Application of magnolin in preparation of anti-HCMV (human cytomegalovirus) infection medicine

Abstract

The invention provides an application of magnolin in preparation of an anti-HCMV drug, and a series of biological experiments show that the magnolin has no obvious cytotoxicity in a concentration range of 20 [mu] M when being used for independently treating an HCMV in-vitro host cell-human embryo lung diploid fibroblast WI-38; the magnolin can be used for remarkably relieving the cytopathic effect caused by HCMV infected cells; the magnolin can be used for effectively inhibiting the expression of immediate early-stage protein IE1 / 2 and early-stage protein p52 of the HCMV in WI-38; the magnolin can be used for effectively inhibiting the DNA copy number of an immediate early gene UL123, an early gene UL44 and a late gene UL32 of the HCMV; drug addition experiments show that magnolin can exert anti-HCMV activity in a plurality of stages before and after viruses enter cells. The results show that magnolin has obvious prevention and treatment effects on HCMV, has small toxic and side effects, is safe and effective, has a multi-stage anti-HCMV effect, and has an application prospect in preparation of anti-HCMV drugs for prevention and / or treatment.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to the use of magnolin in the preparation of a drug for resisting HCMV infection. Background Art

[0002] Human cytomegalovirus (HCMV) is a beta-herpesvirus with a double-stranded DNA (dsDNA) structure. Its genome is approximately 240 kb and encodes at least 165 proteins. In developed countries, the seroprevalence of HCMV among adults is approximately 60%, while in low- and middle-income countries, this figure can be as high as 90%. HCMV shares many similarities with other herpesviruses, including a strict order of gene expression and a lifelong latent state after initial infection. In individuals with healthy immune systems, HCMV infection is typically asymptomatic, but in immunocompromised individuals, it can often lead to dangerous or even life-threatening illness. HCMV infection is a significant contributing factor to microcephaly, cognitive impairment, visual impairment, sensorineural hearing loss, and vascular disease. Elderly individuals are also particularly susceptible to HCMV infection due to underlying medical conditions and age-related immunodeficiency. Currently, there is no effective HCMV vaccine. Ganciclovir (GCV) and its prodrug, valganciclovir (VAV), foscarnet (PFA), cidofovir (CDV), letermovir (LEV), and maribavir (MAV) are approved antiviral drugs for the treatment of HCMV infection. Ganciclovir, valganciclovir, foscarnet, and cidofovir are similar in that they all target viral DNA polymerase, inhibiting viral DNA synthesis. However, long-term clinical use of these drugs has led to the emergence of drug-resistant strains. Furthermore, these drugs can cause side effects such as bone marrow suppression, nephrotoxicity, hepatotoxicity, electrolyte imbalances, carcinogenicity, and decreased fertility. Therefore, active and effective prevention and treatment of HCMV infection is not only essential for improving the quality of life in the elderly population but also a crucial means of enhancing the quality of life for immunocompromised elderly individuals. Therefore, the continued search for low-toxic and highly effective drugs for the prevention and treatment of HCMV infection is of great scientific significance.

[0003] Magnolin is a molecule of the Magnoliaceae plant ( Magnolia biondii A lignan extracted from the dried buds of Pamp. The molecular weight of magnolia oleifera is 416.46, and its molecular formula is C 23 H 28 O7. Modern pharmacological research indicates that magnoliarin has anti-allergic and anti-inflammatory effects, as well as protective effects against renal ischemia-reperfusion injury. However, to date, magnoliarin has not been used in the preparation of drugs for the prevention and / or treatment of HCMV. Systematic research on its antiviral effects will provide a scientific basis for further exploring the potential of Traditional Chinese Medicine (TCM). Summary of the Invention

[0004] In order to address the above-mentioned deficiencies in the prior art, the present invention aims to provide a Chinese medicinal monomer component which has no toxic side effects on diploid fibroblast WI-38 cells and has a significant anti-HCMV effect.

[0005] In order to achieve the above objectives, the present invention is mainly implemented through the following technical solutions: The present invention provides application of magnolin in preparing medicine for inhibiting HCMV infection.

[0006] The inhibition of HCMV refers to the inhibitory effect of magnolin 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, magnolin can exert anti-HCMV effect both before and after the virus enters the cell.

[0008] In the application, magnolia resin can play an antiviral role in the stage before the virus enters the cell - pre-treatment of host cells and pre-treatment of viruses. Magnolia resin 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 above application, magnolin can exert antiviral effects through a certain mechanism after the virus enters the cell.

[0010] In the application, a therapeutically effective amount of magnolin can be combined with a pharmaceutically acceptable carrier, diluent, or excipient, and used alone or with other substances to prepare anti-HCMV drugs.

[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 pure magnolia oil (CAS No. 31008-18-1, HPLC Assay: 99.2%) to conduct a series of biological experiments, providing a theoretical basis for the new use of magnolia oil in preventing and / or treating HCMV infection.

[0013] The magnolin can significantly reduce the cytopathic effect caused by HCMV infection at a non-cytotoxic concentration; can significantly inhibit the expression levels of HCMV immediate early protein IE1 / 2 and early protein p52; effectively inhibit HCMV immediate early gene UL123 , early genes UL44 and late genes UL32 DNA copy number; drug addition experiments showed that magnolia oleate can inactivate HCMV in vitro, block receptors on the surface of host cells, and play an anti-HCMV infection role at different stages after HCMV enters the cell.

[0014] The results of the biological experiments of the present invention show that: 1. HCMV host cells WI-38 were treated with different concentrations (5 μM, 10 μM, 20 μM) of magnolin for five days and detected by CCK8. No obvious cytotoxicity was found.

[0015] 2. Magnoliopsin at the concentrations of 2.5 μM and 10 μM used in the experiment can significantly reduce the WI-38 cell pathological effect caused by HCMV infection.

[0016] 3. Magnoliopsin at concentrations of 5 μM and 10 μM can significantly inhibit the expression of HCMV immediate early protein IE1 / 2 and early protein p52 in WI-38. The concentration of 1.25 μM also has an inhibitory effect on these two proteins, but it is not statistically significant.

[0017] 4. Magnoliopsin at a concentration of 1.25 μM can reduce the expression of early genes UL44 and late genes UL32 The DNA copy number of HCMV immediate early gene was significantly reduced at concentrations of 5 μM and 10 μM. UL123 , early genes UL44 and late genes UL32 DNA copy number.

[0018] 5. Experiments with different drug additions showed that magnolin could significantly reduce the expression of HCMV immediate early genes in pre-treatment of host cells, pre-treatment of HCMV and after HCMV entered cells. UL123 , early genes UL44 and late genes UL32 The results indicate that magnolin can play an antiviral role at multiple stages of HCMV infection.

[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 the administration of the drug, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-retarding agents, and other substances and compounds compatible with the administration of the drug. 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 give magnolia of the present invention outside parenteral administration, it may be necessary to coat magnolia with a material that prevents its inactivation or to give it together with magnolia. The active compound that supplements can also be added to the composition. In specific implementation, magnolia of the present invention is co-formulated and / or co-administered with one or more other therapeutic drugs that can be used for treating diseases. This combination can advantageously utilize therapeutic drugs given at lower doses, thereby avoiding possible toxicity or the complication 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 magnolia linalool, 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 spray drying method, using water as the solvent, and the steps are: taking magnolia oleate, 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 magnolia oleate must generally be sterile and stable under production and storage conditions. The composition can be formulated into a solution, microemulsion, dispersion, liposome or other ordered structure suitable for high drug concentration. A sterile injection is prepared by adding the required amount of magnolia oleate together with one or a combination of the required above-mentioned ingredients to an appropriate solvent and then performing sterile filtration. Generally speaking, a dispersion is prepared by adding the magnolia oleate to a sterile solvent containing a basic dispersion medium and the required other ingredients mentioned above. In the case of sterile powders for the preparation of sterile injections, the recommended preparation methods are vacuum drying and freeze drying agents. For example, the appropriate fluidity of the solution can be maintained by coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption (e.g., monostearate or gelatin) in the composition.

[0029] The advantages and beneficial effects of the present invention are as follows: The present invention provides a new use of magnolin in the preparation of drugs for resisting HCMV infection, magnolin can significantly reduce the cytopathic effect caused by HCMV infection; UL123 , early genes UL44 and late genes UL32The results showed that magnolia resin has a significant inhibitory effect on the DNA copy number of HCMV; drug addition experiments showed that magnolia resin can inhibit the virus from entering the host cell by inactivating the virus in vitro and blocking the viral receptors on the host cell surface. At the same time, after the virus enters the cell, magnolia resin can also exert an anti-HCMV effect, suggesting that magnolia resin has an antiviral effect at multiple stages of the HCMV life cycle. Magnolire resin has few toxic side effects. Its high efficiency, low toxicity, and multi-stage antiviral effects show its unique advantages in the development of antiviral drugs. It has good prospects for the development of applied drugs and is expected to be developed into 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 magnolin; Figure 1 B is the CCK8 assay to determine the effect of magnolianolide on the cytotoxicity of HCMV host cells - human embryonic lung diploid fibroblasts (WI-38). The solvent (DMSO) treatment group was used as the control. Three concentrations of magnolianolide (5 μM, 10 μM, 20 μM) showed no obvious cytotoxicity. ns indicates that magnolianolide has no statistically significant effect on the cytotoxicity of WI-38 cells.

[0031] Figure 2 The cell morphological changes caused by magnolin after HCMV infection of human embryonic lung diploid fibroblast WI-38 cells, among which, Control group: not infected with HCMV, and no magnolin or solvent DMSO was added; HCMV infection group: HCMV infection alone; HCMV+DMSO group: cells were pre-treated with drug solvent DMSO for 2 h before inoculation with HCMV, serving as solvent control; HCMV+PFA group: cells were pre-treated with the positive drug PFA for 2 h before inoculation with HCMV as a positive drug control; the PFA concentration was 200 μg / ml.

[0032] HCMV+magnanol treatment group: cells were pre-treated with different concentrations of magnolin (2.5 μM, 10 μM) for 2 h before inoculation with HCMV; All groups infected with HCMV were inoculated at an MOI of 0.5. HCMV cells were observed and photographed 5 days after infection (5 dpi). Representative images of each group were selected for display.

[0033] Figure 3The effects of different concentrations of magnoliaside (1.25 μM, 5 μM, 10 μM) on the expression of HCMV immediate-early protein IE1 / 2 and early protein p52 in WI-38 cells were determined by Western blot (GAPDH was used as an internal reference). Figure A is a representative image of viral proteins. Figure B is a quantitative analysis of the HCMV immediate-early protein IE1 / 2 and early protein p52 in Figure A.

[0034] The (+) group was infected with HCMV alone and served as the control, with which the other groups were compared; The DMSO group was pretreated with DMSO 2 hours before virus infection and served as the solvent control group; The PFA group was pretreated with PFA 2 hours before virus infection and served as the positive drug control group; All groups infected with HCMV were inoculated at a dose of MOI = 0.5, and samples were collected five days after infection (5 dpi) for protein extraction and detection. All drug-treated groups were statistically compared with the group infected with HCMV alone (+). * P <0.05, ** P <0.01, *** P <0.001. PFA was used at a concentration of 200 μg / ml.

[0035] Figure 4 The qPCR method was used to determine the effects of different concentrations of magnolin (1.25 μM, 5 μM, 10 μM) on the expression of HCMV immediate early genes. UL123 , early genes UL44 and late genes UL32 The effect of PFA on the DNA copy number. The concentration of PFA used was 200 μg / ml. 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 groups treated with different concentrations of magnolia linalool (1.25 μM, 5 μM, 10 μM) were compared with the group infected with HCMV alone. * P <0.05, ** P <0.01, *** P <0.001.

[0036] Figure 5 This is the stage where qPCR method is used to confirm that magnolin exerts anti-HCMV effect. Figure 5 A is a diagram of the drug addition experiment model; Figure 5 B is the qPCR detection of the immediate early genes in the five different drug addition experimental groups. UL123 , early genes UL44 and late genes UL32 The results showed that all five treatments could reduce the DNA copy number, indicating that magnolia resin can inactivate HCMV in vitro and block the receptors on the cell surface before the virus enters the cell, thereby preventing the virus from entering the cell; after the virus enters the cell, magnolia resin can also play an antiviral role 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 magnolia resin used was 10 μM. Compared with the HCMV single infection treatment group (+), * P <0.05, ** P <0.01, *** P <0.001. DETAILED DESCRIPTION

[0037] 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.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] 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).

[0041] Example 1 Toxicity test of magnolin on WI-38 cells WI-38 cells were cultured in DMEM (GIBCO) medium containing 10% fetal bovine serum (FBS, GIBCO) in a 37°C, 5% CO 2 incubator and trypsinized and counted using conventional methods.

[0042] The CCK-8 assay was used to detect the cytotoxicity of magnolia glycosides to host cells. Human embryonic lung diploid fibroblast WI-38 cells in the logarithmic growth phase were trypsinized and seeded into 96-well cell culture plates. The plates were then cultured in a 37°C, 5% CO2 incubator. The cell status and density were observed the next day. After the cells grew into a monolayer, the original culture medium was discarded and magnolia glycosides working solutions of different concentrations (0 μM, 5 μM, 10 μM, 20 μM) were added to DMEM medium containing 0.2% FBS (the 20 mM stock solution was dissolved in DMSO and filtered through a 0.22 μm filter membrane). Six parallel wells were set for each concentration, and a negative control group without magnolia glycosides and a blank control group without cells were also set up. After 5 days of culture, 10 μl of magnolia glycosides were added to each well. CCK-8 solution was prepared. Do not introduce bubbles during this process to avoid interfering with subsequent OD value determination. The 96-well plate was placed at 37°C and 5% CO2 for 1-2 hours. The absorbance at 450 nm was then measured using a microplate reader. The relative cell viability was calculated based on the absorbance of each well. The cell viability of the solvent DMSO-treated group was set as 100%. The experimental results were analyzed as follows: cell viability = [OD (drug added) - OD (blank)] / [OD (0 drug added) - OD (blank)].

[0043] The results of magnolinone's cytotoxicity are shown in Figure 1 B, Within the experimentally tested concentration of 20 μM, WI-38 cells showed no toxicity, demonstrating the advantages of magnoliavine's high efficiency and low toxicity in the development of antiviral drugs.

[0044] Example 2 Effect of magnolin on morphological changes of WI-38 cells after HCMV infection WI-38 cells in the logarithmic growth phase were inoculated into 12-well plates, with HCMV infection alone as the control, DMSO treatment group as the solvent control, and PFA treatment group as the positive drug control. Different concentrations of magnoliacin (2.5 μM, 10 μM) and DMSO and PFA (200 μg / ml) were used to pre-treat the cells 2 hours in advance, and then HCMV was inoculated (MOI=0.5), and the changes in cell morphology were observed at different times. Three days later, the cytopathic effect (CPE) caused by HCMV infection in the HCMV infection alone group was observed under a microscope. The main characteristics were that the virus-infected cells became larger and rounder, and the gaps between cells increased. The cytopathic effect of the solvent DMSO treatment group was similar to that of the HCMV infection alone group. The relief of the cytopathic effect CPE was clearly observed after magnoliacin treatment. This result shows that magnoliacin can protect WI-38 cells from the cytopathic effect caused by HCMV infection. The results are shown in Figure 2 shown.

[0045] Example 3 Effect of magnolin treatment on the expression of HCMV-related proteins To further confirm the inhibitory effect of magnolianes on HCMV infection, we observed the reduction of cytopathic effects of magnolianes on cells infected with HCMV. We also used HCMV immediate early proteins 1 / 2 (IE1 / 2) and early protein p52 as indicators of viral replication. The effects of magnolianes at different concentrations (1.25 μM, 5 μM, and 10 μM) on IE1 / 2 and p52 expression were examined. Cell culture, magnolianes, DMSO, and PFA treatment, and HCMV inoculation were performed as described in Example 2. A blank control group (-) was established without any treatment; a group infected with HCMV alone (+) served as a comparison; a group treated with the solvent DMSO and then inoculated with HCMV (DMSO) served as a solvent control; a group treated with the positive drug PFA and then inoculated with HCMV (PFA) served as a positive drug control; and experimental groups pretreated with magnolianes at different concentrations (1.25 μM, 5 μM, and 10 μM) and then infected with HCMV were also established.

[0046] The dose of HCMV infection was MOI=0.5; the concentration of PFA used was 200 μg / ml. 5 days after HCMV inoculation (5 dpi), the supernatant was discarded, the cells were washed three times with PBS, and the cells were lysed with medium-strength RIPA lysis buffer at 4°C for half an hour. After collecting the samples, they were centrifuged, and then protein electrophoresis and membrane transfer were performed according to conventional methods. They were incubated with commercial IE1 / 2 and p52 antibodies, and finally chemiluminescence detection was performed. The results showed that magnolia oleate at concentrations of 5 μM and 10 μM could significantly reduce the expression of HCMV immediate early protein IE1 / 2 and early protein p52 ( P <0.05) ( Figure 3 ).

[0047] Example 4 Effect of Magnoliopsin Treatment on DNA Copy Number in HCMV-Infected Host Cells Cell culture, magnolia and PFA treatment, and HCMV inoculation were performed as in Example 2. Three magnolia treatment groups (1.25 μM, 5 μM, and 10 μM) were set up. Samples were collected 3 days after HCMV infection (dpi). HCMV DNA copy number was determined by qPCR, and the genes detected included immediate early genes. UL123 , early genes UL44 and late genes UL32Viral DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN). 10 ng of total DNA was used for qPCR experiments using 2× Universal SYBR Green Fast qPCR Mix (Abclonal, Wuhan). The primers used are shown in the following table: Table 1: Primer names and sequences used in the experiment

[0048] 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 was set as 1. The results are as follows Figure 4 The results showed that 1.25 μM magnolin could inhibit the early gene UL44 Magnoliopsin at concentrations of 5 μM and 10 μM could inhibit the DNA copy numbers of the above three genes.

[0049] Example 5 Study on the mechanism of anti-HCMV activity of magnolin Through the above studies, we confirmed that magnolin has good anti-HCMV activity, but it is unclear at which stage of the HCMV life cycle magnolin exerts its effect. We conducted drug addition experiments to clarify this issue. We conducted experiments using five different drug addition methods.

[0050] Experimental group 1: Magnoliopsin was added 2 hours before HCMV inoculation, and then the Magnoliopsin was kept in the cells until the samples were collected 72 hours later; Experimental group 2: magnolin pre-treated cell group, magnolin was added 2 hours before the cells were inoculated with HCMV, and then the magnolin was discarded and the cells were washed three times with PBS before inoculation with HCMV, and the samples were collected 72 hours later; Experimental group 3: Magnoliopsin and HCMV were added simultaneously. Magnoliopsin was added simultaneously with HCMV inoculation. After 2 hours, the cell supernatant was discarded, the cells were washed three times with PBS, and fresh culture medium was added until the samples were collected after 72 hours. Experimental group 4: magnoliavine pre-treated virus group, before HCMV inoculation of cells, magnoliavine and HCMV were mixed and placed in a 4°C refrigerator for 2 hours, and then the magnoliavine and HCMV mixture was added to the cells, discarded after 2 hours, and the cells were washed with PBS three times, and fresh culture medium was added until the samples were collected after 72 hours; Experimental group 5: magnolin-treated group after virus entry into cells. After 2 hours of HCMV infection, cells were washed three times with PBS and culture medium containing magnolin was added until 72 hours later when samples were collected.

[0051] The drug addition experiment model is shown in the figure Figure 5 As shown in A. In the above experimental groups, the HCMV infection dose was MOI = 0.5, and the concentration of magnolin was 10 μM. Viral DNA extraction, primer sequences, and qPCR procedures were the same as those in Example 4 above.

[0052] Figure 5 B is the qPCR detection of the immediate early genes in the five different drug addition experimental groups. UL123 , early genes UL44 and late genes UL32 The results showed that all five treatments could reduce the DNA copy number, indicating that magnolia resin 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, magnolia resin can also exert an antiviral effect through certain mechanisms.

[0053] 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

1. Application of magnolin 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 magnolin 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 Magnoliopsin can exert anti-HCMV effects both before and after the virus enters the cell.

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

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

6. The use according to claim 1, wherein A therapeutically effective amount of magnoliaside is combined with a pharmaceutically acceptable carrier, diluent, and excipient, and is used alone or with other substances to prepare anti-HCMV drugs.

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.

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