Method for preparing tea polysaccharide through fungal fermentation and application of tea polysaccharide in oxidation resistance and HCMV resistance

Abstract

The invention discloses a method for preparing tea polysaccharide through fungal fermentation and application of the tea polysaccharide in oxidation resistance and HCMV resistance. The fermented tea polysaccharide FTP is an acidic polysaccharide, can significantly inhibit expression of immediate early-stage protein IE1 / 2 and early-stage protein p52 of HCMV, and reduces DNA copy numbers of immediate early-stage gene UL123, early-stage gene UL44 and late-stage gene UL32, so that the fermented tea polysaccharide FTP shows clear prevention and treatment effects on HCMV infection; meanwhile, the polysaccharide also shows excellent antioxidant activity, and has relatively high scavenging capacity on DPPH free radicals and ABTS cation free radicals. The fermented tea polysaccharide has antiviral and antioxidant dual biological activities, is high in safety and low in toxic and side effects, provides a new natural source candidate component for developing anti-HCMV medicines, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing tea polysaccharides by fungal fermentation and its application in anti-oxidation and anti-HCMV. Background Technology

[0002] Human cytomegalovirus (HCMV) is one of the largest and most complex viruses known, with a viral particle diameter of approximately 150-200 nm. It contains a double-stranded DNA genome of about 230 kb, encoding over 200 viral proteins. HCMV infection is extremely common, with over 70% of adults worldwide testing positive serologically. Infected individuals can remain latent for life. In healthy individuals, HCMV is usually latent; however, in immunocompromised patients, such as those who have undergone organ or hematopoietic stem cell transplantation, the virus can be activated, leading to serious complications and even death. Furthermore, HCMV can be transmitted vertically across the placenta, causing congenital infection and is the leading infectious cause of birth defects in newborns, resulting in various long-term sequelae such as neurological damage, hearing loss, developmental delays, and cardiac structural abnormalities. Given its high infectivity, latent nature, and serious harm to specific populations, HCMV has been identified as an important subject of global public health and medical research.

[0003] In recent years, breakthroughs have been made in HCMV vaccine development, with several candidate vaccines entering clinical trials. However, their safety and efficacy still need to be verified, and no HCMV vaccine has yet been approved for marketing globally. Currently, clinical treatment still relies on nucleoside analogs such as ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV). However, these drugs generally have serious adverse reactions such as bone marrow suppression and nephrotoxicity, and long-term use can easily induce gene mutations such as UL97, leading to the development of drug-resistant strains, which limits their widespread clinical application. Therefore, developing novel, highly effective, and low-toxicity anti-HCMV drugs is of great significance for preventing birth defects and improving the antiviral capacity of immunocompromised populations.

[0004] Fermented tea is a specialty tea processed through microbial fermentation. Taking post-fermented tea as an example, it primarily relies on the fermentation process of microorganisms to improve tea quality, such as reducing bitterness and astringency, making the taste more mellow and smooth, and developing a unique aged aroma, while also generating various beneficial active ingredients. The selection of fermentation strains is particularly crucial for improving the quality of fermented tea. Ganoderma lucidum (Reishi mushroom) Ganoderma lucidumDuring fermentation, fungi secrete highly active extracellular enzyme systems such as lignin peroxidase and laccase, which can effectively decompose lignin and other components, thus improving the content of amino acids and catechins in tea. Simultaneously, during fungal fermentation of tea, an important class of active tea polysaccharide molecules can be generated, whose biological activity differs significantly from that of unfermented tea polysaccharides. my country has abundant summer and autumn tea resources, but due to their predominantly bitter taste, their utilization rate is low. Considering the growth and metabolic characteristics of summer and autumn tea—namely, the high intensity of sugar metabolism and the accumulation of polysaccharides in tea leaves during these seasons—a new approach is provided for improving the quality of summer and autumn tea through fungal fermentation. Studies have shown that fermented tea polysaccharides possess superior antioxidant, immunomodulatory, and lipid-lowering effects, offering potential applications in the fields of biomedicine and health. Current technologies have not fully revealed their specific uses and value in antiviral treatment, particularly against human cytomegalovirus (HCMV) infection. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a method for preparing tea polysaccharides through fungal fermentation and its application in antioxidation and anti-HCMV activity. The fermented tea polysaccharide FTP of this invention can significantly inhibit the expression of HCMV immediate early proteins IE1 / 2 and p52, and reduce the expression of immediate early genes. UL123 Early genes UL44 and late genes UL32 The method reduces the DNA copy number, thus demonstrating a clear preventive and therapeutic effect against HCMV infection. It exhibits high safety and low toxicity, while also demonstrating highly efficient scavenging capabilities against DPPH and ABTS cationic free radicals, exhibiting significant antioxidant effects. Furthermore, tea leaves are readily available and have low extraction and preparation costs, possessing good potential for clinical development and providing a new and effective option for the preparation of anti-HCMV drugs.

[0006] The present invention discloses a method for preparing tea polysaccharides by fungal fermentation, comprising the following steps:

[0007] Step 1: Take a Ganoderma lucidum mycelium cake with a diameter of about 0.5 cm, inoculate it into 150 ml of PDA liquid medium, and culture it with shaking at 28℃ and 120 rpm for 5-7 days. Repeat the activation process three times, collect the mycelium, wash it three times with sterile physiological saline to remove the culture medium residue, and obtain Ganoderma lucidum seed liquid. Store it at 4℃ for later use.

[0008] Step 2: Take 10 g of roasted green tea raw material and sterilize it at 121℃ for 20 min. In a clean bench, add 4 ml of Ganoderma lucidum seed liquid and 4 ml of sterile water to the sterilized tea leaves, mix thoroughly to evenly moisten the tea leaves, and place it in a 28℃ constant temperature incubator for static fermentation for 10 days, gently turning it once a day during this period. After fermentation, dry the tea leaves in a 65℃ oven to constant weight to obtain the fermented tea sample. Unfermented tea does not undergo steps 1 and 2.

[0009] Step 3: The fermented tea obtained in Step 2 is pulverized by a pulverizer and passed through an 80-mesh sieve. The resulting fermented tea powder is placed in a beaker, and 20,000 mL of anhydrous ethanol is added for every 200g of tea powder. The mixture is extracted at a constant temperature of 70℃ for 2 hours. The extraction is repeated twice. After filtration, the mixture is dried. The resulting residue is then soaked in pure water at a material-to-liquid ratio of 1:20 at 90℃ for 2 hours. The extraction is repeated twice. The extracts are combined and concentrated. The mixture is then precipitated with alcohol at 4℃ overnight. After centrifugation, the crude polysaccharide of the fermented tea is obtained and stored for later use.

[0010] Step 4: Dissolve the fermented tea crude polysaccharide obtained in Step 3 in deionized water, prepare Sevag reagent with n-butanol and chloroform in a volume ratio of 1:4, mix the crude polysaccharide solution and Sevag reagent in equal volumes, shake for 15 min and remove the intermediate protein layer, repeat the above steps until no obvious protein layer appears, collect the polysaccharide solution and concentrate it.

[0011] Step 5: Pour the concentrated solution obtained in Step 4 into a 3500 Da dialysis bag, changing the aqueous solution daily during the process; after dialysis, transfer the solution out of the dialysis bag, centrifuge, concentrate, freeze-dry, collect, and weigh.

[0012] The fermented tea polysaccharide obtained in this invention is an acidic polysaccharide, abbreviated as FTP, with a molecular weight of 3.51 × 10⁻⁶. 5 The monosaccharide composition and ratio of Da are: Xyl : GalA : Glc : Gal : Rha : Man = 4.99 : 4.43 : 3.44 : 2.81 : 1.69 : 1; the unfermented tea polysaccharide is abbreviated as STP, and the molecular weight of the polysaccharide is 6.66 × 10⁻⁶. 5 The monosaccharide composition and ratio of Da are: Xyl : Glc : Gal : Rha : = 1 : 1.4 : 2.59 : 1.14.

[0013] The present invention relates to the application of fermented tea polysaccharides in the preparation of antioxidant agents.

[0014] The fermented tea polysaccharides exhibited significant concentration-dependent scavenging ability against both DPPH free radicals and ABTS cationic free radicals.

[0015] The present invention relates to the application of fermented tea polysaccharides in the preparation of pharmaceutical formulations for the treatment and / or prevention of HCMV.

[0016] The fermented tea polysaccharide targets the immediate early protein IE1 / 2, early protein p52, and the immediate early gene of HCMV. UL123 Early genes UL44 and late genes UL32 The inhibition of DNA copy number is used to suppress HCMV. The results of biological experiments in this invention show that:

[0017] 1. Fermented tea polysaccharides have good scavenging effects on DPPH free radicals and ABTS cationic free radicals, and their IC50... 50 The concentrations were 13.0 µg / ml and 39 µg / ml, respectively, close to the positive control vitamin C, with IC50 values. 50 The concentrations were 6.55 µg / ml and 13.29 µg / ml, respectively.

[0018] 2. Fermented tea polysaccharide FTP and unfermented tea polysaccharide STP, when used alone to treat HCMV host cells WI-38, did not show significant cytotoxicity to WI-38 at concentration gradients of 0.78125 μg / ml, 1.5625 μg / ml, 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml, and 25 μg / ml. However, significant cytotoxicity was observed when the concentrations of FTP or STP reached 50 μg / ml, 100 μg / ml, and 200 μg / ml.

[0019] 3. Fermented tea polysaccharide FTP at concentrations of 2.5 μg / ml, 5 μg / ml, and 10 μg / ml can alleviate the cytopathic effect of HCMV-induced WI-38 cells. The effect of treatment with 10 μg / ml fermented tea polysaccharide FTP is comparable to that of the positive control drug PFA at 200 μg / ml.

[0020] 4. Fermented tea polysaccharide FTP at concentrations of 5 μg / ml and 10 μg / ml significantly inhibited the expression of immediate early HCMV proteins IE1 / 2 and p52 in WI-38 cells. Compared with the group inoculated with HCMV alone, a concentration of 5 μg / ml inhibited the expression levels of IE1 / 2 and p52 by approximately 60%; at a concentration of 10 μg / ml, the inhibition levels of IE1 / 2 and p52 reached 40% and 38%, respectively. Compared with unfermented tea polysaccharide STP, 5 μg / ml of fermented tea polysaccharide FTP showed a similar inhibitory effect on the expression of IE1 / 2 and p52.

[0021] 5. Fermented tea polysaccharide FTP at concentrations of 5 μg / ml and 10 μg / ml can significantly reduce the immediate early stage gene of HCMV. UL123 Early genes UL44 and late genes UL32 DNA copy number. Compared with the group inoculated with HCMV alone, a concentration of 5 μg / ml can inhibit the DNA copy number of the three genes to about 70%; a concentration of 10 μg / ml can inhibit the DNA copy number of the three genes to about 50%.

[0022] The pharmaceutical formulations described in this invention also include pharmaceutically acceptable carriers, diluents, and excipients.

[0023] The pharmaceutical preparations described in this invention are liquid, solid, or semi-solid preparations, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, gels, emulsions, patches, etc.

[0024] The beneficial effects of this invention are reflected in:

[0025] This invention relates to the application of fermented tea polysaccharide FTP in the preparation of drugs for the prevention and treatment of human cytomegalovirus (HCMV) infection. The fermented tea polysaccharide FTP not only inhibits several key aspects of HCMV infection—including the expression of immediate early proteins IE1 / 2 and p52, but also the viral genome... UL123 , UL44 , UL32 It exhibits significant inhibitory effects on viral replication, demonstrating clear antiviral activity. Simultaneously, it also possesses highly efficient scavenging capabilities against DPPH and ABTS cationic radicals, showcasing outstanding antioxidant activity. These two activities together constitute its dual "antiviral-antioxidant" biological function, indicating that this component can exert synergistic protective effects through multiple targets and mechanisms. Benefiting from its natural source, fermented tea polysaccharide FTP exhibits high safety and low cytotoxicity. While effectively inhibiting the virus, it can alleviate infection-induced oxidative stress damage, thus providing a comprehensive and promising candidate drug or functional component for the prevention and treatment of HCMV, possessing significant development prospects and application value. Attached Figure Description

[0026] Figure 1 The image shows the identification results of fermented tea polysaccharide FTP and unfermented tea polysaccharide STP. Figure 1 (A) is a high-performance liquid chromatography (HPLC) analysis of fermented and unfermented tea polysaccharides. Figure 1 (B) is a diagram showing the monosaccharide composition of fermented tea polysaccharides and unfermented tea polysaccharides.

[0027] Figure 2 The scavenging rates of FTP (A, B), STP (C, D), and positive control vitamin C (E, F) on DPPH free radicals and ABTS cationic free radicals are shown.

[0028] Figure 3 This study investigated the effects of fermented tea polysaccharide FTP (A) and unfermented tea polysaccharide STP (B) on the cytotoxicity of HCMV host cells—human embryonic lung fibroblasts (WI-38). The results were compared with the control group (without FTP or STP treatment). * This indicates that P < 0.1. ** This indicates that P < 0.01. *** This indicates that P < 0.001.

[0029] Figure 4 This study investigated the effect of fermented tea polysaccharide (FPP) FTP pretreatment on the morphology of WI-38 cells after HCMV infection. The control group (MOCK) cells were not infected with HCMV and received no FPP FTP treatment. The HCMV group cells were infected with HCMV alone and received no FPP FTP treatment. The HCMV+PFA group cells were infected with HCMV and treated with the positive control drug phosphatidylcholine (PFA, 200 μg / ml). The remaining three groups were infected with HCMV and treated with 2.5, 5, and 10 μg / ml FTP, respectively. All HCMV-infected groups were inoculated at an MOI of 0.5, and observations and photographs were taken five days after infection (5 dpi).

[0030] Figure 5 This study used Western blot to determine the effects of different concentrations of fermented tea polysaccharide FTP (1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml) and the same concentration (5 μg / ml) of fermented tea polysaccharide FTP versus unfermented tea polysaccharide STP on the immediate early proteins IE1 / 2 and p52 of HCMV in WI-38 cells (GAPDH was used as an internal control). Figure 5 (A) represents the expression levels of HCMV proteins IE1 / 2 and p52 in cells treated with fermented tea polysaccharide FTP; Figure 5 (B) is correct. Figure 5 Quantitative analysis and statistics of the results in (A); Figure 5 (C) shows the effect of treating WI-38 cells with the same concentrations of FTP and STP on the immediate early proteins IE1 / 2 and p52 of HCMV. Figure 5 (D) is correct. Figure 5 Quantitative analysis and statistics of the results in (C). ** This indicates that P < 0.01. ***P < 0.001. The HCMV alone group consisted of HCMV infection alone, without the addition of fermented tea polysaccharide FTP or unfermented tea polysaccharide STP treatment. This group served as a control group before and after drug treatment. All HCMV-infected groups were inoculated with an MOI of 0.5, and samples were collected five days after infection (5 dpi) for protein extraction and detection. Compared with the HCMV alone group, ** This indicates that P < 0.01. *** This indicates that P < 0.001.

[0031] Figure 6 The method used was qPCR to determine the effect of different concentrations of fermented tea polysaccharide FTP on the immediate early stage of HCMV genes. UL123 Early genes UL44 and late genes UL32 The effect of DNA copy number. Compared with the group infected with HCMV alone, * This indicates that P < 0.1. ** This indicates that P < 0.01. *** This indicates that P < 0.001. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. The embodiments of the present invention are only used to explain the present invention and do not mean to limit the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0035] The human embryonic lung fibroblast cell line WI-38 and the human cytomegalovirus (HCMV) Towne strain involved in this invention were obtained from the Zhejiang Provincial Key Laboratory of Geriatrics. Ganoderma lucidum strain ( Ganoderma lucidum Purchased from Jinzhai County Liyuan Edible Fungus Planting Professional Cooperative. Summer and autumn teas were purchased from Jinzhai County and are raw materials for roasted green tea.

[0036] Example 1: Preparation of fermented tea polysaccharide FTP

[0037] Take a piece of Ganoderma lucidum mycelium with a diameter of about 0.5 cm, inoculate it into 150 ml of PDA liquid medium, and culture it with shaking at 28℃ and 120 rpm for 5-7 days. Repeat the activation process three times. Collect the mycelium, wash it three times with sterile physiological saline to remove culture medium residue, and obtain Ganoderma lucidum seed liquid, which is stored at 4℃ for later use. Take 10 g of roasted green tea raw material and autoclave it at 121℃ for 20 min. In a clean bench, add 4 ml of Ganoderma lucidum seed liquid and 4 ml of sterile water to the tea leaves, mix thoroughly to evenly moisten the tea leaves. Place it in a constant temperature incubator at 28℃ for static fermentation for 10 days, gently turning it once a day during this period. After fermentation, dry the tea leaves in a 65℃ oven to constant weight to obtain fermented tea samples. The dried fermented tea was pulverized and passed through an 80-mesh sieve. 200 g of the fermented tea powder was weighed and placed in a beaker. 10 times the amount of anhydrous ethanol was added, and the mixture was placed in a constant temperature water bath at 70 ℃ for 2 h. After thorough filtration and drying, the process was repeated twice. The residue was dried and then extracted with pure water at a ratio of 1:20 (w:v) at 90 ℃ for 2 h, which was repeated twice. The extracts were combined, concentrated, and then precipitated with alcohol overnight at 4 ℃. After centrifugation, the crude polysaccharide of the fermented tea was obtained. The obtained fermented tea polysaccharide was then dissolved in deionized water. Sevag reagent was prepared with n-butanol and chloroform at a ratio of 1:4 (v:v). An equal volume of the crude polysaccharide solution was mixed with the reagent and shaken for 15 min to remove the intermediate protein layer. The above steps were repeated until no obvious protein layer appeared. The sugar solution was collected and concentrated. Pour the obtained concentrate into a 3500 Da dialysis bag and change the aqueous solution daily. After three to four days, end the dialysis and transfer the solution out of the dialysis bag. If there is sediment in the dialysate, centrifuge and concentrate it again, freeze-dry it, collect it, and weigh it.

[0038] Example 2: Preparation of unfermented tea polysaccharide STP

[0039] The raw green tea leaves were sterilized at 121℃ for 20 min without fermentation, then dried, pulverized, and passed through an 80-mesh sieve. 200 g of fermented tea powder was weighed and placed in a beaker, and 10 times the amount of anhydrous ethanol was added. The mixture was then placed in a constant temperature water bath at 70℃ for 2 h for extraction. After thorough filtration and drying, the process was repeated twice. The residue was dried and then extracted with pure water at a ratio of 1:20 (w:v) at 90℃ for 2 h, which was repeated twice. The combined extracts were concentrated and then precipitated with alcohol overnight at 4℃ before centrifugation to obtain unfermented tea polysaccharide. The obtained unfermented tea polysaccharide was then dissolved in deionized water, and Sevag reagent was prepared with a ratio of n-butanol to chloroform of 1:4 (v:v). An equal volume of the crude polysaccharide solution was mixed with the reagent, shaken for 15 min, and the intermediate protein layer was removed. The above steps were repeated until no obvious protein layer appeared. The sugar solution was collected and concentrated. Pour the obtained concentrate into a 3500 Da dialysis bag and change the aqueous solution daily. After three to four days, end the dialysis and transfer the solution out of the dialysis bag. If there is sediment in the dialysate, centrifuge and concentrate it again, freeze-dry it, collect it, and weigh it.

[0040] Example 3: Scavenging rate of fermented tea polysaccharide FTP against DPPH free radicals and ABTS cationic free radicals

[0041] DPPH free radical scavenging rate: Different concentrations of fermented tea polysaccharide GLT sample solutions (5, 10, 25, 50, 100 μg / mL) were accurately prepared. 2 mL of each concentration sample solution was added to 2 mL of 0.1 mmol / L DPPH-ethanol solution, mixed thoroughly, and reacted at room temperature in the dark for 30 min. The absorbance was measured at 517 nm (Sample A). Simultaneously, the absorbance of a mixture of 2 mL of sample solution and 2 mL of anhydrous ethanol (Control A), and the absorbance of a mixture of 2 mL of distilled water and 2 mL of DPPH solution (Blank A) were also measured. Vitamin C (Vc) was used as a positive control. The free radical scavenging rate was calculated as follows: Scavenging rate (%) = [1 - (Sample A - Control A) / Blank A] × 100%.

[0042] ABTS radical scavenging rate: An equal volume of 7 mmol / L ABTS solution was mixed with 2.45 mmol / L potassium persulfate solution and reacted at room temperature in the dark for 12-16 h to generate an ABTS cationic radical stock solution. Before use, the solution was diluted with phosphate-buffered saline (PBS, pH 7.4) to obtain an absorbance of 0.70 ± 0.02 at 734 nm, thus obtaining the working solution. 0.2 mL of sample solutions of different concentrations were added to 4 mL of the ABTS working solution, mixed well, and allowed to stand at room temperature for 6 min. The absorbance was then measured at 734 nm. Vitamin C was used as a positive control, and the scavenging rate was calculated using the same method as the DPPH method. Results are shown below. Figure 2 .

[0043] Fermented tea polysaccharides exhibit good DPPH and ABTS cationic free radical scavenging activity, with an IC50 value of [missing information]. 50 The IC50 values ​​for unfermented tea polysaccharides were 13.0 µg / ml and 39 µg / ml, respectively. 50 The concentrations were 22.5 µg / ml and 46.9 µg / ml, respectively. Compared to unfermented tea polysaccharides, fermented tea polysaccharides showed a scavenging effect on DPPH free radicals and ABTS cationic free radicals that was closer to the positive control vitamin C. The IC50 concentration was [missing value]. 50 The concentrations were 6.55 µg / ml and 13.29 µg / ml, respectively.

[0044] Example 4: Effect of fermented tea polysaccharide FTP treatment alone on WI-38 cytotoxicity

[0045] The effect of fermented tea polysaccharide FTP on cytotoxicity was detected by CCK-8 assay.

[0046] The method for determining cytotoxicity is as follows: Human embryonic lung fibroblasts (WI-38) from passage 30 were used, and 5 × 10⁶ cells were extracted per well. 3 Cells were seeded at a density of 1000 μg / mL in 96-well plates and cultured overnight at 37°C with 5% CO2. After cell monolayer formation, the original culture medium was discarded, and fresh culture medium containing different concentrations of fermented tea polysaccharide FTP (0, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg / mL) was added, with 6 replicates per concentration. A cell control group without polysaccharide and a blank control group without cells were also included. After 5 days of culture, 10 μL of CCK-8 solution was added to each well, avoiding air bubbles that could interfere with detection. The cells were incubated for 1-2 hours, and the absorbance of each well was measured at 450 nm using a microplate reader. The relative cell viability was calculated based on the results and normalized to the control group viability. The experimental results are analyzed as follows: Cell viability = [OD(drug-added) - OD(blank)] / [OD(0-drug-added) - OD(blank)]. The results of the effect of fermented tea polysaccharide FTP on cytotoxicity are shown in Figure 3 Fermented tea polysaccharide FTP and unfermented tea polysaccharide STP, when used alone to treat HCMV host cells WI-38, did not show significant cytotoxicity at concentration gradients of 0.78125 μg / ml, 1.5625 μg / ml, 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml, and 25 μg / ml. However, significant cytotoxicity was observed when the concentrations of FTP or STP reached 50 μg / ml, 100 μg / ml, and 200 μg / ml.

[0047] Example 5: HCMV inoculation and FTP treatment of fermented tea polysaccharides

[0048] Human embryonic lung fibroblasts (WI-38) from passage 30 were used, cultured in DMEM medium containing 10% FBS at a rate of 2 × 10⁻⁶. 4 / cm 2 Cells were seeded at the specified density in six-well cell culture plates and cultured at 37 ℃ in a 5% CO2 incubator for 24 hours. The culture medium was then replaced with DMEM containing 0.2% FBS and cultured for another 48 hours. G0 / G1 synchronization was achieved through serum starvation to prepare for subsequent HCMV infection. HCMV infection (Towne strain) was then performed at an MOI of 0.5. The cells were cultured for a specified time before relevant assays were performed. For fermented tea polysaccharide FTP and the positive control drug PFA, different concentrations (2.5, 5, 10 μg / ml) of fermented tea polysaccharide FTP were added to the culture medium 2 hours before HCMV infection. HCMV was then inoculated (MOI=0.5) for viral infection. Finally, changes in cell morphology, viral protein expression, and DNA copy number were observed in the groups treated with and without fermented tea polysaccharide FTP at different time points.

[0049] Example 6: Effect of FTP pretreatment of fermented tea polysaccharides on cell morphology of WI-38 cells after HCMV infection

[0050] After WI-38 cells were inoculated with HCMV (MOI=0.5), a typical cytopathic effect was observed. Figure 4 The cells showed a significant increase in volume and widening of intercellular spaces, with their morphology changing from a normally spread-out shape to round, elongated, or spindle-shaped, indicating that infection led to morphological deterioration. Pretreatment with different concentrations of fermented tea polysaccharides (2.5, 5, and 10 μg / ml) significantly improved the lesion effect, manifested as a reduction in intercellular spaces, restoration of morphology to a near-normal spread-out state, and an increase in cell density. In particular, when the concentration of fermented tea polysaccharides reached 10 μg / ml, its protective effect on infected cells was comparable to that of the positive control group treated with phosphonoformic acid (PFA, 200 μg / ml), with no obvious cytopathic effects observed, suggesting a strong anti-HCMV infection ability.

[0051] Example 7: Effect of Fermented Tea Polysaccharide FTP Pretreatment on HCMV Virus Protein Expression

[0052] To clarify the inhibitory effect of fermented tea polysaccharide FTP on HCMV replication, this study, based on the observation of cytopathic effects, further selected the immediate early proteins IE1 / 2 and p52 of HCMV as viral replication markers, and analyzed the effect of FTP on their expression using Western blotting. Experimental groups included: a blank control group (no HCMV infection, no drug administration), a virus control group (HCMV infection only), four FTP pretreatment groups (1.25, 2.5, 5, 10 μg / ml), and a positive control PFA treatment group. Cells were cultured and pretreated as described above, then inoculated with 0.5 MOI HCMV, and samples were collected 5 days after infection. Sample processing steps were as follows: supernatant was discarded, cells were washed three times with PBS, and cells were lysed using medium-strength RIPA lysis buffer. The lysate was collected and centrifuged. Subsequently, SDS-PAGE electrophoresis was performed, followed by membrane transfer and blocking with TBST containing 5% skim milk powder at room temperature for 2 hours. Primary antibodies against IE1 / 2 and p52 were added separately and incubated overnight at 4°C. After washing the membrane three times with TBST, the corresponding HRP-labeled secondary antibodies were added and incubated at room temperature for 1 hour. Finally, chemiluminescence detection was performed to analyze protein expression levels. The results showed that fermented tea polysaccharide FTP could dose-dependently inhibit the expression of the immediate early proteins IE1 / 2 and p52 in HCMV. Figure 5 Compared with the virus control group (HCMValone), FTP significantly reduced the protein levels of IE1 / 2 and p52 at a concentration of 5 μg / ml (P < 0.001 and P < 0.01, respectively). When the concentration was increased to 10 μg / ml, the inhibitory effect was further enhanced, with the expression levels of IE1 / 2 and p52 decreasing to approximately 40% and 38% of the virus control group, respectively (both P < 0.001), indicating that FTP at this concentration significantly blocked early viral gene expression. These results further confirm that fermented tea polysaccharide FTP has a clear anti-HCMV activity at the protein level, and its inhibitory effect is positively correlated with concentration.

[0053] Example 8: Effect of fermented tea polysaccharide FTP pretreatment on HCMV DNA copy number in host cells

[0054] Cell culture, polysaccharide treatment, and HCMV inoculation were performed as described in Example 4 above. Samples were collected 3 days post-HCMV infection (3 dpi). The copy number of HCMV DNA was determined using qPCR. Viral DNA was extracted using the QIAamp DNA Mini Kit, and 10 ng of total DNA was used for qPCR experiments with 2×Universal SYBR Green Fast qPCR Mix. The primers used are shown in the table below.

[0055]

[0056] Amplification conditions: 95℃ for 5 min, (95℃ for 5 sec, 60℃ for 30 sec) × 40 cycles, for 2... -△△Ct The calculation method uses a sample infected with HCMV alone (HCMV alone) as a reference, with the DNA copy number set to 1. The results are as follows: Figure 6 As shown. Fermented tea polysaccharide FTP at concentrations of 5 μg / ml and 10 μg / ml can immediately reduce HCMV. UL123 , UL44 and UL32 DNA copy number. Compared with the group inoculated with HCMV alone, a concentration of 10 μg / ml can inhibit the DNA copy number of the three genes to about 50%.

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

Claims

1. A method for preparing tea polysaccharides by fungal fermentation, characterized in that... Includes the following steps: Step 1: Inoculate the Ganoderma lucidum mycelium cake into PDA liquid culture medium, and culture it with shaking at 28℃ and 120 rpm for 5-7 days. Repeat the activation three times, collect the mycelium, wash it with sterile physiological saline to remove the culture medium residue, and obtain Ganoderma lucidum seed liquid. Store it at 4℃ for later use. Step 2: Take the roasted green tea raw material and sterilize it at 121℃ for 20 minutes. In a clean bench, add Ganoderma lucidum seed liquid and sterile water to the sterilized tea leaves in sequence, mix thoroughly to make the tea leaves evenly moist, and place it in a 28℃ constant temperature incubator for static fermentation for 10 days, turning it slightly once a day during the period. After the fermentation is completed, dry the tea leaves in a 65℃ oven to constant weight to obtain the fermented tea sample. Step 3: The fermented tea obtained in Step 2 is pulverized by a pulverizer and passed through an 80-mesh sieve. Anhydrous ethanol is added to the obtained fermented tea powder, and the mixture is extracted at a constant temperature of 70 ℃ for 2 h. The extraction is repeated twice. After filtration, the mixture is dried. The obtained filter residue is mixed with pure water and extracted at 90 ℃ for 2 h. The extraction is repeated twice. The extracts are combined and concentrated. After alcohol precipitation and centrifugation, the crude polysaccharide of fermented tea is obtained. Step 4: Dissolve the fermented tea crude polysaccharide obtained in Step 3 in deionized water, prepare Sevag reagent with n-butanol and chloroform in a volume ratio of 1:4, mix the crude polysaccharide solution and Sevag reagent in equal volumes, shake and remove the intermediate protein layer, repeat the above steps until no obvious protein layer appears, collect the polysaccharide solution and concentrate it. Step 5: Pour the concentrated solution obtained in Step 4 into a 3500 Da dialysis bag, changing the aqueous solution daily during the process; after dialysis, transfer the solution out of the dialysis bag, centrifuge, concentrate, freeze-dry, collect, and weigh.

2. A fermented tea polysaccharide, prepared according to the method described in claim 1, characterized in that: The fermented tea polysaccharide is an acidic polysaccharide, abbreviated as FTP, with a molecular weight of 3.51 × 10⁻⁶. 5 Da.

3. The fermented tea polysaccharide according to claim 2, characterized in that: The monosaccharide composition and ratio of the fermented tea polysaccharide are as follows: Xyl : GalA : Glc : Gal : Rha : Man = 4.99 : 4.43 : 3.44 : 2.81 : 1.69 :

1.

4. The use of the fermented tea polysaccharide according to claim 1 or 2 in the preparation of antioxidant agents.

5. The use of the fermented tea polysaccharide according to claim 1 or 2 in the preparation of pharmaceutical formulations for the treatment and / or prevention of HCMV.

6. The application according to claim 5, characterized in that: The pharmaceutical formulation targets the immediate early protein IE1 / 2, the early protein p52, and the immediate early gene of HCMV. UL123 Early genes UL44 and late genes UL32 The inhibition of DNA copy number can suppress HCMV.

7. The application according to claim 5, characterized in that: The pharmaceutical preparation also includes pharmaceutically acceptable carriers, diluents, and excipients.

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