drug PFA (phosphocarboxylic acid) produced the most pronounced CPE, with almost no noticeable CPE.

[0047] Example 4 Effect of 5-O-methylvisamidoside Pretreatment on HCMV Viral Protein Expression To confirm the inhibitory effect of 5-O-methylvisaminol on HCMV infection, HCMV immediate early proteins 1 / 2 (IE1 / 2) and early protein p52 were used as indicators of viral replication. Changes in viral protein expression were measured after pretreatment of cells with 5-O-methylvisaminol at three different concentrations (2.5 μM, 5 μM, and 10 μM). Cell culture, treatment with 5-O-methylvisaminol, the solvent DMSO, the positive agent PFA, and HCMV inoculation were performed as described in Example 2. A blank control group (-) without HCMV inoculation or treatment with either 5-O-methylvisaminol or DMSO was set up; a group infected with HCMV alone (+) served as the experimental control; a group pretreated with the solvent DMSO and then inoculated with HCMV (DMSO) served as the solvent control; and three experimental groups were pretreated with 5-O-methylvisaminol at three different concentrations (2.5 μM, 5 μM, and 10 μM) and then inoculated with HCMV. In all virus infection groups, the HCMV infection dose was MOI=0.5, the infection time was uniformly set at 5 days, and the collection, lysis and extraction of cell samples were carried out according to the instructions of the Biyuntian cell lysis solution. The method is briefly described as follows: First, discard the cell culture supernatant, wash the cells three times with PBS, add 100 μl (according to the amount of 12-well plate) lysis solution (Biyuntian Company) to lyse the cells, place the cells in a 4°C refrigerator for half an hour, then collect and centrifuge. SDS-PAGE protein electrophoresis and transfer were performed using conventional methods, and commercial mouse IE1 / 2 and p52 antibodies were combined with HRP-goat anti-mouse secondary antibodies for incubation and color development. The results showed that 5-O-methylvisamidoside can significantly reduce the expression of HCMV early protein p52 at a concentration of 2.5 μM ( P <0.01) ( Figure 3 ), when the concentration reaches 5 μM, it has a significant inhibitory effect on both immediate early protein IE1 / 2 and early protein p52.

[0048] Example 5 Effect of 5-O-methylvisamidoside Pretreatment on HCMV DNA Copy Number in Host Cells Cell culture, 5-O-methylvisaminol and PFA treatment, and HCMV inoculation were performed as in Example 2. Three treatment groups were set up with different concentrations of 5-O-methylvisaminol (2.5 μM, 5 μM, and 10 μM). Samples were collected 3 days after HCMV infection (dpi). HCMV DNA copy number was determined by qPCR. Viral DNA was extracted using the QIAamp DNA Mini Kit. 10 ng of total DNA was used for qPCR analysis using 2× Universal SYBR Green Fast qPCR Mix. The primers used are shown in the following table: Table 1: Primer names and sequences used in the experiment

[0049] Amplification conditions: 95°C for 5 min, (95°C for 5 sec, 60°C for 30 sec) × 40 cycles, 2 -△△Ct The DNA copy number of the HCMV infection group alone was set as 1. The results are as follows Figure 4 The results showed that 5-O-methylvisaminidine treatment at concentrations of 5 μM and 10 μM had an effect on the expression of immediate early genes. UL23 Has a significant inhibitory effect on early genes UL44 and late genes UL32 , three concentrations (2.5 μM, 5 μM, 10 μM) can exert an inhibitory effect.

[0050] Example 6 Experiments with different drug additions to confirm the stage at which 5-O-methylvisamidoside exerts its anti-HCMV effect Different ways of adding drugs are: 1. Cells were pretreated with 5-O-methylvisamidoside for 2 hours and then infected with HCMV. Samples were collected 3 days later (3 dpi).

[0051] 2. Pretreat the cells with 5-O-methylvisamidoside for 2 hours, discard the drug, and wash the cells three times with PBS. Then, infect the cells with HCMV for 2 hours, discard the HCMV, and wash the cells three times with PBS. Add fresh medium containing 0.2% FBS, and culture for 3 days before collecting the samples.

[0052] 3. After washing the cells three times with PBS, 5-O-methylvisamidoside and HCMV were added to the cells simultaneously. After 2 hours, the culture medium was discarded. After washing the cells three times with PBS, fresh culture medium containing 0.2% FBS was added. The cells were cultured for 3 days and then the samples were collected.

[0053] 4. Add 5-O-methylvisamidoside to the HCMV virus solution 2 hours in advance. Wash the cells three times with PBS, then add the drug and virus mixture to the cells. After 2 hours, discard the mixture, wash the cells three times with PBS, add fresh medium containing 0.2% FBS, and culture for 3 days before collecting the samples.

[0054] 5. After washing the cells three times, infect them with HCMV for 2 hours, then discard the virus solution. After washing the cells three times with PBS, add a solution of 5-O-methylvisamidoside prepared in 0.2% FBS culture medium, culture for 3 days, and then collect the samples.

[0055] 5A is a schematic diagram of the drug addition experiment. In the above experimental groups, the HCMV infection dose was MOI = 0.5, and the concentration of 5-O-methylvisamidoside was 10 μM. Viral DNA extraction, primer sequences, and qPCR procedures were the same as those in Example 5.

[0056] Figure 5 B is the qPCR assay for the expression of HCMV immediate early genes by different addition methods UL123 , early genes UL44 and late genes UL32 The experimental results show that 5-O-methylvisaminol glycoside can inactivate HCMV in vitro and block cell surface receptors before the virus enters the cell, thereby preventing the virus from entering the cell; once the virus enters the cell, 5-O-methylvisaminol glycoside can also exert an antiviral effect through certain mechanisms.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are included in the scope of protection of the present invention.

Claims

Application of 1,5-O-methylvisamidoside in the preparation of drugs for inhibiting HCMV infection.

2. The use according to claim 1, characterized in that The inhibition of HCMV refers to the inhibitory effect of 5-O-methylvisamidoside on HCMV immediate early protein IE1 / 2, early protein p52 and immediate early gene UL123 , early genes UL44 and late genes UL32 The inhibitory effect on DNA copy number.

3. The use according to claim 1, characterized in that 5-O-Methylvisamidoside can exert anti-HCMV effects both before and after the virus enters the cell.

4. The use according to claim 3, characterized in that 5-O-Methylvisaminol glycoside can exert an antiviral effect in the stage before the virus enters the cell - pre-treatment of host cells and pre-treatment of viruses. 5-O-Methylvisaminol glycoside can inactivate virus particles in vitro and block receptors on the surface of host cells, preventing the virus from entering the host cells.

5. The use according to claim 3, characterized in that 5-O-Methylvisamidoside can exert an antiviral effect through a certain mechanism after the virus enters the cell.

6. The use according to claim 1, characterized in that The medicine also includes pharmaceutically acceptable carriers, diluents, and excipients.

7. The use according to claim 1, characterized in that The preparation of the medicine is liquid preparation, granule, tablet, granule, capsule, sustained-release preparation or injection.

Images