Use of ginkgo biloba leaf polysaccharide in preparation of medicine for treating or preventing hcmv

Abstract

This invention provides the application of Ginkgo biloba polysaccharide in the preparation of drugs for treating and / or preventing HCMV. Ginkgo biloba L. GP) for HCMV immediate early protein IE1 / 2, early protein p52, and immediate early gene UL123 Early genes UL44 and late genes UL32 It has a significant inhibitory effect on DNA copy number, and has significant preventive and therapeutic effects on HCMV infection. Moreover, GP has few toxic side effects, is safe and effective, and can be used for HCMV infection, showing good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of ginkgo leaf polysaccharide in the preparation of drugs for the treatment or prevention of HCMV. Background Technology

[0002] Human cytomegalovirus (HCMV) belongs to the beta herpesvirus subfamily. HCMV is the largest herpesvirus group, with a viral diameter of approximately 200 nm. Its genome consists of 235 kb of double-stranded DNA (dsDNA), encoding at least 165 virus-associated proteins. HCMV is universally contagious. Like other human herpesviruses, HCMV is not completely cleared after infection but remains latent in the host. In immunocompromised individuals or organ transplant recipients, secondary infections are highly likely to occur, exacerbating the condition. Furthermore, HCMV infection is a major cause of miscarriage in pregnant women and congenital diseases in newborns, leading to microcephaly, jaundice, hepatitis, intellectual disability, blindness, and cognitive impairment. Therefore, HCMV infection imposes a significant social and medical burden.

[0003] Although some progress has been made in HCMV vaccine development, no mature vaccine has yet been approved for market release. Antiviral drugs remain the only clinical option. Ganciclovir (GCV), foscarnet (FOS or PFA), and cidofovir (CDV) are the main clinically approved drugs for treating HCMV infection. These drugs primarily exert their anti-HCMV effect by inhibiting viral DNA polymerase. However, these drugs have adverse reactions such as bone marrow suppression, nephrotoxicity, and electrolyte disturbances. Furthermore, the emergence of drug-resistant strains has significantly reduced the effectiveness of these drugs, limiting their clinical application. In conclusion, effective prevention of human cytomegalovirus infection and active and effective treatment of infected individuals are crucial means to improve the quality of life of newborns and elderly people with weakened immune systems.

[0004] ginkgo( Ginkgo biloba L.Ginkgo biloba, also known as maidenhair tree or ginkgo nut, is a deciduous tree belonging to the genus Ginkgo in the family Ginkgoaceae within the phylum Gymnosperm. It is endemic to China and widely distributed, especially abundant in Taixing City, Jiangsu Province. Ginkgo trees have ornamental, economic, and medicinal value. The Ming Dynasty medical text *Compendium of Materia Medica* records that cooked ginkgo "warms the lungs and benefits qi, relieves asthma and cough, reduces urination, and stops leukorrhea; raw ginkgo reduces phlegm, disinfects, and kills parasites," indicating that ginkgo fruit has good effects in treating coughs, asthma, seminal emission, enuresis, and leukorrhea. Ginkgo fruit also has anti-allergic, antifungal, brain-aging-delaying, and memory-enhancing effects. Modern research shows that ginkgo leaf extract has good effects in treating coronary heart disease, angina pectoris, and hyperlipidemia, and can significantly improve symptoms such as dizziness, chest tightness, palpitations, shortness of breath, and fatigue in patients with coronary heart disease. However, to date, ginkgo leaves, including the active polysaccharides derived from them, have not been used in the preparation of drugs for the prevention and / or treatment of HCMV. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention extracts polysaccharide GP from Ginkgo biloba leaves and conducts a series of biological experiments, aiming to provide a safe and effective drug for inhibiting HCMV with minimal side effects.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides the use of Ginkgo biloba polysaccharide GP in the preparation of drugs for the treatment and / or prevention of HCMV infection.

[0007] The preparation method of the Ginkgo biloba polysaccharide includes the following steps: 1) Pick fresh ginkgo leaves, cut off the branches and stems, wash them repeatedly until clean, and then drain them through a sieve. 2) Place the dried ginkgo leaves on a tray to air dry naturally, and then put the ginkgo leaves into a drying oven to dry at a constant temperature of 70℃; 3) After the ginkgo leaves are completely dried, they are crushed and passed through an 80-mesh sieve to obtain powder; 4) Weigh the ginkgo leaf powder and add it to a three-necked round-bottom flask. Add petroleum ether. Assemble the reflux condenser and place the three-necked round-bottom flask in a water bath at 70°C. Reflux once at a constant temperature for degreasing. This process takes 1 hour. 5) After decolorization, add the ginkgo leaf powder to distilled water and boil at 100℃ for 2 hours to extract; 6) Filter the liquid through nylon cloth to remove the residue and prepare for extraction again. The extraction process should be the same as the first time. Repeat the extraction for 1 hour and then filter again through nylon cloth. Combine the two filtrates. 7) The combined filtrate was then filtered using a circulating water vacuum pump. The filtrate was concentrated using a rotary evaporator, placed into a dialysis bag with a molecular weight cutoff of 500 Da, and dialyzed. After being concentrated using a rotary evaporator, alcohol was added at a volume ratio of 1:4 to begin alcohol precipitation. The mixture was allowed to stand overnight, filtered, dried, and weighed.

[0008] In the application described above, the weight-to-volume ratio of ginkgo leaf powder to petroleum ether in step 4) is 1:5.

[0009] In the application described, the weight-to-volume ratio of ginkgo leaf powder to distilled water in step 5) is 1:15.

[0010] Ginkgo biloba polysaccharide GP targets the immediate early protein IE1 / 2, the early protein p52, and the immediate early gene in HCMV. UL123 Early genes UL44 and late genes UL32 The inhibition of DNA copy number is used to suppress HCMV.

[0011] Ginkgo biloba polysaccharide GP can significantly protect against cytopathic effects induced by HCMV infection.

[0012] The results of the biological experiments of this invention show that: 1. Treatment of HCMV host cells WI-38 with Ginkgo biloba polysaccharide GP alone did not show significant cytotoxicity at concentration gradients of 1.5625 μg / ml, 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml, and 25 μg / ml. However, cytotoxicity was observed when the concentration of Ginkgo biloba polysaccharide GP reached 50 μg / ml, 100 μg / ml, and 200 μg / ml.

[0013] 2. Ginkgo biloba polysaccharide GP at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml can alleviate the cytopathic effect of HCMV-induced WI-38 cells, with the most significant effects observed at concentrations of 10 μg / ml and 20 μg / ml. The effect of treatment with 20 μg / ml Ginkgo biloba polysaccharide GP is comparable to that of the positive control drug PFA at 200 μg / ml.

[0014] 3. Ginkgo biloba polysaccharide GP at concentrations of 10 μg / ml and 20 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 up to 84% (…). p >0.05, no significant difference) and 78% ( p >0.05, no significant difference); a concentration of 10 μg / ml could inhibit the expression levels of IE1 / 2 and p52 to 66% ( ** p <0.01, the difference is extremely significant) and 59% ( ** p<0.01, the difference was extremely significant; when the GP concentration reached 20 μg / ml, it could inhibit the expression levels of IE1 / 2 and p52 by up to 44% ( * p <0.05, significant difference) and 30% ( *** p <0.001, the difference is extremely significant.

[0015] 4. Ginkgo biloba polysaccharide GP at concentrations of 5 μg / ml, 10 μg / ml, and 20 μg / ml can 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 could inhibit early gene generation. UL44 Copy count up to 78% ** p <0.01, extremely significant difference), suppressing late-stage genes. UL32 DNA copy number up to 81% ( * p <0.05, significant difference); a concentration of 10 μg / ml can inhibit early gene... UL123 Copy count up to 80% * p <0.05, significant difference), inhibiting early genes UL44 DNA copy number up to 71% ( ** p <0.01, extremely significant difference), suppressing late-stage genes. UL32 DNA copy number up to 65% ( ** p <0.01, highly significant difference); a concentration of 20 μg / ml can inhibit early gene... UL123 Copy number up to 72% ( * p <0.05, significant difference), inhibiting early genes UL44 DNA copy number up to 64% ( *** p <0.001, the difference is extremely significant), suppressing late-stage genes. UL32 DNA copy number up to 66% ( ** p <0.01, the difference is extremely significant.

[0016] The drug described in this invention also includes pharmaceutically acceptable carriers, diluents, and excipients.

[0017] The formulations of the drugs described in this invention are liquid, solid, or semi-solid formulations, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, gels, emulsions, patches, etc.

[0018] The beneficial effects of this invention are as follows: This invention provides the application of Ginkgo biloba polysaccharide GP in the preparation of drugs for the treatment and / or prevention of HCMV. Ginkgo biloba polysaccharide GP has significant anti-HCMV activity at doses without obvious cytotoxicity, manifested in its effect on HCMV immediate early protein IE1 / 2, early protein p52, and the immediate early gene. UL123 Early genes UL44 and late genes UL32 It has a significant inhibitory effect on DNA copy number, is safe and effective, and can be used for HCMV infection, showing good application prospects. Attached Figure Description

[0019] Figure 1 This study investigated the effect of Ginkgo biloba polysaccharide GP on the cytotoxicity of HCMV host cells—human embryonic lung fibroblasts (WI-38). The Control group consisted of cells not treated with Ginkgo biloba polysaccharide GP, serving as the experimental control group. Comparisons and statistical analyses were performed between the various Ginkgo biloba polysaccharide GP concentration groups and the Control group. *** P <0.001 indicates that the difference is extremely significant.

[0020] Figure 2 Ginkgo biloba polysaccharide GP alleviates the cytopathic effect of HCMV infection in human embryonic lung fibroblasts WI-38. Control group cells, not infected with HCMV, and not treated with Ginkgo biloba polysaccharide GP; The HCMV group cells were infected with HCMV alone, without the addition of Ginkgo biloba polysaccharide GP treatment; The PFA group cells were treated with the positive control drug phosphonoformic acid (PFA, 200 μg / ml) alone and were not infected with HCMV. All groups infected with HCMV were vaccinated with a dose of MOI=0.5, and were observed and photographed five days after infection (5 dpi).

[0021] Figure 3 shows the effects of different concentrations of Ginkgo biloba polysaccharide GP (5 μg / ml, 10 μg / ml, 20 μg / ml) on the immediate early proteins IE1 / 2 and p52 of HCMV in WI-38 cells as determined by Western blot (GAPDH was used as an internal control and was homogenized). Figure 3(A) shows representative images of the expression levels of immediate early proteins IE1 / 2 and p52 after treatment with Ginkgo biloba polysaccharide GP. Figure 3(B) shows the quantitative analysis and statistics of the results in Figure 3(A).

[0022] In Figure 3(B), the HCMV alone group was HCMV infection alone without the addition of Ginkgo biloba polysaccharide GP treatment. This group was set to 1 in the statistical analysis as a control group before and after drug treatment. Three concentrations of Ginkgo biloba polysaccharide (GP) and the positive control drug (PFA) were used to infect cells with HCMV at an MOI of 0.5. Samples were collected five days post-infection (5 dpi), and cells were lysed using lysis buffer for protein analysis. Compared with the group infected with HCMV alone, * express P <0.05, ** express P <0.01, *** express P <0.001.

[0023] Figure 4 The qPCR method was used to determine the effect of different concentrations of Ginkgo biloba polysaccharide GP on the immediate early HCMV gene. UL123 Early genes UL44 and late genes UL32 The effect of HCMV on DNA copy number. All three concentrations of Ginkgo biloba polysaccharide (GP) treatment groups and the positive control drug (PFA) treatment group were inoculated at an MOI of 0.5. Samples were collected three days after HCMV infection (3 dpi), viral DNA was extracted, and qPCR was performed. Compared with the HCMV-infected cells alone group, * express P <0.05, ** express P <0.01, *** express P <0.001. Detailed Implementation

[0024] 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.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials, antibodies, and chemical reagents used in the following examples can be purchased from the respective companies.

[0027] The human embryonic lung fibroblast cell line WI-38 and the human cytomegalovirus (HCMV) Towne strain involved in this invention are both from the American Type Culture Collection (ATCC).

[0028] Example 1: Preparation of Ginkgo biloba polysaccharide GP Fresh ginkgo leaves were harvested, stems and branches were removed, and the leaves were repeatedly rinsed until clean before being air-dried. The dried ginkgo leaves were placed on trays to air dry naturally, and then placed in a drying oven at a constant temperature of 70℃. The completely dried ginkgo leaves were pulverized and passed through an 80-mesh sieve to obtain powder. The ginkgo leaf powder was weighed to 100 g and added to a three-necked round-bottom flask, along with 500 mL of petroleum ether (ratio 1:5 (g / mL)). A reflux condenser was assembled, and the three-necked round-bottom flask was placed in a water bath at a constant temperature of 70℃ for one reflux degreasing cycle, which took 1 hour. After decolorization, the ginkgo leaf powder was added to distilled water at a ratio of 1:15, and the mixture was boiled at 100℃ for 2 hours. The extract was then filtered through nylon cloth to remove the residue and prepared for another extraction (the experimental procedure was the same as the first). This extraction was repeated for 1 hour, followed by filtration through nylon cloth again. The two filtrates were combined. The combined filtrates were then filtered using a circulating water vacuum pump. The filtrate was concentrated using a rotary evaporator and dialyzed into a dialysis bag with a molecular weight cutoff of 500 Da. After concentration using a rotary evaporator, alcohol was added at a ratio of 1:4 (by volume) to initiate alcohol precipitation. The mixture was allowed to stand overnight, filtered, dried, and weighed. This yielded Ginkgo biloba polysaccharide GP.

[0029] Example 2: Effect of Ginkgo biloba polysaccharide GP treatment alone on WI-38 cell cytotoxicity The effect of Ginkgo biloba polysaccharide GP on cytotoxicity was detected using the conventional CCK-8 assay.

[0030] The method for determining cytotoxicity is briefly described below: Human embryonic lung fibroblasts (WI-38) cells (passage 30, PD30) were selected. After the cells reached confluence in the cell culture flask, they were digested with trypsin and counted. Cells were seeded at a density of 5000 cells / well in 96-well cell culture plates and incubated at 37°C with 5% CO2. Cells were observed on the second day. Once a confluence of cells was achieved, the original culture medium was discarded and replaced with fresh culture medium containing different concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg / ml) of Ginkgo biloba polysaccharide (GP). Three parallel wells were set up for each concentration. A negative control group without GP and a cell-free blank control group were also set up, each with three parallel wells. The cells were incubated at 37°C with 5% CO2 for another 5 days. Care was taken to avoid introducing air bubbles when adding culture medium. Then, add 10 μl of CCK-8 solution to each well, taking care not to introduce air bubbles into the wells to avoid affecting subsequent OD value measurements. Continue culturing at 37℃ and 5% CO2 for 1-2 hours. When the culture medium turns dark brown, remove it from the incubator and measure the absorbance at 450 nm. Calculate the relative cell viability based on the measured data, setting the cell viability of the control group (without Ginkgo biloba polysaccharide GP) as 1. The experimental results are analyzed as follows: Cell viability = [OD (with added drug) - OD (blank)] / [OD (control) - OD (blank)]. The results of the effect of Ginkgo biloba polysaccharide GP on cytotoxicity are shown in […]. Figure 1 .

[0031] Example 3: HCMV inoculation and Ginkgo biloba polysaccharide GP treatment

[0032] WI-38 cells in the logarithmic growth phase (PD30) were selected, digested with trypsin, and counted. Cells were diluted with standard DMEM medium containing 10% FBS, and then cultured at a rate of 2 × 10⁻⁶ cells / cells. 4 / cm 2Cells were seeded at the specified density in six-well cell culture plates and cultured at 37°C with 5% CO2 for 24 hours. Once a monolayer had formed, the medium was replaced with DMEM containing 0.2% FBS, and the cells were cultured for another 36-48 hours to synchronize G0 / G1 cell proliferation, which is beneficial for subsequent HCMV infection experiments. HCMV virus (Towne strain) was inoculated at an MOI of 0.5 and cultured at 37°C with 5% CO2 for the specified time for relevant assays (DNA copy number assay at 3 days post-HCMV infection, protein assay at 5 days post-HCMV infection). For treatment with Ginkgo biloba polysaccharide GP and the positive control drug PFA, the medium was replaced with different concentrations (5, 10, 20 μg / ml) of Ginkgo biloba polysaccharide GP two hours before HCMV infection, followed by inoculation with HCMV (MOI=0.5). Finally, changes in cell morphology, viral protein expression, and DNA copy number were observed regardless of whether Ginkgo biloba polysaccharide GP treatment was used.

[0033] Example 4: Effect of Ginkgo biloba polysaccharide GP treatment on cytopathic effects of WI-38 cells after HCMV infection. Three days after WI-38 cells were inoculated with HCMV (MOI=0.5), cytopathic effect (CPE) caused by HCMV infection was observed in the HCMV-infected-only group. The main characteristic was that virus-infected cells became larger and rounder, with increased intercellular spaces. By day five, this cytopathic effect was more pronounced, forming a distinct net-like structure, and cells began to detach. Pretreatment of cells with Ginkgo biloba polysaccharide GP (5, 10, 20 μg / ml) significantly alleviated CPE induced by HCMV infection, especially when the concentration of Ginkgo biloba polysaccharide GP reached 10 μg / ml and 20 μg / ml, the effect was more significant, and no severe CPE occurred. Figure 2 Meanwhile, treatment of cells with Ginkgo biloba polysaccharide GP at concentrations of 5, 10, and 20 μg / ml alone did not cause significant changes in cell morphology. Figure 2 This result is consistent with Figure 1 The results of the CCK8 assay for the effect of Ginkgo biloba polysaccharide GP on cell viability were consistent.

[0034] Example 5: Effect of Ginkgo biloba polysaccharide GP treatment on HCMV virus protein expression

[0035] To further confirm the anti-HCMV effect of Ginkgo biloba polysaccharide GP, we selected HCMV immediate early protein 1 / 2 (IE1 / 2) and early protein p52 as indicators for virus detection, and examined the effect of different concentrations (5, 10, 20 μg / ml) of Ginkgo biloba polysaccharide GP pretreatment on the expression of IE1 / 2 and p52. Cell culture and plating, polysaccharide treatment, and HCMV inoculation were as described in Example 3 above. We established a negative control group (-) without HCMV infection and without polysaccharide treatment; a control group alone with HCMV inoculation and without polysaccharide treatment; and three experimental groups with different concentrations (5 μg / ml, 10 μg / ml, 20 μg / ml) of Ginkgo biloba polysaccharide pretreatment and HCMV inoculation. We also established a positive control group with PFA pretreatment and HCMV inoculation, for a total of six experimental groups. The HCMV dose was set at MOI=0.5. Cell samples were collected 5 days after HCMV infection, and cells were lysed for virus-related protein detection. SDS-PAGE protein electrophoresis and membrane transfer were performed using standard methods. After blocking with TBST containing 5% skim milk powder at room temperature for 2 hours, the membrane was incubated overnight at 4°C with mouse-derived anti-IE1 / 2 and p52 antibodies. The membrane was washed three times with TBST for five minutes each time, and then incubated with HRP-conjugated goat anti-mouse secondary antibody at room temperature for 1 hour. Finally, chemiluminescence detection was performed. The results showed that Ginkgo biloba polysaccharide GP at a concentration of 5 μg / ml could reduce the expression of HCMV immediate early proteins IE1 / 2 and p52, but the difference was not statistically significant (Figure 3). When the concentration reached 10 μg / ml and above, the inhibitory effect on IE1 / 2 and p52 proteins was significant, showing a statistically significant difference.

[0036] Example 6: Effect of Ginkgo biloba polysaccharide GP pretreatment on HCMV DNA copy number in host cells Cell culture, polysaccharide treatment, and HCMV inoculation were performed as described in Example 3 above. HCMV was also infected at an MOI of 0.5, and samples were collected 3 days post-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×UniversalSYBR Green Fast qPCR Mix. The primers used are shown in the table below. Table 1: Primer names and sequences used in the experiment

[0037] Amplification conditions: 95℃ for 5 min, (95℃ for 5 sec, 60℃ for 30 sec) × 40 cycles, for 2... -△△CtThe 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 4 As shown.

[0038] 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. The application of Ginkgo biloba polysaccharide in the preparation of drugs for treating or preventing human cytomegalovirus infection, wherein the preparation method of Ginkgo biloba polysaccharide includes the following steps: 1) Pick fresh ginkgo leaves, cut off the branches and stems, wash them repeatedly until clean, and then drain them through a sieve. 2) Place the dried ginkgo leaves on a tray to air dry naturally, and then put the ginkgo leaves into a drying oven to dry at a constant temperature of 70℃; 3) After the ginkgo leaves are completely dried, they are crushed and passed through an 80-mesh sieve to obtain powder; 4) Weigh the ginkgo leaf powder and add it to a three-necked round-bottom flask. Add petroleum ether. Assemble the reflux condenser and place the three-necked round-bottom flask in a water bath at 70°C. Reflux once at a constant temperature for degreasing. This process takes 1 hour. 5) After decolorization, add the ginkgo leaf powder to distilled water and boil at 100℃ for 2 hours to extract; 6) Filter the liquid through nylon cloth to remove the residue and prepare for extraction again. The extraction process should be the same as the first time. Repeat the extraction for 1 hour and then filter again through nylon cloth. Combine the two filtrates. 7) The combined filtrate was then filtered using a circulating water vacuum pump. The filtrate was concentrated using a rotary evaporator, placed into a dialysis bag with a molecular weight cutoff of 500 Da, and dialyzed. After being concentrated using a rotary evaporator, alcohol was added at a volume ratio of 1:4 to begin alcohol precipitation. The mixture was allowed to stand overnight, filtered, dried, and weighed.

2. The application as described in claim 1, characterized in that... The drug also includes pharmaceutically acceptable carriers and excipients.

3. The application as described in claim 1, characterized in that... The drug also includes pharmaceutically acceptable diluents.

4. The application as described in claim 1, characterized in that... The drug is formulated as a liquid, solid, or semi-solid preparation.

5. The application as described in claim 1, characterized in that... In step 4), the weight-to-volume ratio of ginkgo leaf powder to petroleum ether is 1:

5.

6. The application as described in claim 1, characterized in that... In step 5), the weight-to-volume ratio of ginkgo leaf powder to distilled water is 1:15.

Images