Application of Trametes sanguinea polysaccharide in preparing drug for inhibiting HCMV infection

Abstract

The present invention provides the use of Trametes sanguinea polysaccharide in the preparation of a drug for inhibiting HCMV infection. The Trametes sanguinea polysaccharide refers to the total Trametes sanguinea polysaccharide TSL-TP or the Trametes sanguinea polysaccharide TSL1. The total Trametes sanguinea polysaccharide TSL-TP is a mixture of the Trametes sanguinea polysaccharides TSL1 and TSL2 in any proportion. The present invention has a significant preventive and therapeutic effect on HCMV, with small toxic and side effects, being safe and effective. It can be used for HCMV infection and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of polysaccharide from Trametes sanguinea in the preparation of a drug for inhibiting HCMV infection. Background Art

[0002] Human cytomegalovirus (HCMV) is a member of the beta subfamily of herpesviruses, with a 235-kb double-stranded DNA (dsDNA) genome encoding at least 165 proteins. HCMV can infect a variety of cell types, including fibroblasts, epithelial cells, macrophages, endothelial cells, dendritic cells, and smooth muscle cells, by using its surface pentamers and corresponding cellular receptors. HCMV infection in immunocompetent individuals is mostly asymptomatic or has very mild symptoms. However, in immunocompromised individuals, including organ transplant and HIV-infected patients, HCMV infection can lead to a high mortality rate. In pregnant women, due to changes in endocrine and metabolic functions, latent HCMV is easily activated, resulting in recurrent HCMV infection. At the same time, HCMV is the main pathogen causing congenital diseases in newborns, mainly manifested as microcephaly, cognitive impairment, vision loss, sensorineural hearing loss, etc. In addition, due to the decline of immune function with age in the elderly, the probability of recurrent HCMV infection increases. At the same time, due to various underlying diseases in the elderly themselves, HCMV infection is likely to exacerbate the condition, posing a major threat to the life and quality of life of the elderly. Although great progress has been made in the research and development of HCMV vaccines in recent years, unfortunately, no HCMV vaccine has been approved for marketing so far. Antiviral drugs such as foscarnet, cidofovir, fomivirsen, ganciclovir and its prodrug valganciclovir used clinically mainly target DNA polymerase and act on the replication and translation stages after the virus invades target cells. However, long-term use of these drugs has obvious adverse reactions such as bone marrow suppression, nephrotoxicity, and electrolyte disorders. In addition, the emergence of drug-resistant strains and mutant strains has also reduced the efficacy of these drugs.

[0003] Trametes sanguinea is a medicinal fungus belonging to the genus Trametes of the family Polyporaceae, growing on fallen logs, stumps, and rotten woods of various broad-leaved forests. Its fruiting body is laterally sessile, corky, the pileus is flat to depressed-globose, solitary to imbricate, and the color is mostly orange to red, with a few wrinkles. The dried fruiting body can be used as medicine, with the effects of dispelling wind and dampness, clearing heat and detoxifying. Polysaccharide is an important active ingredient in fungi, with characteristics such as safety, non-toxicity, and diverse biological activities. Polysaccharides can affect the intestinal ecology and physiology of the body, and have effects such as anticoagulation, anti- Alzheimer's disease, and prevention of Parkinson's disease. However, so far, there has been no application of polysaccharide from Trametes sanguinea in the preparation of drugs for preventing and / or treating HCMV. Summary of the Invention

[0004] To solve the deficiencies in the above-mentioned existing technologies, the object of the present invention is to provide a drug with low toxicity and side effects, which is safe and effective in inhibiting HCMV.

[0005] The object of the present invention is achieved through the following technical solutions:

[0006] The present invention provides the application of Trametes sanguinea polysaccharide in the preparation of a drug for inhibiting HCMV infection. The Trametes sanguinea polysaccharide refers to the total polysaccharide TSL-TP of Trametes sanguinea or the polysaccharide TSL1 of Trametes sanguinea. The total polysaccharide TSL-TP of Trametes sanguinea is a mixture of polysaccharide TSL1 and TSL2 of Trametes sanguinea in any proportion.

[0007] The polysaccharide TSL1 of Trametes sanguinea is composed of mannose, glucose, galactose and fucose, and the molar ratio is 1.03:1.57:2.54:1.00. The polysaccharide TSL2 of Trametes sanguinea is composed of mannose, glucuronic acid, glucose and galactose, and the molar ratio is 1.87:1.00:94.20:4.22.

[0008] The preparation method of the polysaccharide TSL1 of Trametes sanguinea includes the following steps: Take the fruiting body of Trametes sanguinea, add 5-6 times the volume of 95% ethanol and reflux for defatting 3 times at a frequency of 1 h / time. After boiling, keep it in a slightly boiling state. After drying the defatted Trametes sanguinea, crush it. Take the powder, add pure water, stir magnetically for 4 h and then centrifuge. After centrifugation, concentrate the supernatant to 1 / 10 of the original volume, stir and slowly add it to 5 times the volume of 95% ethanol, place it in a 4°C refrigerator overnight, centrifuge at 6000 rpm for 10 min. Take the precipitate, add 3 times the volume of anhydrous ethanol, ether and acetone, stir and wash it thoroughly and then centrifuge again. Dry the obtained precipitate at 50°C. After drying, place the sample in a dialysis bag with a molecular weight cut-off of 3000, dialyze with running water until the volume no longer changes, centrifuge at 6000 rpm for 10 min, take the supernatant, concentrate it and then freeze-dry it to obtain the cold-water extracted polysaccharide TSL1 of Trametes sanguinea.

[0009] The preparation method of the polysaccharide TSL2 of Trametes sanguinea includes the following steps: Take the residue of the cold-water extraction of the powder of Trametes sanguinea, add pure water, stir magnetically in a water bath at 100°C for 1 h, cool and then filter by suction. Repeat the extraction 2 times, combine the filtrates, concentrate to 1 / 10 of the original volume, stir and slowly add 95% ethanol to 5 times the volume, place it in a 4°C refrigerator overnight, centrifuge at 6000 rpm for 10 min. Take the precipitate, add 2 times the volume of anhydrous ethanol, ether and acetone, stir and wash it thoroughly and then centrifuge again. Dry the obtained precipitate at 50°C. After drying, place the sample in a dialysis bag with a molecular weight cut-off of 3000, dialyze with running water until the volume no longer changes, centrifuge at 6000 rpm for 10 min, take the supernatant, concentrate it and then freeze-dry it to obtain the hot-water extracted polysaccharide TSL2 of Trametes sanguinea.

[0010] The inhibition of HCMV refers to the inhibitory effect on the DNA copy numbers of HCMV immediate-early proteins IE1 / 2, early protein UL44, and immediate-early gene UL123, early gene UL44, and late gene pp150.

[0011] The medicament of the present invention further includes pharmaceutically acceptable carriers, diluents, and excipients. Pharmaceutically acceptable carriers include any and all substances compatible with the administration of the medicament, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other substances and compounds compatible with the administration of the medicament. Unless a particular conventional medium or reagent is incompatible with the active compound, its use in the compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0012] Useful pharmaceutical carriers for preparing its compositions can be solid, liquid, or gaseous; thus, the compositions can be in the form of tablets, pills, capsules, suppositories, powders, enteric-coated or other protected formulations (such as bound to ion exchange resins or encapsulated in lipoprotein vesicles), sustained-release formulations, solutions, suspensions, elixirs, aerosols, etc. The carriers can be selected from different oils, including petroleum, animal oils, vegetable oils, or oils of synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water, saline, aqueous dextrose, and ethylene glycol are preferred liquid carriers, especially (when isotonic with blood) for injectable solutions. For example, a formulation for intravenous administration includes a sterile aqueous solution of the active ingredient, which is produced by dissolving the solid active ingredient in water and rendering the solution sterile. Suitable pharmaceutical excipients include starch, cellulose, talc, talc, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, anhydrous skim milk, glycerol, propylene glycol, water, ethanol, etc. Conventional pharmaceutical additives such as preservatives, stabilizers, wetting agents or emulsifiers, salts for adjusting osmotic pressure, buffers, etc. can be added to the compositions. In any case, such compositions will contain an effective amount of the active compound together with a suitable carrier to prepare a proper dosage form for proper administration to a recipient.

[0013] The medicament of the present invention can be administered in any convenient form of administration, such as, for example, tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, gels, emulsions, gels, patches, etc. Such compositions can contain conventional ingredients in pharmaceutical formulations, such as, for example, diluents, carriers, pH regulators, sweeteners, fillers, and other active agents.

[0014] To administer the Trametes cinnabarina polysaccharide of the present invention other than parenterally, it may be necessary to coat the Trametes cinnabarina polysaccharide with a material that prevents its inactivation or administer it together with the Trametes cinnabarina polysaccharide. Supplementary active compounds may also be added to the composition. In a specific implementation, the Trametes cinnabarina polysaccharide of the present invention is co-formulated and / or co-administered with one or more other therapeutic drugs that can be used to treat diseases. Such combination use can advantageously utilize the therapeutic drugs administered at a lower dose, thus avoiding possible toxicity or complications associated with various single therapies.

[0015] The preparation of the present invention can be made into one or more of liquid preparations such as aqueous solutions, oil suspensions or other liquid preparations, such as one or more of syrups or elixirs, etc.; when used for parenteral administration, it can be made into one or more of injection solutions, aqueous solutions or oily suspensions, etc.

[0016] Among the above-mentioned forms of use, the preferred forms are one or more of tablets, coated tablets, capsules, suppositories or injections, etc., and further preferably one or more of tablets, capsules or injections, etc.

[0017] As an alternative, the dosage form can be a powder for injection. Generally, the powder for injection is prepared by the conventional freeze-drying method using water as a solvent. The steps are as follows: take Trametes cinnabarina polysaccharide, add excipients, dissolve in water, add activated carbon, filter and sterilize, fill, semi-cork, freeze-dry, and then cork and crimp the cap. The excipients used are selected from one or several of mannitol, hydrolyzed gelatin, glucose, lactose, dextran, etc.

[0018] As an alternative, the powder for injection of the present invention can also be prepared by the spray-drying method using water as a solvent. The steps are as follows: take Trametes cinnabarina polysaccharide, with or without excipients, dissolve in water, add activated carbon, filter and sterilize, spray-dry, aseptically sub-pack, and then cork and crimp the cap.

[0019] As an alternative of the present invention, the dosage form can be a small injection. The small injection can be prepared by formulating with injection water as a solvent, or appropriate excipients can be added. The excipients are selected from one or several of ethanol, propylene glycol, glycerol, polyethylene glycol, benzyl benzoate, dimethylacetamide.

[0020] The pharmaceutical compositions of the polysaccharides from Trametes sanguinea generally must be sterile and stable under the conditions of production and storage. The compositions can be formulated into solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions are prepared by adding the required amount of the polysaccharides from Trametes sanguinea to a suitable solvent together with one or a combination of the above-mentioned required ingredients and then subjecting the mixture to sterile filtration. Generally, dispersions are prepared by adding the polysaccharides from Trametes sanguinea to a sterile vehicle containing a basic dispersion medium and the above-mentioned other required ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the recommended methods of preparation are vacuum drying and freeze-drying. For example, the appropriate fluidity of the solution can be maintained by coating with, such as lecithin, by maintaining the required particle size in the case of dispersions and by using surfactants. Prolonged absorption of the injectable compositions can be achieved by including agents that delay absorption (such as monostearate or gelatin) in the compositions.

[0021] The present invention utilizes a total polysaccharide TSL-TP from Trametes sanguinea and its components, polysaccharides TSL1 and TSL2 from Trametes sanguinea, to conduct a series of biological experimental studies, providing a scientific basis for the use of the total polysaccharide TSL-TP from Trametes sanguinea and its components, polysaccharides TSL1 and TSL2 from Trametes sanguinea, in preventing and treating human cytomegalovirus infection.

[0022] The research results of the biological experiments of the present invention show that:

[0023] 1. When the total polysaccharide TSL-TP from Trametes sanguinea is used to treat the HCMV host cells WI-38 alone, it does not show obvious cytotoxicity and inhibitory cell proliferation activity against WI-38 at concentration gradients of 1 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 50 μg / ml, and 100 μg / ml; polysaccharides TSL1 and TSL2 from Trametes sanguinea do not show cytotoxicity and inhibitory cell proliferation activity against WI-38 at concentration gradients of 1 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml, and will show cytotoxicity and inhibitory cell proliferation activity when the concentration reaches 50 μg / ml and above.

[0024] 2. The total polysaccharide TSL-TP from Trametes sanguinea and polysaccharide TSL1 from Trametes sanguinea at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml can alleviate the cytopathic effect of WI-38 cells caused by HCMV; polysaccharide TSL2 from Trametes sanguinea at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml cannot alleviate the cytopathic effect of WI-38 cells caused by HCMV.

[0025] 3. The total polysaccharide TSL-TP of Trametes sanguinea can significantly inhibit the expression of HCMV immediate-early protein IE1 / 2 and early protein UL44 in WI-38 at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml; the polysaccharide TSL1 of Trametes sanguinea can significantly inhibit the expression of HCMV immediate-early protein IE1 / 2 and early protein UL44 in WI-38 at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml, while the polysaccharide TSL2 of Trametes sanguinea can inhibit the expression of HCMV immediate-early protein IE1 / 2 to 70% level at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml, and has no significant effect on the expression of early protein UL44.

[0026] 4. The total polysaccharide TSL-TP of Trametes sanguinea can significantly reduce the DNA copy numbers of HCMV immediate-early gene UL123, early gene UL44, and late gene pp150 at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml. At a concentration of 20 μg / ml, its inhibition rate can reach 85%; the polysaccharide TSL1 of Trametes sanguinea can significantly inhibit the DNA copy numbers of HCMV immediate-early gene UL123, early gene UL44, and late gene pp150 at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml. At a concentration of 20 μg / ml, its inhibition rate can reach 85%; while the polysaccharide TSL2 of Trametes sanguinea can inhibit HCMV immediate-early gene UL123 at concentrations of 10 μg / ml and 20 μg / ml, but there is no significant difference in the DNA copy numbers of early gene UL44 and late gene pp150.

[0027] The advantages and beneficial effects of the present invention are as follows: The present invention provides the application of the polysaccharide of Trametes sanguinea in the preparation of a drug for inhibiting HCMV infection. The total polysaccharide TSL-TP of Trametes sanguinea and the polysaccharide TSL1 of Trametes sanguinea have significant inhibitory effects on the HCMV immediate-early protein IE1 / 2, early protein UL44, and the DNA copy numbers of immediate-early gene UL123, early gene UL44, and late gene pp150, have significant preventive and therapeutic effects on HCMV, have small toxic and side effects, are safe and effective, can be used for HCMV infection, and have good application prospects. Brief Description of the Drawings

[0028] Figure 1 It shows the effects of the total polysaccharide TSL-TP of Trametes sanguinea, the polysaccharide TSL1 of Trametes sanguinea, and the polysaccharide TSL2 of Trametes sanguinea on the cytotoxicity and cell proliferation activity of WI-38 cells measured by the CCK8 method. Among them, A shows the effect of the total polysaccharide TSL-TP of Trametes sanguinea on the cell proliferation activity of WI-38 cells;

[0029] Effect of Total Polysaccharide TSL-TP from Trametes sanguinea on the Cytotoxicity of WI-38 Cells;

[0030] Effect of Polysaccharide TSL1 from Trametes sanguinea on the Proliferation Activity of WI-38 Cells;

[0031] Effect of Polysaccharide TSL1 from Trametes sanguinea on the Cytotoxicity of WI-38 Cells;

[0032] Effect of Polysaccharide TSL2 from Trametes sanguinea on the Proliferation Activity of WI-38 Cells;

[0033] Effect of Polysaccharide TSL2 from Trametes sanguinea on the Cytotoxicity of WI-38 Cells;

[0034] Compared with the control group without polysaccharide treatment, *P<0.05, **P<0.01, ***P<0.001.

[0035] Figure 2 This shows the effects of total polysaccharide TSL-TP from Trametes sanguinea, polysaccharide TSL1 from Trametes sanguinea, and polysaccharide TSL2 from Trametes sanguinea on the cytopathic effects caused by HCMV-infected human embryonic lung fibroblasts WI-38. Among them, 1-Control group cells (control), not infected with HCMV;

[0036] 2-Cells infected with HCMV (MOI 0.5) for 5 days (5 dpi);

[0037] 3-Foscarnet (PFA, 200 μg / ml) + Cells infected with HCMV for 5 days (5 dpi);

[0038] 4-TSL-TP (5 μg / ml) + Cells infected with HCMV for 5 days (5 dpi);

[0039] 5-TSL-TP (10 μg / ml) + Cells infected with HCMV for 5 days (5 dpi);

[0040] 6-TSL-TP (20 μg / ml) + Cells infected with HCMV for 5 days (5 dpi);

[0041] 7-TSL1 (5 μg / ml) + Cells infected with HCMV for 5 days (5 dpi);

[0042] 8-TSL1 (10 μg / ml) + Cells infected with HCMV for 5 days (5 dpi);

[0043] 9-TSL1 (20 μg / ml) + Cells infected with HCMV for 5 days (5 dpi);

[0044] 10-TSL2 (5 μg / ml) + Cells infected with HCMV for 5 days (5 dpi);

[0045] 11 - TSL2 (10 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi);

[0046] 12 - TSL2 (20 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi).

[0047] Figure 3 Effect of different concentrations of TSL-TP on immediate-early protein IE1 / 2 and early protein UL44 of HCMV determined by Western-blot method (GAPDH as internal reference); in Figure A:

[0048] 1 - Cells of negative control group (control), not infected with HCMV, without TSL-TP treatment;

[0049] 2 - HCMV (MOI 0.5) alone-infected cells 5 days post-infection (5 dpi);

[0050] 3 - Foscarnet (PFA, 200 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi);

[0051] 4 - TSL-TP (5 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi);

[0052] 5 - TSL-TP (10 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi);

[0053] 6 - TSL-TP (20 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi).

[0054] In Figure B:

[0055] 1 - HCMV (MOI 0.5) alone-infected cells 5 days post-infection (5 dpi);

[0056] 2 - Foscarnet (PFA, 200 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi);

[0057] 3 - TSL-TP (5 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi);

[0058] 4 - TSL-TP (10 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi);

[0059] 5 - TSL-TP (20 μg / ml) + HCMV-infected cells 5 days post-infection (5 dpi).

[0060] Compared with the group of cells infected with HCMV alone, *P<0.05, **P<0.01, ***P<0.001.

[0061] Figure 4 It is a figure showing the effects of different concentrations of TSL1 and TSL2 on the immediate-early protein IE1 / 2 and early protein UL44 of HCMV determined by Western-blot method (GAPDH as the internal reference); among them, Panel A:

[0062] 1 - Cells in the negative control group (control), not infected with HCMV and without polysaccharide treatment;

[0063] 2 - Cells infected with HCMV (MOI 0.5) alone for 5 days (5 dpi);

[0064] 3 - Cells infected with phosphonoformic acid (PFA, 200 μg / ml) + HCMV for 5 days (5 dpi);

[0065] 4 - Cells infected with TSL1 (5 μg / ml) + HCMV for 5 days (5 dpi);

[0066] 5 - Cells infected with TSL1 (10 μg / ml) + HCMV for 5 days (5 dpi);

[0067] 6 - Cells infected with TSL1 (20 μg / ml) + HCMV for 5 days (5 dpi);

[0068] 7 - Cells infected with TSL2 (5 μg / ml) + HCMV for 5 days (5 dpi);

[0069] 8 - Cells infected with TSL2 (10 μg / ml) + HCMV for 5 days (5 dpi);

[0070] 9 - Cells infected with TSL2 (20 μg / ml) + HCMV for 5 days (5 dpi).

[0071] Panel B:

[0072] 1 - Cells infected with HCMV (MOI 0.5) alone for 5 days (5 dpi);

[0073] 2 - Cells infected with phosphonoformic acid (PFA, 200 μg / ml) + HCMV for 5 days (5 dpi);

[0074] 3 - Cells infected with TSL1 (5 μg / ml) + HCMV for 5 days (5 dpi);

[0075] 4 - Cells infected with TSL1 (10 μg / ml) + HCMV for 5 days (5 dpi);

[0076] 5-TSL1 (20 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi);

[0077] 6-TSL2 (5 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi);

[0078] 7-TSL2 (10 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi);

[0079] 8-TSL2 (20 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi).

[0080] Compared with the group of cells infected with HCMV alone, **P < 0.01, ***P < 0.001.

[0081] Figure 5 This is Figure A in the determination of the effect of different concentrations of Trametes sanguinea polysaccharide on the copy number of HCMV DNA by qPCR method; the effects of different concentrations of TSL-TP on the immediate-early gene UL123, early gene UL44, and late gene pp150 of HCMV, where,

[0082] 1-HCMV (MOI 0.5) alone-infected cells at 5 days post-infection (5 dpi);

[0083] 2-Foscarnet (PFA, 200 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi);

[0084] 3-TSL-TP (5 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi);

[0085] 4-TSL-TP (10 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi);

[0086] 5-TSL-TP (20 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi).

[0087] Compared with the group of cells infected with HCMV alone, *P < 0.05, **P < 0.01, ***P < 0.001.

[0088] Figure B: The effects of different concentrations of TSL1 and TSL2 on the immediate-early gene UL123, early gene UL44, and late gene pp150 of HCMV, where,

[0089] 1-HCMV (MOI 0.5) alone-infected cells at 5 days post-infection (5 dpi);

[0090] 2-Foscarnic acid (PFA, 200 μg / ml) + HCMV-infected cells at 5 days post-infection (5 dpi);

[0091] 5 days after HCMV-infected cells were treated with 3-TSL1 (5 μg / ml) (5 dpi);

[0092] 5 days after HCMV-infected cells were treated with 4-TSL1 (10 μg / ml) (5 dpi);

[0093] 5 days after HCMV-infected cells were treated with 5-TSL1 (20 μg / ml) (5 dpi);

[0094] 5 days after HCMV-infected cells were treated with 6-TSL2 (5 μg / ml) (5 dpi);

[0095] 5 days after HCMV-infected cells were treated with 7-TSL2 (10 μg / ml) (5 dpi);

[0096] 5 days after HCMV-infected cells were treated with 8-TSL2 (20 μg / ml) (5 dpi).

[0097] Compared with the group of cells infected with HCMV alone, *P<0.05, **P<0.01, ***P<0.001. Detailed implementation mode

[0098] The present invention will be further described below in conjunction with specific embodiments. The embodiments of the present invention are only used to explain the present invention and do not mean to limit the protection scope of the present invention.

[0099] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0100] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0101] The human embryonic lung fibroblast cell line WI-38 and human cytomegalovirus (HCMV) Towne strain involved in the present invention are both from the American Type Culture Collection (ATCC).

[0102] Example 1 Preparation of polysaccharide from Trametes sanguinea

[0103] Prepare polysaccharide TSL1 extracted from Trametes sanguinea with cold water:

[0104] Take 2 kg of the fruiting bodies of Trametes sanguinea, add 5 - 6 times the volume of 95% ethanol, and reflux for defatting 3 times at a frequency of 1 h / time. After boiling, maintain a slightly boiling state. After drying the defatted Trametes sanguinea, crush it, take 150 g of the powder, add 3 L of pure water, stir magnetically for 4 h, then centrifuge. After centrifugation, concentrate the supernatant to 1 / 10 of the original volume, stir and slowly add it to 5 times the volume of 95% ethanol, and place it in a 4°C refrigerator overnight. Centrifuge at 6000 rpm for 10 min, take the precipitate, add 3 times the volume of absolute ethanol, ether, and acetone, stir well for washing, and centrifuge again. Dry the obtained precipitate at 50°C. Place the dried sample in a dialysis bag with a molecular weight cut-off of 3000, dialyze with running water until the volume no longer changes, centrifuge at 6000 rpm for 10 min, take the supernatant, concentrate it, and then freeze-dry to obtain the cold-water extracted polysaccharide TSL1, with a yield of 2.06%.

[0105] Preparation of the hot-water extracted polysaccharide TSL2 from Trametes sanguinea:

[0106] Take the residue of the cold-water extraction of the powder of Trametes sanguinea, add 3 L of pure water, stir magnetically in a water bath at 100°C for 1 h, cool and then filter by suction. Repeat the extraction 2 times, combine the filtrates, concentrate to 1 / 10 of the original volume, stir and slowly add 95% ethanol to 5 times the volume, and place it in a 4°C refrigerator overnight. Centrifuge at 6000 rpm for 10 min, take the precipitate, add 2 times the volume of absolute ethanol, ether, and acetone, stir well for washing, and centrifuge again. Dry the obtained precipitate at 50°C. Place the dried sample in a dialysis bag with a molecular weight cut-off of 3000, dialyze with running water until the volume no longer changes, centrifuge at 6000 rpm for 10 min, take the supernatant, concentrate it, and then freeze-dry to obtain the hot-water extracted polysaccharide TSL2, with a yield of 8.05%.

[0107] Example 2 Analysis of the monosaccharide composition of the polysaccharides from Trametes sanguinea

[0108] Accurately weigh 2 mg each of the polysaccharides TSL1 and TSL2 from Trametes sanguinea. Using a decasaccharide standard (mannose, rhamnose, glucosamine, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, fucose) prepared in equimolar amounts as the standard, determine the monosaccharide composition of TSL1 and TSL2 by PMP-HPLC method. The determination results are shown in Table 1 below.

[0109] Table 1: Determination results of the monosaccharide composition of the polysaccharides from Trametes sanguinea

[0110]

[0111] As can be seen from Table 1, both of the two polysaccharides from Trametes sanguinea contain mannose, glucose, and galactose; among them, the four monosaccharides in polysaccharide TSL1 have an average proportion. Compared with TSL2, it contains an additional 20% fucose, while polysaccharide TSL2 mainly contains glucose, with a proportion of 94%.

[0112] Effect of polysaccharide from Trametes sanguinea alone on cytotoxicity and proliferation activity of WI-38 cells

[0113] The effects on cytotoxicity and proliferation activity were detected by CCK-8 method.

[0114] The method for determining cytotoxicity was as follows: Human embryonic lung fibroblast WI-38 cells at PD30 were seeded into 96-well cell culture plates at 5000 cells per well. The next day, after the cells grew to confluence, different concentrations of polysaccharide from Trametes sanguinea were added. Three parallel wells were set for each concentration, and a blank control group without polysaccharide and a cell-free control group were set. After culturing for 5 days, 10 μl of CCK-8 solution was added to each well, taking care not to introduce air bubbles into the wells to avoid interfering with the OD value measurement. The plates were cultured at 37 °C and 5% CO2 for 1 - 4 h. Before measuring with a microplate reader, the 96-well plates were gently mixed on a shaker, and then the absorbance was measured at 450 nm. The relative cell viability was calculated, with the cell viability of the blank group without polysaccharide set as 1.

[0115] The method for determining cell proliferation activity was as follows: Human embryonic lung fibroblast WI-38 cells at PD30 were seeded into 96-well cell culture plates at 2500 cells per well. The next day, after the cells grew to 25% confluence, different concentrations of polysaccharide from Trametes sanguinea were added. Three parallel wells were set for each concentration, and a blank control group without polysaccharide and a cell-free control group were set. After culturing for 5 days, 10 μl of CCK-8 solution was added to each well, taking care not to introduce air bubbles into the wells to avoid interfering with the OD value measurement. The plates were cultured at 37 °C and 5% CO2 for 1 - 4 h. Before measuring with a microplate reader, the 96-well plates were gently mixed on a shaker, and then the absorbance was measured at 450 nm. The relative cell viability was calculated, with the cell viability of the blank group without polysaccharide set as 1. The experimental results were analyzed as follows: Cell viability = [OD (drug added) - OD (blank)] / [OD (0 drug added) - OD (blank)]. The results of the effects of total polysaccharide of Trametes sanguinea TSLTP and polysaccharides TSL1 and TSL2 of Trametes sanguinea on cytotoxicity and cell proliferation activity are shown in Figure 1 .

[0116] Example 4 Inoculation of HCMV and treatment with total polysaccharide TSL-TP of Trametes sanguinea and polysaccharides TSL1 and TSL2 of Trametes sanguinea

[0117] Human embryonic lung fibroblast WI-38 cells at PD30 were used and cultured in a medium containing 10% FBS at 2×10 4 / cm 2Plate a six-well cell culture plate with the cell quantity, and after 24 hours, replace the medium with 0.2% FBS and continue culturing for 48 hours. Through the method of serum starvation, the cells are synchronized at G0 / G1 at this time, which is beneficial to the infection of HCMV. Then, inoculate with the HCMV virus (Towne virus strain), and the inoculation dose is 0.5 MOI (multiplicity of infection), and continue culturing until the specified time for relevant detections. When detecting the anti-HCMV activity of the polysaccharide from Trametes sanguinea, add a certain concentration of the polysaccharide from Trametes sanguinea to the medium 2 hours in advance, and then inoculate 0.5 MOI HCMV, and then observe the changes in cell morphology, viral protein expression, DNA copy number, etc. at different times.

[0118] Example 5 Effects of the total polysaccharide TSL-TP from Trametes sanguinea and the polysaccharides TSL1 and TSL2 from Trametes sanguinea on the morphology of WI-38 cells after HCMV inoculation

[0119] After WI-38 cells were inoculated with 0.5 MOI HCMV, the cytopathic effect caused by HCMV infection could be observed in the wells infected with HCMV alone three days later. By the fifth day, the cytopathic phenomenon was significant. The total polysaccharide TSL-TP from Trametes sanguinea and the polysaccharide TSL1 from Trametes sanguinea could significantly reduce the cytopathic effect caused by HCMV infection, and the cell morphology was similar to that of the cells treated with the positive drug foscarnet (PFA, 200 μg / ml), and no serious cytopathic results occurred; while the polysaccharide TSL2 from Trametes sanguinea could not reduce the cytopathic effect caused by HCMV infection ( Figure 2 ).

[0120] Example 6 Effects of the total polysaccharide TSL-TP from Trametes sanguinea and the polysaccharides TSL1 and TSL2 from Trametes sanguinea on the expression of HCMV viral proteins

[0121] To confirm the anti-HCMV effects of the total polysaccharide TSL-TP from Trametes sanguinea, as well as the polysaccharides TSL1 and TSL2 from Trametes sanguinea, while observing the cytopathic effects after virus infection, we further detected the effects of these three polysaccharides on the expression of HCMV immediate-early protein 1 / 2 (IE1 / 2) and early protein UL44. Cell culture, polysaccharide treatment, and HCMV inoculation were the same as described in Example 4 above. A blank control group (control) without HCMV inoculation and polysaccharide treatment, a control group with only HCMV inoculation and no polysaccharide treatment, and experimental groups with polysaccharide (5 μg / ml, 10 μg / ml, 20 μg / ml) treatment and HCMV inoculation were established. Cell samples were collected for detection 5 days after HCMV inoculation. First, the supernatant was discarded, the cells were washed three times with PBS, and the cells were lysed with medium-strength RIPA lysis buffer and then the samples were collected. SDS-PAGE protein electrophoresis and membrane transfer were performed by conventional methods, and after blocking with 5% skim milk powder, the corresponding antibodies were used for detection. The results showed that the total polysaccharide TSL-TP from Trametes sanguinea could significantly reduce the expression of HCMV immediate-early protein IE1 / 2 and early protein UL44 at the lowest detected concentration of 5 μg / ml (P<0.05 and P<0.01)( Figure 3 ); the polysaccharide TSL1 from Trametes sanguinea could also significantly reduce the expression of HCMV immediate-early protein IE1 / 2 and early protein UL44 at the three detected concentrations (P<0.001)( Figure 4 ); while the polysaccharide TSL2 from Trametes sanguinea could down-regulate the expression of immediate-early protein IE1 / 2 to 70-80% of the level of single HCMV infection (P<0.05), and the down-regulating effect on early protein UL44 was not significant (P>0.05)( Figure 4 ).

[0122] Example 7 Effects of the total polysaccharide TSL-TP from Trametes sanguinea, as well as the polysaccharides TSL1 and TSL2 from Trametes sanguinea on the DNA copy number of HCMV in host cells

[0123] Cell culture, polysaccharide treatment, and HCMV inoculation treatment were the same as described in Example 4 above. The copy number of HCMV DNA was determined by qPCR. Viral DNA was extracted using the QIAamp DNA Mini Kit, and qPCR was performed using 2× Universal SYBR Green Fast qPCR Mix. The primers used were as follows: HCMV UL123 - forward 5-TCTGCCAGGACATCTTTCTC-3 and reverse 5-GTGACCAAGGCCACGACGTT-3; HCMV UL44 - forward 5-ACTGCCGTGCACGTTGCGTA-3 and reverse 5-ACTTGCCGCTGTTCCCGACG-3; HCMV pp150 - forward 5-GGTTTCTGGCTCGTGGATGTCG-3 and reverse 5-CACACAACACCGTCGTCCGATTAC-3; GAPDH - forward 5-CTGTTGCTGTAGCCAAATTCGT-3 and reverse 5-ACCCACTCCTCCACCTTTGAC-3. Amplification conditions: 95°C for 5 min, (95°C for 5 sec, 60°C for 30 sec) × 40 cycles. After 2 -△△Ct calculations by the Figure 5 method, the DNA copy number of the sample infected with HCMV alone was set to 1, and the results are as

[0124] The specific embodiments of the present invention have been described above, but are not intended to limit the present invention. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. Use of Trametes sanguinea polysaccharide in the preparation of a drug for inhibiting HCMV infection. The Trametes sanguinea polysaccharide refers to Trametes sanguinea polysaccharide TSL1. The Trametes sanguinea polysaccharide TSL1 is composed of mannose, glucose, galactose and fucose, and the molar ratio is 1.03:1.57:2.54:1.

00. The preparation method of the Trametes sanguinea polysaccharide TSL1 includes the following steps: Take the fruiting body of Trametes sanguinea, add 5-6 times the volume of 95% ethanol and reflux for defatting 3 times at a frequency of 1 h / time. After boiling, keep it in a slightly boiling state. After drying the defatted Trametes sanguinea, crush it. Take the powder, add pure water, stir magnetically for 4 h and then centrifuge. After centrifugation, concentrate the supernatant to 1 / 10 of the original volume, stir and slowly add it to 5 times the volume of 95% ethanol, place it in a refrigerator at 4 °C overnight, centrifuge at 6000 rpm for 10 min. Take the precipitate, add 3 times the volume of absolute ethanol, ether and acetone, stir and wash thoroughly and centrifuge again. Dry the obtained precipitate at 50 °C. Place the dried sample in a dialysis bag with a molecular weight cut-off of 3000, dialyze with running water until the volume no longer changes, centrifuge at 6000 rpm for 10 min, take the supernatant, concentrate and freeze-dry to obtain Trametes sanguinea cold-water extract polysaccharide TSL1.

2. The application according to claim 1, wherein The inhibition of HCMV refers to the inhibitory effect on the DNA copy numbers of HCMV immediate-early protein IE1 / 2, early protein UL44, and immediate-early gene UL123, early gene UL44 and late gene pp150.

3. The application according to claim 1, characterized in that The drug also includes a pharmaceutically acceptable carrier, diluent, excipient.

4. The application according to claim 1, wherein The preparation of the drug is a liquid preparation, solid preparation or semi-solid preparation.

Images