Application of cudrania tricuspidata fruit polysaccharide in preparation of medicine for treating or preventing HCMV infection

Abstract

The invention provides an application of cudrania tricuspidata polysaccharide in preparation of a medicine for treating or preventing HCMV infection, the cudrania tricuspidata polysaccharide ZGs-1 shows a remarkable inhibition effect on expression of immediate early protein IE1 / 2 and early protein p52 and copy numbers of virus genes UL123, UL44 and UL32 in a plurality of key links of HCMV infection, and has definite antiviral activity. Due to the natural source characteristic of the cudrania tricuspidata polysaccharide ZGs-1, the cudrania tricuspidata polysaccharide ZGs-1 is high in safety and low in cytotoxicity, and the cudrania tricuspidata raw material is easy to obtain, so that a candidate drug or functional component with remarkable potential is provided for preventing and treating the HCMV, and the cudrania tricuspidata polysaccharide ZGs-1 has a good development prospect and application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a polysaccharide in a fruit of a rattan tree in preparation of a medicine for treating or preventing HCMV infection. BACKGROUND

[0002] Human cytomegalovirus (HCMV) is a typical representative of the herpes virus family, and the genome includes 230 kb of double-stranded DNA, which encodes more than 165 viral proteins. HCMV infection is extremely common, and can be long-term latent in the host body. In the case of low immunity of the host, especially in the elderly, it is extremely easy to be activated. Due to its wide cell tropism, HCMV infection can cause a variety of symptoms and can lead to organ failure. HCMV infection is also one of the main causes of birth defects in newborns, leading to severe sequelae such as nervous system development retardation and neural deafness.

[0003] In recent years, HCMV vaccine research and development has continued to make breakthroughs, and many candidate vaccines have entered the clinical research stage, but their safety and effectiveness still need to be verified. So far, there is no HCMV vaccine approved for marketing worldwide. At present, clinical treatment still relies on nucleoside analogs represented by 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 UL97 gene mutation to produce drug-resistant strains, limiting their wide application in the clinic. Therefore, the development of new anti-HCMV drugs with high efficiency and low toxicity is of great significance for preventing neonatal defects, improving the antiviral ability of the elderly and people with low immune function, and improving the quality of life of the elderly and the quality of the population.

[0004] Polysaccharides are a class of macromolecular substances existing in living organisms, and have biological activities such as immunomodulation, antitumor, anti-inflammatory, antiviral, antibacterial, and antioxidant. The fruit of the rattan tree is a medicinal plant, and can be used as medicine. At present, the research on the rattan tree mainly focuses on flavonoids, and there is little research on polysaccharides from the rattan tree. The currently available research on polysaccharides of the rattan tree is the invention patent with the publication number CN117843820A, which mainly relates to an extraction method of polysaccharides in the fruit of the rattan tree and liver protection activity. At present, there is no report on the antiviral effect of polysaccharides in the fruit of the rattan tree, especially the anti-HCMV effect. SUMMARY

[0005] In view of the deficiencies of the prior art, the application provides application of a polysaccharide in a fruit of a rattan tree in preparation of a medicine for treating and / or preventing HCMV infection.

[0006] The object of the application is achieved by the following technical solutions. The application provides application of a polysaccharide in a fruit of a genus of Cudrania in preparation of a medicine for treating or preventing HCMV infection.

[0007] The preparation method of the polysaccharide in the fruit of the genus of Cudrania comprises the following steps: drying a Cudrania fruit, crushing the dried Cudrania fruit into powder, placing the powder in water for ultrasonic extraction, obtaining water extract after centrifugation; adding diatomite into the water extract for stirring and then standing and filtering, collecting the filtrate and then concentrating; then placing the filtrate in ethanol for alcohol precipitation, collecting the alcohol precipitate after filtration; placing the alcohol precipitate in a chloroform-n-butanol mixed solution with a volume ratio of 5:1 for oscillation, performing secondary alcohol precipitation after filtration, washing with ethanol after centrifugation, and drying to obtain crude polysaccharide; dissolving the crude polysaccharide in water, purifying the crude polysaccharide by using a macroporous adsorption resin, eluting with ultrapure water, collecting the fraction for freeze-drying to obtain the preliminary purified polysaccharide; dissolving the polysaccharide in water, and then performing two-stage classification treatment by using a DEAE cellulose anion exchange column and a Sephadex S-100 propylene glycan gel column in sequence, finally eluting with ultrapure water, collecting the elution components, and concentrating and freeze-drying to obtain the final polysaccharide in the fruit of the genus of Cudrania, which is recorded as ZGs-1.

[0008] The polysaccharide in the fruit of the genus of Cudrania has a molecular weight of 94.551 KDa, and the molar ratio of arabinose (Ara), glucose (Glc), galactose (Gal), rhamnose (Rha) and xylose (Xyl) in the polysaccharide is 0.544:0.189:0.178:0.048:0.041.

[0009] The polysaccharide in the fruit of the genus of Cudrania is mainly composed of a pyranose ring Glc as a main skeleton, and the main structure is composed of Araf-(1→, Xylp-(1→,→3)-Xylp-(1→,→5)-Araf-(1→,→4)-Xylp-(1→, Glcp-(1→,→3,5)-Araf-(1→,→4)-Galp-(1→,→4)-Glcp-(1→,→6-Glcp-(1→,→6)-Galp-(1→,→3,4)-Glcp-(1→,→4,6)-Glcp-(1→,→4,6)-Galp-(1→,→3,6)-Galp-(1→,→2,4,6)-Galp-(1→.

[0010] The treatment or prevention of HCMV is achieved by inhibiting the DNA copy number of HCMV immediate early protein IE1 / 2, early protein p52 and immediate early gene UL123 , early gene UL44 and late gene UL32 .

[0011] The biological experiment research results of the application show that: 1. The HCMV host cell WI-38 treated by Zhegu polysaccharide ZGs-1 alone does not show obvious cytotoxicity at the concentration gradient of 5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 80 μg / ml and 100 μg / ml, indicating high safety.

[0012] 2. Zhegu polysaccharide ZGs-1 can relieve the cytopathic effect of WI-38 cells caused by HCMV infection at the concentrations of 10 μg / ml, 50 μg / ml and 100 μg / ml, wherein the cytopathic effect of the group treated by 100 μg / ml Zhegu polysaccharide ZGs-1 is equivalent to that of the group treated by 200 μg / ml positive drug PFA, indicating the protective effect of Zhegu polysaccharide ZGs-1 on the HCMV-infected host cells.

[0013] 3. Zhegu polysaccharide ZGs-1 can significantly inhibit the expression of HCMV immediate early protein IE1 / 2 and early protein p52 in WI-38 at the concentrations of 10 μg / ml, 50 μg / ml and 100 μg / ml, and the effect is close to that of the group treated by positive drug PFA, and the effect is very significant.

[0014] 4. Zhegu polysaccharide ZGs-1 can significantly reduce the DNA copy number of HCMV immediate early gene UL123 , early gene UL44 and late gene UL32 at the concentrations of 25 μg / ml, 50 μg / ml and 100 μg / ml. Compared with the group inoculated with HCMV alone, the inhibition rate is about 50%.

[0015] The preparation of the drug also includes a pharmaceutically acceptable carrier, diluent and excipient.

[0016] The preparation of the drug is a liquid preparation, a solid preparation or a semi-solid preparation.

[0017] The beneficial effects of the present application are as follows: the present application relates to the application of Zhegu polysaccharide ZGs-1 in the preparation of a drug for preventing and treating human cytomegalovirus (HCMV) infection, and the Zhegu polysaccharide ZGs-1 shows significant inhibitory effect on the expression of immediate early protein IE1 / 2 and early protein p52, and the copy number of viral gene UL123, UL44, UL32 , and has clear antiviral activity. Due to its natural source characteristics, Zhegu polysaccharide ZGs-1 has high safety and low cytotoxicity, and the raw material of Zhegu is easy to obtain, thereby providing a potential candidate drug or functional component for the prevention and treatment of HCMV, and having good development prospect and application value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 1 is the identification result of Zizyphus jujuba Mill. polysaccharide ZGs-1, wherein Figure 1 (A) is the ion chromatogram of monosaccharide composition of Zizyphus jujuba Mill. polysaccharide ZGs-1; Figure 1 (B) is the infrared spectrum of Zizyphus jujuba Mill. polysaccharide ZGs-1; Figure 1 (C) is the one-dimensional nuclear magnetic H spectrum of Zizyphus jujuba Mill. polysaccharide ZGs-1; Figure 1 (D) is the one-dimensional nuclear magnetic C spectrum of Zizyphus jujuba Mill. polysaccharide ZGs-1.

[0019] Figure 2 Figure 2 is the effect of Zizyphus jujuba Mill. polysaccharide ZGs-1 on cytotoxicity of HCMV host cells-human embryonic lung fibroblast (WI-38) cells by CCK8 method, wherein ns represents no significant difference compared with the Control group without Zizyphus jujuba Mill. polysaccharide ZGs-1 treatment.

[0020] Figure 3 Figure 3 is the effect of Zizyphus jujuba Mill. polysaccharide ZGs-1 pretreated WI-38 cells on cytopathic effect after HCMV infection, wherein: the control group of cells (Mock) is that WI-38 cells are not treated with Zizyphus jujuba Mill. polysaccharide ZGs-1 and are not infected with HCMV; the HCMV only group is that WI-38 cells are not treated with Zizyphus jujuba Mill. polysaccharide ZGs-1 but are infected with HCMV only; the HCMV+PFA group is that WI-38 cells are pretreated with a positive drug phosphonoformic acid (PFA, 200 μg / ml) and then are infected with HCMV; the remaining three experimental groups are that WI-38 cells are pretreated with Zizyphus jujuba Mill. polysaccharide ZGs-1 (10, 50, 100 μg / ml) and then are infected with HCMV. All groups infected with HCMV are inoculated at a dose of MOI=0.5, and are observed and photographed at five days after infection (5 dpi).

[0021] Figure 4 Figure 4 is the effect of Zizyphus jujuba Mill. polysaccharide ZGs-1 pretreated WI-38 cells on expression of HCMV immediate early protein IE1 / 2 and early protein p52 (GAPDH is an internal reference) by Western-blot method, wherein all groups infected with HCMV are inoculated with virus at a dose of MOI=0.5, and are sampled at five days after infection (5 dpi), and the protein is extracted by a conventional method for Western-blot detection. Phosphonoformic acid PFA is used as a positive drug control.

[0022] Figure 5 Figure 5 is the effect of Zizyphus jujuba Mill. polysaccharide ZGs-1 (25 μg / ml, 50 μg / ml, 100 μg / ml) on HCMV immediate early gene IE1 / 2 by qPCR method. UL123DNA copy number of early genes UL44 and late genes UL32 compared to the HCMV only infected cell group, * indicates P <0.1, ** indicates P <0.01, *** indicates P <0.001. DETAILED DESCRIPTION

[0023] The present application will be further described below in connection with specific examples, which are only intended to explain the present application and are not meant to limit the scope of the present application.

[0024] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0025] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0026] The human embryonic lung fibroblast cell line WI-38 and human cytomegalovirus (HCMV) Towne strain used in the present application are from American type culture collection (ATCC).

[0027] Example 1: Preparation of polysaccharide ZGs 1 from the fruit of Cudrania tricuspidata The dried fruit of the plant is ground and the powder is passed through a 60 mesh screen. The powder is then extracted with water using ultrasonic extraction. The ratio of the fruit powder to water is 1:20 and the extraction is carried out for 2 hours at a power setting of 60 W. The mixture is then centrifuged at 4000 rpm for 10 minutes to obtain the water extract. Silica is then added to the water extract and stirred. The mixture is then allowed to stand and the filtrate is collected. The filtrate is then concentrated. The concentrated filtrate is then precipitated with ethanol for 48 hours. The precipitate is then collected by filtration. The precipitate is then dissolved in a mixture of chloroform and n-butanol in a volume ratio of 5:1 and shaken. The mixture is then filtered and the precipitate is re-precipitated with ethanol for 12 hours. The precipitate is then washed with ethanol and dried to obtain the crude polysaccharide. The crude polysaccharide is then dissolved in water and purified using a macroporous adsorption resin. The purified polysaccharide is then eluted with ultrapure water and the fractions are collected and freeze-dried to obtain the preliminary purified polysaccharide. The preliminary purified polysaccharide is then dissolved in water and fractionated using a DEAE cellulose anion exchange column. The column is then eluted with ultrapure water, 0.1 M, 0.3 M and 0.5 M sodium chloride solutions in sequence. The fractions are then collected and the fraction eluted with 0.1 M sodium chloride solution is freeze-dried to obtain the first fraction of the polysaccharide. The first fraction of the polysaccharide is then dissolved in water and fractionated using a Sephadex S-100 propylene glycan gel column. The column is then eluted with ultrapure water and the fractions are collected, concentrated and freeze-dried to obtain the final polysaccharide from the fruit of the plant, which is designated as ZGs-1.

[0028] Example 2: Effect of ZGs-1 on the toxicity of WI-38 cells The effect of ZGs-1 on the toxicity of cells was determined by CCK-8 method.

[0029] The method for determining cytotoxicity is briefly described as follows: human embryonic lung fibroblasts WI-38 were digested with trypsin, then dispersed with DMEM medium containing 10% FBS, and plated in a 96-well plate, and cultured at 37°C, 5% CO2 for overnight. The next day, after observing the cell monolayer, the original culture medium was discarded, and fresh culture medium containing different concentrations of Zhegu polysaccharide ZGs-1 (0, 5, 10, 20, 30, 40, 50, 80, 100 μg / ml) was added, with 6 replicates for each concentration, and a cell control group without polysaccharide and a blank control group without cells were set up for subsequent numerical calculation. The cell morphology was observed daily and recorded. The culture was continued at 37°C, 5% CO2 for 5 days, and the CCK-8 working solution was prepared in advance according to the amount of 10 μl / well. The culture medium in the 96-well plate was aspirated, and the freshly prepared CCK-8 working solution was added. Incubate in the incubator for 1-2 hours, and use the microplate reader to measure the absorbance value of each well at 450 nm wavelength. According to the results of the absorbance value, the relative viability of the cells was calculated, and the control group was used as the reference for normalization. The experimental results are analyzed as follows: cell viability = [OD (drug) - OD (blank)] / [OD (0 drug) - OD (blank)]. The results of the effect of Zhegu polysaccharide ZGs-1 on cell toxicity are shown in Figure 2 Zhegu polysaccharide ZGs-1 alone showed no obvious cytotoxicity to WI-38 cells at a concentration gradient of 100 μg / ml or less.

[0030] Example 3: Inoculation of HCMV and treatment of Zhegu polysaccharide ZGs 1 and positive drug PFA Human embryonic lung fibroblasts WI-38 were routinely digested with trypsin, diluted and dispersed with DMEM medium containing 10% FBS, plated in a 12-well cell culture plate, and incubated at 37°C, 5% CO2 for 24 h, then replaced with DMEM medium containing 0.2% FBS, and incubated for another 48 h to synchronize G0 / G1, so as to facilitate subsequent HCMV infection. Then, HCMV virus (Towne strain) infection was performed, and the inoculation dose of HCMV was MOI=0.5. Finally, the culture was incubated to the specified time for sample collection and related detection. When Zhegu polysaccharide ZGs-1 and positive drug phosphonoformic acid PFA were treated, different concentrations (10, 50, 100 μg / ml) of Zhegu polysaccharide ZGs-1 or positive drug phosphonoformic acid PFA were added to the culture medium 2 h before HCMV infection (MOI=0.5), and then the virus was infected. Then, the cell morphology, viral protein expression, and viral gene DNA copy number of Zhegu polysaccharide ZGs-1 and phosphonoformic acid PFA treatment groups were observed daily.

[0031] Example 4: Effect of Zhegufu polysaccharide ZGs 1 pretreatment of WI-38 cells on cytopathic effect after HCMV infection After WI-38 cells were inoculated with HCMV (MOI = 0.5), typical cytopathic effects were observed Figure 3 - HCMV only group): Compared with the cells of the Mock group, the HCMV-infected cells were swollen in volume, the intercellular space was widened, and the cell morphology changed from fibrous to round, elongated or spindle-shaped. After pretreatment with different concentrations of Zhegufu polysaccharide ZGs-1 (10, 50, 100 μg / ml), the cytopathic effect was significantly improved, and the protective effect of Zhegufu polysaccharide ZGs-1 on infected cells was comparable to that of the positive control phosphonoformic acid (PFA, 200 μg / ml) treatment group, with only a small cytopathic effect, which suggests that Zhegufu polysaccharide ZGs-1 has strong anti-HCMV infection ability.

[0032] Example 5: Effect of Zhegufu polysaccharide ZGs 1 pretreatment on HCMV viral protein expression To further confirm the inhibitory effect of Zhegufu polysaccharide ZGs-1 on HCMV infection, based on the observation of cytopathic effect, the effect of Zhegufu polysaccharide ZGs-1 treatment on the expression of HCMV immediate early protein IE1 / 2 and early protein p52 was further verified by Western-blot experiment. Four groups were set up, respectively: negative control group (-), HCMV infection alone group (+), positive drug phosphonoformic acid PFA treatment group (PFA), and three Zhegufu polysaccharide ZGs-1 pretreatment groups (10, 50, 100 μg / ml). Cell culture, drug treatment and HCMV infection were carried out according to the previous Example 3, with 0.5 MOI of HCMV inoculation, and the samples were collected after 5 days of infection. SDS-PAGE electrophoresis and membrane transfer were carried out according to the conventional method, and the membrane was blocked with 5% skim milk powder in TBST at room temperature for 2 hours. Specific mouse monoclonal antibodies for IE1 / 2 and p52 were added respectively, and incubated at 4°C overnight. The next day, the corresponding HRP-labeled goat anti-mouse secondary antibody was added and incubated at room temperature for 1 hour, and finally the protein expression level was detected by chemiluminescence. The results showed that Zhegufu polysaccharide ZGs-1 could significantly inhibit the expression of HCMV immediate early protein IE1 / 2 and early protein p52, and the effect was very significant at a concentration of 10 μg / ml Figure 4 ). This result shows that Zhegufu polysaccharide ZGs-1 has clear anti-HCMV activity at the protein level, which is consistent with the observed cytopathic effect. Figure 3

[0033] Example 6: Effect of Zhegufu polysaccharide ZGs 1 pretreatment on DNA copy number of HCMV in host cells ​Cell culture, ZGs-1 treatment and HCMV inoculation were performed as described in Example 3 above. ZGs-1 was set at three concentrations (25, 50, 100 μg / ml), and HCMV was inoculated at a dose of 0.5 MOI. Samples were collected at 3 days post-infection (3 dpi). Viral DNA was extracted using QIAamp DNA Mini Kit, and the copy number of HCMV DNA was determined by qPCR. 10 ng of total DNA was used for qPCR with 2x Universal SYBR Green Fast qPCR Mix. The primers used are shown in Table 1: Table 1: Primer names and sequences used in the experiment Gene name Primer name Sequence -forward-reverse 5'-TCTGCCAGGACATCTTTCTC-3' 5'-GTGACCAAGGCCACGACGTT-3' -forward-reverse 5'-ACTGCCGTGCACGTTGCGTA-3' 5'-ACTTGCCGCTGTTCCCGACG-3' -forward-reverse 5'-GGTTTCTGGCTCGTGGATGTCG-3' 5'-CACACAACACCGTCGTCCGATTAC-3' -forward-reverse 5'-CTGTTGCTGTAGCCAAATTCGT-3' 5'-ACCCACTCCTCCACCTTTGAC-3' Amplification conditions: 95°C for 5 min, (95°C for 5 sec, 60°C for 30 sec) x 40 cycles, 2 -△△Ct The results are shown in Figure 6. The copy number of DNA of HCMV immediate-early gene (IE1), early gene (UL123) and late gene (UL44) was reduced at concentrations of 25 μg / ml, 50 μg / ml and 100 μg / ml of ZGs-1. Figure 5 UL123 UL44 UL32 Compared with the HCMV only group, ZGs-1 inhibited the copy number of the three genes to about 40%.

[0034] The above description is merely preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall fall within the scope of the present application.​​​

Claims

1. Use of a polysaccharide from the fruit of Smilax china L. in the preparation of a drug for treating or preventing HCMV infection.

2. Use according to claim 1, wherein The preparation method of the polysaccharide from the fruit of Smilax china L. comprises the following steps: drying the fruit of Smilax china L. and crushing it into powder, placing the powder in water and performing ultrasonic extraction, centrifuging to obtain a water extract; adding diatomite to the water extract and stirring, then standing and filtering, collecting the filtrate and concentrating; then placing the filtrate in ethanol and performing alcohol precipitation, filtering and collecting the alcohol precipitate; placing the alcohol precipitate in a chloroform-n-butanol mixed solution with a volume ratio of 5:1 and performing oscillation, performing secondary alcohol precipitation, washing with ethanol after centrifuging, drying to obtain crude polysaccharide; dissolving the crude polysaccharide in water, purifying with macroporous adsorption resin, eluting with ultrapure water, collecting the fractions and freeze-drying to obtain preliminarily purified polysaccharide; dissolving the polysaccharide in water, and performing two-stage fractionation with a DEAE cellulose anion exchange column and a Sephadex S-100 propylene glycol gel column in sequence, finally eluting with ultrapure water, collecting the elution components, concentrating and freeze-drying to obtain the final polysaccharide from the fruit of Smilax china L., denoted as ZGs-1.

3. The use according to claim 1, characterized in that The polysaccharide from the fruit of Smilax china L. has a molecular weight of 94.551 KDa, and the molar ratio of arabinose (Ara), glucose (Glc), galactose (Gal), rhamnose (Rha) and xylose (Xyl) in the polysaccharide is 0.544:0.189:0.178:0.048:0.

041.

4. The use according to claim 1, characterized in that The treatment or prevention of HCMV is by the inhibitory effect on the DNA copy number of the HCMV immediate early proteins IE1 / 2, early protein p52 and immediate early gene UL123 , early gene UL44 and late gene UL32 .

5. Use according to claim 1, characterized in that The preparation of the drug further comprises a pharmaceutically acceptable carrier, diluent or excipient.

6. The use according to claim 1, characterized in that The preparation of the drug is a liquid preparation, a solid preparation or a semi-solid preparation.

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