A uniform polysaccharide of russula sanguinea with anti-human cytomegalovirus effect and a preparation method and application thereof

Abstract

This invention discloses a homogeneous polysaccharide from *Rhizoctonia solani* that exhibits antiviral activity against human cytomegalovirus in both the virus-cell binding stage and the post-virus invasion stage, along with its preparation method and applications. This polysaccharide is a homogeneous polysaccharide Ts1-1A extracted, isolated, and purified from the fruiting bodies of *Rhizoctonia solani*. *Rhizoctonia solani* homogeneous polysaccharide Ts1-1A is a novel heteropolymer containing partially branched chains, with its main chain consisting of →3)-α-Fuc p -(1→and→6)-α-Gal p -(1→ Composed in a 1:1 ratio, with T-α-Man branches p -(1→, connected to the 2nd and 3rd positions of the same Gal on the main chain, with an average of 2 branches replacing every 16 sugar residues in the main chain. During the viral attachment period, it can prevent the virus from adsorbing onto cell surface receptors by binding to the virus. At the same time, after entering the cell, it can inhibit viral replication through antioxidant effects, thereby exerting an antiviral effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically a homogeneous polysaccharide of Hemoglobinobacterium with anti-human cytomegalovirus activity, its preparation method and application. Background Technology

[0002] Human cytomegalovirus (HCMV), also known as human herpesvirus 5, belongs to the β-herpesvirus subfamily of DNA viruses, and its genetic material is double-stranded DNA. HCMV is widespread worldwide. Although it usually manifests as asymptomatic infection in healthy individuals, in newborns and immunocompromised individuals, HCMV can infect various types of cells and organs, developing into overt end-organ diseases with inflammation as the main pathological manifestation, such as gastrointestinal ulcers, hepatitis, pneumonia, and retinitis. It can further induce autoimmune diseases, cardiovascular diseases, and cancer, leading to a high mortality rate after HCMV infection.

[0003] Currently, there is no vaccine for the prevention and treatment of HCMV; drug therapy is the only treatment option. Ganciclovir (GCV), foscarnet (FOS or PFA), and cidofovir (CDV) are clinically approved anti-HCMV drugs that inhibit viral replication by interfering with viral DNA synthesis. However, the toxicity and drug resistance exhibited by these approved drugs in long-term use limit their use, thus necessitating new treatment methods. In recent years, natural polysaccharides have received increasing attention due to their diverse biological activities and low toxicity. Increasing evidence suggests that natural sugar components from plants, animals, and microorganisms, especially sulfated polysaccharides from algae, exhibit good antiviral activity against various pathogenic viruses, with mechanisms including direct viral killing, interference with viral replication, and activation of antiviral immune responses. Fungal polysaccharides, important active components in fungal fruiting bodies, hold great potential for the discovery of highly effective and low-toxicity antiviral substances.

[0004] *Trametes sanguinea*, a medicinal fungus belonging to the genus *Trametes* in the family Polyporaceae, is used medicinally as its dried fruiting body. It has a slightly pungent and astringent taste, and is warm in nature, possessing properties such as dispelling wind and dampness, clearing heat and detoxifying. Modern pharmacological studies have shown that *Trametes sanguinea* extract possesses various biological activities, such as antitumor, immune enhancement, antibacterial, and antiviral activity. Polysaccharides are the main active substances responsible for its antitumor effects. Our research group has been dedicated to the study of the active substances and mechanisms of pharmacologically active fungi such as *Trametes sanguinea* for many years, and has conducted relatively systematic research on the pharmacological basis of *Trametes sanguinea*. Previously, a novel glucan was identified from the hot water extract of *Trametes sanguinea*, which can exert an immune-enhancing effect by activating Toll-like receptor 4 (TLR4). In this study, using activity-directed separation technology, a novel heteropolysaccharide was purified from the cold water extract of *Trametes sanguinea*, exhibiting good anti-human cytomegalovirus activity. Research on the structure and activity of Hemoglobin polysaccharides can promote the application of such substances in antiviral drugs, further explore medicinal fungal resources, broaden the screening range of antiviral drugs, and provide a scientific basis for the development and utilization of Hemoglobin polysaccharide resources. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a homogeneous polysaccharide of *Hemibacillus erythropoietinus* with antiviral activity, its preparation method, and its applications. The homogeneous polysaccharide of *Hemibacillus erythropoietinus* in this invention exhibits significant antiviral activity and shows no cytotoxicity to human diploid fibroblast WI-38 cells; its high efficiency and low toxicity demonstrate its unique advantages as an antiviral drug.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0007] A homogeneous polysaccharide of *Hemothorax*, characterized by a structure as shown in formula (I):

[0008]

[0009] The homogeneous polysaccharide of *Hemiglossum* is a heterosaccharide containing a small number of branched chains. Its main chain is composed of →3)-α-Fucp-(1→ and →6)-α-Galp-(1→) in a 1:1 ratio, and the branched chain is T-α-Manp-(1→), which is attached to the 2nd and 3rd positions of Gal on the main chain. On average, there are 2 branched chains replacing every 16 sugar residues on the main chain. The homogeneous polysaccharide of *Hemiglossum* obtained in this invention contains heterosaccharides with equal proportions of Fuc and Gal in the main chain structure. Such a structure has not been reported in the literature of *Hemiglossum*.

[0010] The homogeneous polysaccharide from *Hemiglossum* can be prepared through the following steps:

[0011] (1) The fruiting bodies of Hemothromyces are defatted, dried and pulverized to obtain defatted powder of Hemothromyces;

[0012] (2) The defatted powder of Hemothromyces is extracted by cold soaking in distilled water. The extract is then concentrated, precipitated with alcohol, dialyzed, centrifuged and freeze-dried to obtain the total sugar TsL1 extracted from Hemothromyces by cold water.

[0013] (3) The homogeneous polysaccharide Ts1-1A of Hemoglobinococcus was obtained after separation and purification by anion exchange chromatography and high performance gel permeation chromatography.

[0014] Preferably, in step (1), the fruiting bodies of Hemoglobinus are taken and refluxed with 5 to 6 times the amount of 95% ethanol three times, 1 hour each time.

[0015] Preferably, in step (2), the Hemoglobinus thrombocytogenetic powder is extracted with 20 times the amount of pure water at room temperature by magnetic stirring for 4 hours, filtered to obtain a cold water extract, and then concentrated, precipitated with alcohol, dialyzed, centrifuged and freeze-dried to obtain Hemoglobinus thrombocytogenetic polysaccharide TsL1. The concentration operation is to concentrate the original volume to 1 / 10 of the original volume, and the dialysis bag used in the dialysis operation is a dialysis bag with a molecular weight cutoff of 2000.

[0016] Preferably, in step (3), the polysaccharide TsL1 of *Hemanthotomyces rubrum* is initially separated and purified by an anion exchange column. The obtained component is then dialyzed, concentrated, and lyophilized to obtain the initially separated polysaccharide Ts1-1. It is then further separated and purified by a gel chromatography column and lyophilized to obtain the homogeneous polysaccharide Ts1-1A of *Hemanthotomyces rubrum*. The concentration operation is to concentrate the original volume to 1 / 10 of the original volume. The dialysis bag used in the dialysis operation is a dialysis bag with a molecular weight cutoff of 2000. The packing material used in the anion exchange column is DEAE-agarose. The mobile phase used is an aqueous solution. The packing material used in the gel column is dextran G-50 gel with a separation range of 1500-30000.

[0017] The present invention also provides the application of the aforementioned Hemolymphoidus homogeneous polysaccharide, which has anti-human cytomegalovirus activity.

[0018] The application of the aforementioned Hemoglobinus homogeneous polysaccharide allows it to exert antiviral effects in both the viral adsorption stage and the stage after viral entry into cells.

[0019] The application of the aforementioned Hemoglobin homogeneous polysaccharide is that the Hemoglobin homogeneous polysaccharide prevents the virus from adsorbing onto the host cell surface receptor by binding to the virus.

[0020] The polysaccharide and pharmaceutical composition of the present invention can be used to prepare antiviral drugs. Using human diploid fibroblast WI-38 cells inoculated with human cytomegalovirus as an experimental model, the antiviral activity of *Heliotropium indicum* homogeneous polysaccharide was evaluated. The results showed that *Heliotropium indicum* homogeneous polysaccharide had no cytotoxic effect on human diploid fibroblast WI-38 cells and exerted antiviral effects both before and after viral entry into the cells. By detecting changes in virus-related protein and DNA copy numbers after pretreatment of cells with *Heliotropium indicum* homogeneous polysaccharide or after virus pretreatment, it was found that virus pretreatment with *Heliotropium indicum* homogeneous polysaccharide significantly reduced viral protein and DNA copy numbers, indicating that the antiviral effect exerted before viral entry into cells is related to the interaction between *Heliotropium indicum* homogeneous polysaccharide and the virus. Simultaneously, by measuring the expression of downstream proteins quinone NADH dehydrogenase 1 (NQO1) and heme oxygenase 1 (HO-1) in the Keap1-Nrf2 pathway, it was found that both *Hemanthotomyces hemangiomae* homogeneous polysaccharide and the positive control N-acetylcysteine ​​downregulated the levels of NQO1 and HO-1 proteins, indicating that *Hemanthotomyces hemangiomae* homogeneous polysaccharide has antioxidant activity, and its antiviral effect after viral entry into cells is related to antioxidant activity. These results demonstrate that *Hemanthotomyces hemangiomae* homogeneous polysaccharide can exert antiviral effects through different mechanisms at different stages of the viral life cycle, and as a multi-stage antiviral substance, it has significant advantages in the development of antiviral drugs. All cell lines and experimental animals used were commercially available products.

[0021] The hemolymphoid thrombocytogenetic polysaccharide of the present invention can simultaneously block viral attachment and viral replication after entering cells to exert antiviral effects. It can be combined with one or more pharmaceutically acceptable carriers and used alone or with other substances to prepare antiviral drugs and their adjuvants.

[0022] The pharmaceutically acceptable carriers mentioned above refer to drug carriers in the pharmaceutical field. Examples include: diluents and excipients such as water, physiological saline, glucose, mannitol, glycerol, ethanol, and other mixtures; fillers such as starch and sucrose; binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; wetting agents such as glycerol; disintegrants such as calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as Tween-80; and lubricants such as talc, calcium stearate, magnesium stearate, and polyethylene glycol. Other excipients such as flavoring agents and sweeteners may also be added to the composition. The drug can be in the form of decoctions, pills, powders, ointments, granules, oral liquids, capsules, tablets, injections, etc.

[0023] The beneficial effects of this invention are as follows:

[0024] The homogeneous polysaccharide Ts1-1A of *Hemangioblastus* of this invention is a novel heteropolysaccharide containing partial branches. Its main chain is composed of →3)-α-Fucp-(1→ and →6)-α-Galp-(1→) in a 1:1 ratio, with T-α-Manp-(1→) branches attached to the 2nd and 3rd positions of the same Gal on the main chain. During viral attachment, it covalently binds to the virus, preventing viral adsorption to receptors on the host cell surface. After viral invasion, it inhibits viral replication through antioxidant activity, thereby exerting an antiviral effect against human cytomegalovirus. No antiviral polysaccharides with this structure have been reported in the literature. This homogeneous polysaccharide has various potential applications in the biomedical field and is expected to serve as a novel carbohydrate-based antiviral drug or its adjuvant for the prevention and treatment of various diseases.

[0025] Compared with existing drugs, the advantages of the *Heliotropium indicum* homogeneous polysaccharide described in this invention as an antiviral drug or its adjuvant are that the polysaccharide is simple to prepare, has a novel structure, is non-cytotoxic, and exhibits significant antiviral activity. The solvents used in the preparation of the *Heliotropium indicum* polysaccharide are inexpensive, readily available, and non-toxic; the resulting homogeneous polysaccharide has a novel structure, no cytotoxic activity, and significant antiviral activity, making it an ideal lead compound for developing antiviral drugs with promising application prospects. Attached Figure Description

[0026] Figure 1 The structural formula of the homogeneous polysaccharide structure of the *Hemothrombus* is shown below;

[0027] Figure 2 is a schematic diagram of the molecular weight and monosaccharide composition analysis results of the homogeneous polysaccharide of *Hemanthotomyces hemanthotomyces*; where (A) is the high-performance gel permeation chromatogram of the homogeneous polysaccharide of *Hemanthotomyces hemanthotomyces*; (B) is the pre-column derivatization high-performance liquid chromatogram of the homogeneous polysaccharide of *Hemanthotomyces hemanthotomyces*; and (C) is the infrared spectrum of the homogeneous polysaccharide of *Hemanthotomyces hemanthotomyces*.

[0028] Figure 3 shows schematic diagrams of various NMR spectra of the homogeneous polysaccharide of *Hemibacillus thuringiensis*; where (A) is the NMR spectrum of the homogeneous polysaccharide of *Hemibacillus thuringiensis*. 1 (A) ¹H NMR spectrum; (B) shows the homogeneous polysaccharide of the *Hemangioblastus* strain. 13 (C) C NMR and partial DEPT spectra; (C) represents the homogeneous polysaccharide of the *Hemangioblastus*. 1 H- 1 H COSY spectrum; (D) represents the homogeneous polysaccharide of the *Hemangioblastus*. 1 H- 13 C HSQC spectrum; (E) represents the homogeneous polysaccharide of the *Hemangioblastus*. 1 H- 1 H NOESY spectrum;

[0029] Figure 4 is a schematic diagram of the high-order structure of the homogeneous polysaccharide of *Hemangiospermum erythrorhizon*; wherein, (A) is a plan view of the homogeneous polysaccharide of *Hemangiospermum erythrorhizon* under an electron mechanical microscope; and (B) is a three-dimensional view of the homogeneous polysaccharide of *Hemangiospermum erythrorhizon* under an electron mechanical microscope.

[0030] Figure 5 shows the effect of Ts1-1A on normal WI-38 cells and HCMV-infected cells; where (A) is the effect of Ts1-1A on the viability of WI-38 cells, and ns indicates that Ts1-1A has no significant effect on the viability of WI-38 cells; (B) is the effect of Ts1-1A on the morphological changes of cells with or without HCMV infection (top figure) or with infection (bottom figure).

[0031] Figure 6 shows the results of the antiviral activity evaluation of Ts1-1A; (A) shows the levels of HCMV protein IE1 / 2 and UL44 in Ts1-1A-treated cells; (B) shows the quantitative analysis of the results in (A); (C) shows the change in the copy number of HCMV gene UL123 in Ts1-1A-treated cells; (D) shows the results of the IE1 / 2 protein in Ts1-1A-treated cells in the indirect immunofluorescence experiment; (E) shows the quantitative analysis of the results in (D); (F) shows the viral titer in Ts1-1A-treated cells; ns indicates no significant effect; * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001;

[0032] Figure 7 shows the results of the study on the antiviral activity stages and mechanisms of Ts1-1A; (A) is a schematic diagram of the polysaccharide addition time of Ts1-1A against HCMV infection; (B) is the level of HCMV protein IE1 / 2 and UL44 in cells treated with Ts1-1A at different addition times; (C) is a quantitative analysis of the results in (B); (D) is the copy number level of HCMV gene UL123, UL44, and pp150 in cells treated with Ts1-1A; (E) is the level of NQO1 and HO-1 proteins in WI-38 cells infected with HCMV after NAC treatment; (F) is a quantitative analysis of the results in (E); (G) is the level of NQO1 and HO-1 proteins in WI-38 cells infected with HCMV after Ts1-1A treatment; (H) is a quantitative analysis of the results in (G). Detailed Implementation

[0033] Example 1: Preparation of homogeneous polysaccharide from Hemoglobinococcus spp.

[0034] (1) Preparation of TsL1 polysaccharide extracted from Hemoglobinococcus faecium via cold water

[0035] Take 2 kg of *Hemibarbus erythropoietinus* fruiting bodies, add approximately 5-6 times the volume of 95% ethanol, and reflux to defatt the bacteria three times, 1 hour each time. After boiling, maintain a gentle simmer to avoid excessive temperature. After defatting, dry the *Hemibarbus erythropoietinus* and pulverize it. Take 150 g of the powder, add 3000 mL of purified water, and magnetically stir for 4 hours. Repeat the extraction three times. Combine the filtrates, concentrate to 1 / 10 of the original volume, stir, and slowly add to 5 times the volume of 95% ethanol. Let stand overnight at 4°C. Centrifuge at 6000 rpm for 10 minutes to obtain the precipitate. Add 2 times the volume of anhydrous ethanol, ether, and acetone respectively, stir thoroughly to wash the precipitate, and centrifuge. Dry the precipitate in a 50°C oven. After drying, place the sample in a dialysis bag with a molecular weight cutoff of 2000 and dialyze with running water until the volume no longer changes. Centrifuge at 6000 rpm for 10 minutes, collect the supernatant, concentrate, and freeze-dry to obtain the cold-water extracted polysaccharide TsL1, with a yield of 2.06%.

[0036] (2) Preparation of homogeneous polysaccharide Ts1-1A from Hemoglobinococcus spp.

[0037] The polysaccharide TsL1 extracted from cold water was initially separated using a DEAE-Sepharose Fast Flow anion exchange column. Gradient elution was performed with 0, 0.05, 0.1, and 0.2 mol / L NaCl solutions, and the 0 mol / L NaCl solution was collected. The eluent was dialyzed to remove salt, concentrated, and then freeze-dried to obtain the preliminarily separated polysaccharide Ts1-1. Further purification was achieved using a Sephadex G-50 gel chromatography column with pure water as the mobile phase at a flow rate of 1 mL / min, and the fraction was collected using an automated collector. Detection was performed using the phenol-sulfuric acid method, and the corresponding peak tip was collected and freeze-dried to obtain the homogeneous polysaccharide Ts1-1A from *Hemibarbus erythrocyte*.

[0038] Example 2: Molecular weight and monosaccharide composition analysis of homogeneous polysaccharides from *Hemangioblastus*

[0039] (1) Determination of purity and molecular weight of homogeneous polysaccharide from Hemoglobinococcus suis

[0040] A 5 mg / mL solution of homogeneous polysaccharide Ts1-1A from *Hemibarbus erythrocyte* was accurately prepared. The purity and molecular weight of the dextran (Mw: 2700 Da, 5250 Da, 9750 Da, 13050 Da, 36800 Da, 64650 Da) were determined using high-performance gel permeation chromatography (HPGPC) as standards. The results are shown in Figure 2(A).

[0041] As shown in Figure 2(A), the homogeneous polysaccharide Ts1-1A of *Hemiberlesia lataniae* appears as a single component in the HPGPC chromatogram, indicating its high purity; the retention time (t) of the sample... RSubstituting the equation into the standard regression equation y = -0.3575x + 9.1797, we can obtain its molecular weight. The molecular weight of the homogeneous polysaccharide Ts1-1A of Hemoglobinobacterium is calculated to be 12.8 kDa.

[0042] (2) Determination of the monosaccharide composition of homogeneous polysaccharide from Hemoglobinococcus

[0043] Accurately weigh 2 mg of Hemoglobinococcus homogeneous polysaccharide Ts1-1A, and use equimolarly prepared decasaccharide standards (mannose, rhamnose, glucosamine, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose) as standards. The monosaccharide composition of each polysaccharide is determined by PMP-HPLC. The results are shown in Figure 2(B).

[0044] As can be seen from the comparison of the high performance gel permeation chromatogram (a) of Ts1-1A in Figure 2(B) with the pre-column derivatization high performance liquid chromatogram (b) of the monosaccharide standard, the homogeneous polysaccharide Ts1-1A of Hemoglobin contains mannose, galactose, fucose and a small amount of glucose, and is a heterosaccharide.

[0045] Example 3: Structural Study of Homogeneous Polysaccharide from Hemoglobinobacterium

[0046] (1) Determination of the glycosidic bond linkage mode of homogeneous polysaccharide of Hemoglobin

[0047] Methylation analysis of the homogeneous polysaccharide Ts1-1A from *Strombus haematobium* was performed using a modified Hakomori method. Gas chromatography-mass spectrometry (GC-MS) peaks were analyzed using the glycosidic mass spectrometry library (CCSD) from the Center for Glycocomplex Research (CCRC) at the University of Georgia to determine the glycosidic bond linkages in each polysaccharide structure. The results are shown in Table 1.

[0048] Table 1: Methylation data analysis of Hemoglobin polysaccharides

[0049] Methylated sugars Mass spectrometry fragments Area ratio Connection method <![CDATA[2,4-Me2-Fucp]]> 89,101,117,131,233,247 41 1,3-linked Fucp <![CDATA[2,3,4,6-Me4-Manp]]> 71,87,101,117,129,145,161,205 11 1-linked Manp 2-Me-Fucp 87,99,117,129,173,275 1 1,3,4-linked Fucp <![CDATA[2,3,6-Me3-Manp]]> 71,87,101,113,117,129,161,173,233 1 1,4-linked Manp <![CDATA[2,3,4-Me3-Galp]]> 71,87,99,101,117,129,161,173,189,233 39 1,6-linked Galp <![CDATA[2,3-Me2-Galp]]> 85,101,117,127,261 1 1,4,6-linked Galp 4-Me-Galp 87,99,129,159,189,233 6 1,2,3,6-linked Galp

[0050] As shown in Table 1, the homogeneous polysaccharide Ts1-1A exhibits diverse linkage modes, containing a total of 7 linkage modes, including →3)-Fucp-(1→, Manp-(1→, →3,4)-Fucp-(1→, →4)-Manp-(1→, →6)-Galp-(1→, →4,6)-Galp-(1→ and →2,3,6)-Galp-(1→), with an area ratio of 41:11:1:1:39:1:6. Among them, →3)-Fucp-(1→ and →6)-Galp-(1→ are the main linkage modes, indicating that the homogeneous polysaccharide Ts1-1A of Hemothorax is a branched heterosaccharide.

[0051] (2) Identification of the primary structure of homogeneous polysaccharide from Hemoglobinococcus spp.

[0052] Using pure KBr pellets as a blank, an appropriate amount of sample was mixed with KBr powder and pelleted to determine the infrared spectrum of the sample. Figure 2(C) shows the infrared spectrum of the homogeneous polysaccharide of *Hemangiospermum erythrorhizon*. As shown in Figure 2(C), the infrared spectrum of *Hemangiospermum erythrorhizon* homogeneous polysaccharide Ts1-1A contains the characteristic absorption peak of polysaccharides, at 3449.9 cm⁻¹. -1 The strong absorption peak at 2928.0 cm⁻¹ is due to the OH stretching vibration. -1 The peak at 1644.7 cm⁻¹ represents the CH stretching vibration peak. -1 This is a typical OH bending vibration peak; 1378.6 cm⁻¹ -1 The peak at 1200-1000 cm⁻¹ is the CH-angle vibration peak. -1 This is the peak of COC stretching vibration.

[0053] 50 mg of homogeneous polysaccharide sample from *Hemibacillus thrombocytopenia* was dissolved in D2O, lyophilized, and the process was repeated twice. After lyophilization, the sample was dissolved in 0.5 mL of D2O and transferred to an NMR tube. The NMR spectrometer was used to determine the sample's composition. 1 H NMR, 13 C NMR, 1 H- 1 H COSY、 1 H- 1 HTOCSY 1 H- 13 C HSQC, 1 H- 13 C HMBC and 1 H- 1 HNOESY spectrum. Figure 3(A) shows the homogeneous polysaccharide of the *Hemangiosporium* species. 1 1H NMR spectrum; Figure 3(B) shows the homogeneous polysaccharide of the *Hemangioblastus*. 13 Figure 3(C) shows the C NMR and partial DEPT spectra of the *Hemangiosporus* homogeneous polysaccharide. 1 H- 1 HCl COSY spectrum; Figure 3(D) shows the homogeneous polysaccharide of the *Hemangioblastus* strain. 1 H- 13 C HSQC spectrum; Figure 3(E) shows the homogeneous polysaccharide of the aforementioned Hemoglobinobacterium. 1 H- 1 H NOESY spectra. Comprehensive analysis of the NMR spectra of *Hemibacillus thuringiensis* homogeneous polysaccharide (Figure 3) yields information such as glycosidic bond configuration and the linkage sequence of each linker segment. The NMR spectra of *Hemibacillus thuringiensis* homogeneous polysaccharide Ts1-1A... 11H NMR spectroscopy revealed four terminal hydrogen signals in the anomeric proton region, labeled A, B, C, and D. The hydrogen signal for segment A was at 5.04 ppm, segment B at 5.01 ppm, segment C at 4.95 ppm, and segment D at 4.90 ppm, indicating that it represents an α-configuration. 13 In the C NMR and DEPT spectra, there are four anodic carbon signals at 102.4, 101.5, 98.4, and 98.0 ppm in the anodic region, which are consistent with... 1 The anomeric hydrogen signals in the HNMR spectrum correspond to those in the polysaccharide. Two-dimensional spectra can provide further information about the polysaccharide structure. 1 H- 1 H COSY spectra can provide correlation signals for hydrogens on adjacent carbons, and by sequential assignment, complete signals for hydrogens on the same sugar ring can be obtained. 1 H- 13 C HSQC spectroscopy can provide carbon-hydrogen correlation signals on the same carbon atom, combined with 1 H- 1 H COSY spectra can be attributed to carbon and hydrogen signals on the same sugar ring. 1 H- 13 In the C HSQC spectrum, the anomeric carbon signals at 102.4, 101.5, 98.4, and 98.0 ppm were correlated with the anomeric hydrogen signals at 5.04, 5.01, 4.95, and 4.90 ppm, respectively; the signals at 77.7, 77.5, and 66.5 ppm were assigned to C-2, C-3, and C-6 signals of →2,3,6)-α-Galp-(1→), the signal at 77.6 ppm was assigned to C-3 signal of →3)-α-Fucp-(1→), and the signal at 66.6 ppm was assigned to C-6 signal of 6)-α-Galp-(1→. Compared with the unsubstituted C-2, C-3, and C-6, their chemical shift values ​​are significantly shifted to the lower field. Based on the fragment substitution information and fragment ratios, combined with the methylation results, it can be preliminarily inferred that fragment A represents →2,3,6)-α-Galp-(1→), fragment B represents α-Manp-(1→), fragment C represents →3)-α-Fucp-(1→), and fragment D represents 6)-α-Galp-(1→. Compared with methylation analysis, NMR analysis failed to detect all signals of →3,4)-Fucp-(1→, →4)-Manp-(1→, and →4,6)-Galp-(1→). Acetyl esterification determination showed an acetyl esterification degree of 0.06, consistent with the sum of the area ratios of these three residues in the methylation analysis. Furthermore, in Ts1-1A... 13In the C NMR, three signals were observed at 20.6, 20.5, and 20.3 ppm and 174.0, 173.6, and 173.4 ppm, respectively. These three signals are attributed to three acetylated residues. In summary, we infer that Ts1-1A contains three acetylated residues, all of which are acetylated at the 4-position.

[0054] (3) High-order structure analysis of homogeneous polysaccharide from Hemoglobinobacterium tumefaci

[0055] The homogeneous polysaccharide sample of *Hemibarbus erythrocyte* was dissolved in ultrapure water to prepare a solution with a concentration of approximately 5 μg / mL. A small amount of the sample solution was pipetted onto a mica sheet using a dropper. After drying, the sample was scanned under an atomic force microscope. The results are shown below. Figure 4(A) and 4(B) .

[0056] Figure 4(A) is a plan view of the homogeneous polysaccharide of *Hemanthotomyces rubrum* under an electron mechanical microscope; Figure 4(B) is a three-dimensional view of the homogeneous polysaccharide of *Hemanthotomyces rubrum* under an electron mechanical microscope.

[0057] The AFM spectrum of the homogeneous polysaccharide Ts1-1A from *Hemiberlesia lataniae* provides a clear view of its molecular morphology. As shown in Figures 4(A) and (B), the Ts1-1A polysaccharide chains exhibit irregular flame-shaped particles with a height of approximately 7.22 nm, significantly higher than the height of a single polysaccharide chain. This indicates that Ts1-1A does not exist as a single-chain molecule, but rather as a branched polysaccharide chain or an aggregate of multiple sugar chains. The numerous hydroxyl groups present in the polysaccharide chains, along with intermolecular or intramolecular hydrogen bonds, cause the sugar chains to intertwine, forming nonlinear elliptical or cylindrical structures.

[0058] Example 4: Verification of the anti-HCMV activity of homogeneous polysaccharide from *Helicobacter pylori*

[0059] (1) Toxicity experiment of Hemoglobinuria homogeneous polysaccharide on WI-38 cells

[0060] Human cytomegalovirus (Towne strain) and human diploid fibroblasts (WI-38 cells) were derived from the American Tissue Culture Collection (ATCC). Cells were cultured in a medium containing 10% fetal bovine serum and 1× penicillin-streptomycin.

[0061] The effect of *Rhizoctonia solani* homogenized polysaccharide on WI-38 cell viability was detected using the CCK-8 assay. WI-38 cells were seeded in 96-well plates and incubated at 37°C with 5% CO2. After incubation, the culture medium was discarded, and fresh culture medium containing different concentrations (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 15.625, 31.25, 62.5, 125, 250, 500 μg / mL) of *Rhizoctonia solani* homogenized polysaccharide was added to each well. Three parallel wells were set up for each concentration. A negative control group without *Rhizoctonia solani* homogenized polysaccharide and a cell-free blank control group were also included. Cells were cultured for another 5 days. After discarding the drug-containing culture medium, 10 μL of CCK-8 was added to each well, taking care not to introduce air bubbles into the wells to avoid interfering with OD value measurement. After incubation at 37℃ and 5% CO2 for 1-2 hours, the culture was terminated. The optical density (OD) value of each well was measured at 450 nm using a microplate reader, and the relative cell viability was calculated. The cell viability of the negative control group without added hemoglobin was set as 1.

[0062] Data analysis was performed on the effect of the compound on the cell viability of WI-38 cells, and the results are shown in Figure 5(A). The cell viability results showed that within the polysaccharide concentration range of 0-500 μg / mL, the cell viability of WI-38 cells was not significantly different from that of the negative control group, indicating that the homogeneous polysaccharide from *Hemangiospermum erythrorhizon* had no cytotoxic activity against WI-38 cells. This demonstrates that its highly effective and low-toxicity antiviral activity has significant advantages in the development of antiviral drugs as an active lead compound.

[0063] (2) Protective effect of Hemoglobinuria homogeneous polysaccharide on WI-38 cells infected with human cytomegalovirus

[0064] WI-38 cells were seeded into 12-well plates. They were cultured in fresh DMEM medium containing 10% fetal bovine serum and 1× penicillin-streptomycin at 37°C in a 5% CO2 incubator. Before inoculation with human cytomegalovirus (HCMV), the medium was discarded, and low-serum medium containing 0.2% FBS was added. The cells were cultured for two days to synchronize them to the G0 phase, which is beneficial for HCMV infection. Then, equal volumes of different concentrations of Hemoglobinoblastic thrombectomycin Ts1-1A (0, 50, 200, 500 μg / mL) or positive control phosphonoformic acid (PFA) were added to each well. After culturing for another 2 hours, the cells were inoculated with HCMV at an MOI of 0.5. Cell morphology was observed under a microscope at different time points. The results are shown in Figure 5(B).

[0065] As shown in Figure 5(B), within the concentration range of 0-500 μg / mL of homogeneous polysaccharide from *Helicobacter pylori*, Ts1-1A had no significant effect on the morphology of WI-38 cells, consistent with the cell viability results. In the wells inoculated with HCMV, cytopathic effect (CPE) induced by HCMV infection was observed five days later in wells infected solely with HCMV, specifically manifested as cell rounding and enlarged inclusion bodies. However, the PFA positive control group showed a significant protective effect against HCMV-infected cells, with WI-38 cell morphology consistent with the negative control group; simultaneously, Ts1-1A, similar to the positive control group, protected WI-38 cells from the cytopathic effect induced by human cytomegalovirus infection.

[0066] (3) Homocystis jirovecii polysaccharide significantly inhibited the expression of HCMV-related proteins.

[0067] To further confirm the anti-HCMV effect of *Helicobacter pylori* homogeneous polysaccharide, we investigated its influence on the expression of HCMV immediate early proteins IE1 / 2 and UL44 in HCMV-infected WI-38 cells. Cell culture and HCMV inoculation methods were as described in Example 4(2). An HCMV-only group, a positive control group (HCMV + PFA), and multiple experimental groups (HCMV + different drug concentrations) were established. The positive control drug and *Helicobacter pylori* homogeneous polysaccharide were added to the culture medium two hours before HCMV inoculation. Samples were collected five days after HCMV inoculation for analysis. The supernatant was discarded, cells were washed three times with PBS, and cells were lysed with moderate-strength RIPA lysis buffer before sample collection. SDS-PAGE protein electrophoresis and transfer were performed using standard methods. After blocking with 5% skim milk powder, IE1 / 2, UL44, and the internal control antibody GAPDH were detected, followed by imaging and quantitative analysis. Results are as follows: Figure 6(A) and 6(B) As shown, the 50 μg / mL Hemoglobin homogenate group had no statistically significant effect on IE1 / 2 protein, but significantly reduced the expression of UL44 protein; when the concentration of Hemoglobin homogenate reached 200 μg / mL or above, it showed a significant inhibitory effect on both IE1 / 2 and UL44 proteins.

[0068] (4) Treatment with homogenized polysaccharide from Hemoglobin significantly reduced the DNA copy number of HCMV in host WI-38 cells.

[0069] Cell culture, HCMV inoculation, and drug treatment methods were the same as described in Example 4(2). The DNA copy number of HCMV in its in vitro host WI-38 cells was determined using qPCR. The steps were as follows: Cells were harvested after five days of culture, and viral DNA was extracted using the QIAampDNA mini kit. DNA was amplified using 2×Universal SYBR Green Fast qPCR Mix. The HCMV UL123 primers used were 5′-TCTGCCAGGACATCTTTCTC-3′ upstream and 5′-GTGACCAAGGCCACGACGTT-3′ downstream. Amplification conditions: 95℃ for 5 min, (95℃ for 5 s and 60℃ for 30 s) × 40 cycles. Through 2 -ΔΔct The calculations, as shown in Figure 6(C), revealed that the copy number of HCMV UL123 was significantly inhibited by *Hemangiospermum erythrorhizon* homogeneous polysaccharide at concentrations of 200 μg / mL and above, exhibiting a concentration-dependent effect similar to its inhibitory effect on HCMV-related proteins. These results further confirm the anti-HCMV effect of *Hemangiospermum erythrorhizon* homogeneous polysaccharide.

[0070] (5) Homocystis jirovecii polysaccharide significantly inhibited the expression of HCMV immediate early protein IE1 / 2 in the cell nucleus.

[0071] Cell culture, HCMV inoculation method, and drug treatment method were the same as described in Example 4(2). Cells were collected after five days of culture and fixed in pre-cooled acetone / methanol (1:1) solution at -20℃. They were then incubated with IE1 / 2 antibody and FITC-conjugated secondary antibody. Samples were observed under a fluorescence microscope and quantitatively analyzed. The results are shown in Figure 6(D). The number of fluorescent signals in the cell nuclei of the 200 μg / mL and 500 μg / mL Hemolysin-containing homogeneous polysaccharide experimental groups was significantly reduced in a concentration-dependent manner. Statistical analysis (Figure 6(E)) also showed that Hemolysin-containing homogeneous polysaccharide significantly reduced the number of IE1 / 2 fluorescent signals. These results further confirm the inhibitory effect and anti-HCMV effect of Hemolysin-containing homogeneous polysaccharide on HCMV-related proteins.

[0072] (6) Hemoglobinuria homogeneous polysaccharide significantly inhibited the viral titer of HCMV.

[0073] Cell culture, HCMV inoculation method, and drug treatment method were the same as described in Example 4(2). WI-38 cells were inoculated into 96-well plates one day prior to inoculation to form a monolayer. Then, the collected viral solutions of each experimental group (treated with homogenized polysaccharide of different concentrations of Hemoglobin thrombocytopenia before inoculation with HCMV) were serially diluted 10-fold with fresh medium containing 0.2% FBS. Eight parallel wells were set up for each experimental group and added to the 96-well plates. The plates were incubated at 37°C in a 5% CO2 incubator. The cytopathic effect was observed and recorded daily until the number of diseased wells no longer increased. The half-maximal dose (TICD) of tissue culture was calculated according to the Reed-Muench method. 50 The results, as shown in Figure 6(F), were similar to those for HCMV-related proteins and DNA copy number. The 50 μg / mL *Hemibacillus erythropoietin* homogenized polysaccharide group showed no statistically significant effect on viral titer. However, when the concentration of *Hemibacillus erythropoietin* homogenized polysaccharide reached 200 μg / mL or higher, it exhibited a significant inhibitory effect on viral titer in a concentration-dependent manner. These results further confirm the anti-HCMV effect of *Hemibacillus erythropoietin* homogenized polysaccharide.

[0074] Example 5: Study on the mechanism of antiviral activity of homogeneous polysaccharide from *Hemiberlesia lataniae*

[0075] (1) Hemoglobinuria homogeneous polysaccharide exerts anti-HCMV activity in two stages: before and after the virus enters the cell.

[0076] Cell culture and HCMV inoculation were performed according to the steps in Example 4 (2). The difference was that the HCMV used was inoculated into WI-38 cells at an MOI of 5, and the concentration of Hemolymphoid polysaccharide used was 200 μg / mL. The antiviral effect of Hemolymphoid polysaccharide was studied by treating the cells with Hemolymphoid polysaccharide at different times. Five groups were set up in this experiment. Experimental group 1: The Hemolymphoid polysaccharide was administered 2 hours before cell inoculation, and the cells were pretreated with Hemolymphoid polysaccharide for 2 hours. Then, the culture medium was kept in the Hemolymphoid polysaccharide throughout the experiment until harvest 72 hours after inoculation. Experimental group 2: Hemolymphoid polysaccharide and cell pretreatment group. The Hemolymphoid polysaccharide was administered 2 hours before cell inoculation, and the cells were pretreated with Hemolymphoid polysaccharide for 2 hours. Then, the culture medium containing Hemolymphoid polysaccharide was discarded, the cells were washed with PBS, and inoculation was performed. After 2 hours, the culture medium containing the virus was discarded, and the cells were washed three times with PBS to remove unbound viruses. Then, fresh culture medium was added until 72 hours before collection. Experimental Group 3: Cells were given *Hemangiosporinus* homogeneous polysaccharide simultaneously with virus inoculation. Infection was terminated after 2 hours, the culture medium containing the virus and *Hemangiosporinus* homogeneous polysaccharide was discarded, and the cells were washed three times with PBS to remove unbound viruses. Then, fresh culture medium was added until 72 hours before collection. Experimental Group 4: *Hemangiosporinus* homogeneous polysaccharide and virus pretreatment group. After 2 hours of pretreatment with *Hemangiosporinus* homogeneous polysaccharide and virus, the mixture was added to the cells for virus infection. After 2 hours, the culture medium containing the virus and *Hemangiosporinus* homogeneous polysaccharide was discarded, and the cells were washed three times with PBS to remove unbound viruses. Then, fresh culture medium was added until 72 hours before collection. Experimental Group 5: Viral infection was performed first, and the cells were washed three times with PBS to remove unbound viruses. Then, fresh culture medium containing *Hemangiosporinus* homogeneous polysaccharide was added until 72 hours before collection. See Figure 7(A) for a detailed illustration of the treatment process. The antiviral activity stage of *Hemangiospermum erythrocyte* homogeneous polysaccharide was determined by detecting the expression of virus-related proteins and DNA copy number in cells. The specific operation was the same as described in (3) and (4) of Example 4, with the following additional primers: UL44-upstream primer 5′-ACTGCCGTGCACGTTGCGTA-3′, UL44-downstream primer 5′-ACTTGCCGCTGTTCCCGACG-3′, pp150-upstream primer 5′-GGTTTCTGGCTCGTGGATGTCG-3′, pp150-downstream primer 5′-CACACAACACCGTCGTCCGATTAC-3′. The results are as follows: Figure 7(B) , 7(C)As shown in Figure 7(D), the expression of virus-related proteins and DNA copy number in experimental group 1 were significantly decreased, consistent with the evaluation results of the antiviral activity of *Hemibacillus erythropoietin* homogeneous polysaccharide. The results for experimental group 3 were similar to those for experimental group 1, indicating that *Hemibacillus erythropoietin* homogeneous polysaccharide exerts an antiviral effect in the early stages of viral infection in cells. Pretreatment of cells with *Hemibacillus erythropoietin* homogeneous polysaccharide (experimental group 2) had no significant effect on virus-related proteins and DNA copy number, while pretreatment of viruses with *Hemibacillus erythropoietin* homogeneous polysaccharide (experimental group 4) significantly reduced the levels of virus-related proteins and DNA copy number in cells. This indicates that the antiviral effect of *Hemibacillus erythropoietin* in the early stages of viral infection is related to its interaction with the virus; *Hemibacillus erythropoietin* homogeneous polysaccharide exerts its antiviral effect by binding to the virus, thereby preventing the virus from adsorbing onto the host cell surface. Experimental group 5 also showed an inhibitory effect on virus-related proteins and DNA copy number, indicating that *Hemibacillus erythropoietin* can also exert an antiviral effect after the virus enters the cell.

[0077] (2) Hemoglobinuria homogeneous polysaccharide interferes with viral replication after viral entry into cells by reducing intracellular oxidative stress levels.

[0078] Cell culture, HCMV inoculation, and drug treatment were performed according to the steps in Example 4 (2). NAC was used as the positive control drug. The antiviral mechanism of *Hemangiospermum erythrocyte* homogeneous polysaccharide was determined by detecting the expression of downstream proteins NQO1 and HO-1 in the Keap1-Nrf2 pathway in cells, following the same procedures as described in Example 4 (3). The results are shown in the figure. Figure 7(E) , 7(F) As shown in Figures 7(G) and 7(H), the homogeneous polysaccharide of *Hemibium hemangiosum* exhibits the same effect as the positive control NAC in reducing cellular oxidative stress. Previous literature has indicated that increased oxidative stress induced by exogenous hydrogen peroxide stimulation promotes HCMV replication, while NAC can inhibit HCMV replication by reducing oxidative stress levels. Therefore, the antiviral effect of *Hemibium hemangiosum* homogeneous polysaccharide after viral infection may be mediated through its reduction of intracellular oxidative stress levels.

[0079] In summary, the homogeneous polysaccharide of *Hemiglossum* of the present invention is simple to prepare, has a highly effective anti-human cytomegalovirus activity, and is non-cytotoxic. It is an ideal lead compound for the development of immune adjuvants and has good application prospects.

[0080] The specific embodiments described in this invention are merely illustrative of the spirit of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0081] Although this invention uses a great deal of terminology, the possibility of using other terms is not excluded. These terms are used merely for the convenience of describing and explaining the essence of this invention; interpreting them as any additional limitation would be contrary to the spirit of this invention.

Claims

1. A homogeneous polysaccharide of *Hemibarbus erythrocyte*, characterized in that, The structure is shown in equation (I): (I)， The homogeneous polysaccharide of *Hemoglobinobacterium* is a heteropolymer containing a small number of branched chains, and its main chain consists of →3)-α-Fuc p -(1→and→6)-α-Gal p -(1→ Composed in a 1:1 ratio, with T-α-Man branches p -(1→, connected to the 2nd and 3rd positions of Gal on the main chain, with an average of 2 branch substitutions per 16 sugar residues on the main chain, and the molecular weight of the homogeneous polysaccharide of the blood thrombus bacteria is 12.8 kDa.

2. The homogeneous polysaccharide of *Hemiglossum* as described in claim 1, characterized in that... It can be prepared through the following steps: (1) The fruiting bodies of Hemothorax are defatted, dried and pulverized to obtain defatted powder of Hemothorax; (2) The defatted powder of Hemothromyces was extracted by cold soaking in distilled water. The extract was concentrated, precipitated with alcohol, dialyzed, centrifuged and freeze-dried to obtain the total sugar TsL1 of Hemothromyces cold water extraction. (3) After separation and purification by anion exchange chromatography and high performance gel permeation chromatography, the homogeneous polysaccharide Ts1-1A of Hemoglobinus was obtained.

3. The homogeneous polysaccharide of *Hemiglossum* as described in claim 2, characterized in that... In step (1), take the fruiting body of Hemoglobinus thrombus, add 5-6 times the amount of 95% ethanol and reflux to defatt it 3 times, 1 hour each time.

4. The homogeneous polysaccharide of *Hemiglossum* as described in claim 2, characterized in that... In step (2), the Hemoglobinus thrombocytogenetic powder was extracted with 20 times the amount of pure water at room temperature by magnetic stirring for 4 hours. The cold water extract was obtained by filtration, concentration, alcohol precipitation, dialysis, centrifugation and freeze drying to obtain Hemoglobinus thrombocytogenetic polysaccharide TsL1. The concentration operation was to concentrate the original volume to 1 / 10 of the original volume. The dialysis bag used in the dialysis operation was a dialysis bag with a molecular weight cutoff of 2000.

5. The homogeneous polysaccharide of *Hemangioblastus* as described in claim 2, characterized in that... In step (3), the polysaccharide TsL1 of *Hemanthotomyces rubrum* was initially separated and purified by an anion exchange column. The obtained component was dialyzed, concentrated, and lyophilized to obtain the initially separated polysaccharide Ts1-1. It was then further separated and purified by gel chromatography and lyophilized to obtain the homogeneous polysaccharide Ts1-1A of *Hemanthotomyces rubrum*. The concentration operation was to concentrate the original volume to 1 / 10 of the original volume. The dialysis bag used in the dialysis operation was a dialysis bag with a molecular weight cutoff of 2000. The packing material used in the anion exchange column was DEAE-agarose. The mobile phase used was an aqueous solution. The packing material used in the gel column was dextran G-50 gel with a separation range of 1500-30000.

6. The application of the homogeneous polysaccharide of *Hemiberlesia lataniae* as described in claim 1, characterized in that, A therapeutically effective amount of Hemoglobinoblastic homogeneous polysaccharide, combined with a pharmaceutically acceptable carrier, can be used alone or with other substances to prepare anti-human cytomegalovirus drugs.

7. The application as described in claim 6, characterized in that, The medicine is in the form of decoction, pills, powder, ointment, granules, oral liquid, capsule or tablet.

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