Application of crocus sativus petal polysaccharide in preparation of anti-HCMV (human cytomegalovirus) infection medicine

Abstract

The invention provides application of the saffron petal polysaccharide APPCS-1a in preparation of drugs for preventing and treating human cytomegalovirus (HCMV) infection, the saffron petal polysaccharide APPCS-1a can significantly reduce expression of immediate early-stage protein IE1 / 2 and early-stage protein p52 of HCMV and inhibit DNA copy numbers of virus genes UL123, UL44 and UL32, has definite anti-HCMV infection activity, and can be used for preparing drugs for preventing and treating HCMV infection. Therefore, the compound shows good prevention and treatment effects on HCMV infection, is low in toxic and side effects, has good development prospects and application values, and can provide a new effective choice for preparation of anti-HCMV infection drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of saffron petal polysaccharide in the preparation of anti-HCMV infection drugs. Background Technology

[0002] Human cytomegalovirus (HCMV) is a double-stranded DNA (dsDNA) virus belonging to the β-herpesvirus family. HCMV infection is widespread in the population and can cause severe symptoms and even death in newborns or adults with immature or compromised immune systems (such as HIV patients and organ transplant recipients). Numerous studies have shown that HCMV's most significant characteristic is its ability to evade recognition and killing by host immune cells, thus establishing a latent infection within the host. When host immunity is weakened, the latent HCMV can rapidly reactivate. Although significant progress has been made in HCMV vaccine research, no HCMV vaccine has yet been approved for HCMV prevention. Currently, clinical treatment still relies on nucleoside analogs such as ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV). However, the long-term use of these drugs has severely hampered their efficacy due to serious adverse reactions and the emergence of drug-resistant strains. Therefore, finding highly effective and low-toxicity anti-HCMV drugs remains a key focus of virological research.

[0003] Saffron ( Crocus sativus Saffron (L.), also known as crocus, is a perennial herb belonging to the genus Crocus in the Iridaceae family. It is a valuable traditional Chinese medicine with both medicinal and edible uses. It possesses effects such as promoting blood circulation, removing blood stasis, cooling the blood, detoxifying, relieving depression, and calming the mind. Currently, many saffron products have been developed and are used in Tibetan medicine, health products, and cosmetics. However, these applications are limited to the stigma of the pistil, a characteristic that restricts its large-scale commercial application in terms of yield. Polysaccharides, as macromolecular compounds with various activities, have wide applications in medicine, cosmetics, and anti-aging. Currently, there is little research on saffron petal polysaccharides. Patent publication number CN114751995A discovered a saffron petal polysaccharide with anti-inflammatory effects, and patent publication number CN119930851A mainly relates to an extraction method and antioxidant activity of alkali-extracted saffron petal polysaccharide APPCS-1a. However, to date, saffron, including its active polysaccharides, has not been found to be used in the preparation of anti-HCMV drugs. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides the application of alkali-extracted saffron petal polysaccharide APPCS-1a in the preparation of drugs for treating and / or preventing HCMV infection.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides the application of saffron petal polysaccharide in the preparation of drugs against HCMV infection.

[0006] The extraction method of saffron petal polysaccharide includes the following steps: Fresh saffron petals are dried, ground into powder, and filtered after adding 95% ethanol. The residue is collected and dried for later use. Then, 8% NaOH solution is added, and the mixture is heated in a water bath at 80℃ and stirred with a magnetic stirrer for 2 hours. Extraction is performed twice, and the extract is immediately neutralized to neutral after extraction. The mixture is centrifuged at 4000 rpm and filtered for 10 minutes. The supernatant is collected, compressed under reduced pressure, and dialyzed in a 3500 Da dialysis bag for 3 days to obtain the polysaccharide product. A small amount of KCl is added to the concentrated polysaccharide to promote polysaccharide aggregation. After precipitation with anhydrous ethanol, the product is filtered and dried to obtain crude polysaccharide. The crude polysaccharide is separated using a DEAE-52 cellulose column (5.5×50cm) and continuously eluted with deionized water and 0.1, 0.3, and 0.5 M NaCl. Finally, it is purified using an S-100 gel column. The eluents are combined and dialyzed continuously in a 500 Da dialysis bag at 4℃ for 3 days. After concentration, the polysaccharide is dried using a vacuum freeze dryer. The obtained polysaccharide is designated APPCS-1a.

[0007] The anti-HCMV infection described in this invention refers to the inhibition of HCMV immediate early proteins IE1 / 2, p52, and the immediate early gene. UL123 Early genes UL44 and late genes UL32 It inhibits the DNA copy number.

[0008] The results of the biological experiments of this invention show that: 1. Saffron petal polysaccharide APPCS-1a, when used alone to treat HCMV host cells WI-38, did not show significant cytotoxicity to WI-38 at concentration gradients 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.

[0009] 2. Saffron petal polysaccharide APPCS-1a at concentrations of 10 μg / ml, 50 μg / ml, and 100 μg / ml can alleviate the cytopathic effect of WI-38 cells caused by HCMV infection.

[0010] 3. Saffron petal polysaccharide APPCS-1a at concentrations of 25 μg / ml, 50 μg / ml, and 100 μg / ml significantly inhibited the expression of HCMV immediate early protein IE1 / 2 and early protein p52 in WI-38. In particular, the effect of the 100 μg / ml concentration was close to that of the positive drug PFA group, indicating its inhibitory effect on HCMV viral proteins.

[0011] 4. Saffron petal polysaccharide APPCS-1a at concentrations of 25 μg / ml, 50 μg / ml, and 100 μg / ml can significantly reduce the immediate early stage gene of HCMV. UL123 Early genes UL44 and late genes UL32 The DNA copy number indicates its inhibitory effect on HCMV virus genes.

[0012] The pharmaceutical formulations described in this invention also include pharmaceutically acceptable carriers, diluents, and excipients.

[0013] The formulation of the drug described in this invention is a liquid formulation, a solid formulation, or a semi-solid formulation.

[0014] The beneficial effects of this invention are as follows: This invention provides the application of saffron petal polysaccharide APPCS-1a in the preparation of drugs for preventing and treating human cytomegalovirus (HCMV) infection. The saffron petal polysaccharide APPCS-1a can significantly reduce the expression of HCMV immediate early proteins IE1 / 2 and p52, inhibiting viral gene expression. UL123, UL44, UL32 The DNA copy number is low, and it has clear anti-HCMV infection activity, thus showing good preventive and therapeutic effects against HCMV infection. Moreover, it has low toxicity and side effects, and has good development prospects and application value. It can provide a new and effective option for the preparation of anti-HCMV infection drugs. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of saffron petal polysaccharide APPCS-1a and an analysis of its physicochemical properties. Figure 1 (A) is a schematic diagram of the structure of saffron petal polysaccharide APPCS-1a; Figure 1 (B) is the UV full-wavelength scanning analysis of saffron petal polysaccharide APPCS-1a; Figure 1 (C) is the infrared spectrum analysis of saffron petal polysaccharide APPCS-1a; Figure 1 (D) is a diagram showing the composition of monosaccharide APPCS-1a from saffron petal polysaccharide.

[0016] Figure 2The effect of saffron petal polysaccharide APPCS-1a on the cytotoxicity of HCMV host cells—human embryonic lung fibroblasts (WI-38) was detected by CCK8 assay. Among them, compared with the control group without saffron petal polysaccharide APPCS-1a treatment, ns indicates no significant difference.

[0017] Figure 3 This study investigated the effect of saffron petal polysaccharide APPCS-1a pretreatment on cytopathic effects in WI-38 cells after HCMV infection. The cells were categorized into three groups: a control group (Mock), where WI-38 cells were not treated with saffron petal polysaccharide APPCS-1a and were not infected with HCMV; an HCMV-only group, where WI-38 cells were infected with HCMV alone without APPCS-1a treatment; an HCMV+PFA group, where WI-38 cells were pretreated with the positive control drug phosphatidylcholine (PFA, 200 μg / ml) for 2 h before HCMV infection; and three experimental groups, where WI-38 cells were pretreated with saffron petal polysaccharide APPCS-1a (10, 50, and 100 μg / ml) for 2 h before HCMV infection. All HCMV infection groups were infected at an MOI of 0.5. Cell morphological changes were observed daily after infection, and images were taken and recorded five days post-infection (5 dpi).

[0018] Figure 4 This study investigated the effects of different concentrations of saffron petal polysaccharide APPCS-1a (10 μg / ml, 50 μg / ml, and 100 μg / ml) pretreatment on the expression of immediate early HCMV proteins IE1 / 2 and p52 (GAPDH was used as an internal control) in WI-38 cells using Western blotting. All HCMV-infected groups were inoculated with the virus at an MOI of 0.5, and samples were collected five days after infection (5 dpi). Proteins were extracted using standard methods and analyzed by Western blotting. Phosphoric acid (PFA) was used as a positive control.

[0019] Figure 5 The study used qPCR to determine the effect of different concentrations of saffron petal polysaccharide APPCS-1a (25 μg / ml, 50 μg / ml, 100 μg / ml) on the immediate early HCMV gene. UL123 Early genes UL44 and late genes UL32 The effect of DNA copy number on the control of HCMV-only cell infection compared to the control group. ** express P <0.01, *** express P <0.001. Detailed Implementation

[0020] 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.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] 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).

[0024] Example 1: Preparation of saffron petal polysaccharide APPCS-1a Fresh saffron petals were dried, ground into powder, and filtered after adding 95% ethanol. The residue was collected, dried, and set aside. Then, 8% NaOH solution was added, and the mixture was heated in a water bath at 80°C and stirred with a magnetic stirrer for 2 hours. This process was repeated twice, and the extract was immediately neutralized to neutral after each extraction. The mixture was centrifuged at 4000 rpm and filtered for 10 minutes. The supernatant was collected, compressed under reduced pressure, and dialyzed in a 3500 Da dialysis bag for 3 days to obtain the polysaccharide product. A small amount of KCl was added to the concentrated polysaccharide to promote polysaccharide aggregation. The polysaccharide was then precipitated with anhydrous ethanol, filtered, and dried to obtain crude polysaccharide. The crude polysaccharide was separated using a DEAE-52 cellulose column (5.5×50cm) and eluted continuously with deionized water and 0.1, 0.3, and 0.5 M NaCl. Finally, the polysaccharide was purified using an S-100 gel column. The eluents were combined and dialyzed continuously in a 500 Da dialysis bag at 4°C for 3 days. After concentration, the polysaccharide was dried using a vacuum freeze dryer. The obtained polysaccharide was designated APPCS-1a.

[0025] Example 2: Effect of saffron petal polysaccharide APPCS-1a alone on WI-38 cell cytotoxicity The effect of saffron petal polysaccharide APPCS-1a on cytotoxicity was detected by CCK-8 assay.

[0026] The method for determining cytotoxicity was based on the CCK-8 reagent instructions and is briefly described below: Human embryonic lung fibroblasts (WI-38) were cultured using standard methods in DMEM medium supplemented with 10% FBS. Depending on the cell volume used, cells were seeded into 96-well plates one day in advance and cultured overnight at 37°C and 5% CO2. On the second day, cells were observed until a monolayer formed. The original medium was discarded, and fresh medium containing different concentrations of saffron petal polysaccharide APPCS-1a (0, 5, 10, 20, 30, 40, 50, 80, 100 μg / ml) was added, with six replicates for each concentration. A control group without polysaccharide and a blank control group without cells were also included. Cell morphology was observed and recorded daily. Cells were cultured for another 5 days at 37°C and 5% CO2. CCK-8 solution was added at a rate of 10 μl / well, taking care not to introduce air bubbles that could affect subsequent absorbance measurements. The cells were placed in an incubator for 1-2 hours, and the color changes in the wells were observed continuously. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. The relative cell viability was calculated based on the absorbance values, and normalized to the control group viability. The experimental results are analyzed as follows: Cell viability = [OD (drug-treated) - OD (blank)] / [OD (0 drug-treated) - OD (blank)]. The effects of saffron petal polysaccharide APPCS-1a on cytotoxicity are shown in the following figures. Figure 2 As shown, saffron petal polysaccharide APPCS-1a, when used alone to treat HCMV host cells WI-38, did not exhibit significant cytotoxicity to WI-38 cells within the detection concentration range (5, 10, 20, 30, 40, 50, 80, 100 μg / ml).

[0027] Example 3: HCMV inoculation and treatment with saffron petal polysaccharide APPCS-1a and positive control drug PFA Human embryonic lung fibroblasts (WI-38) were routinely digested with trypsin, diluted and dispersed in DMEM medium containing 10% FBS, plated into 12-well cell culture plates, and incubated at 37 °C with 5% CO2 for 24 h. The medium was then replaced with DMEM medium containing 0.2% FBS, and cultured for another 48 h to synchronize G0 / G1 cells for subsequent HCMV infection. HCMV infection (Towne strain) was then performed, with an MOI of 0.5. Finally, samples were collected at the designated time for relevant assays. When treating saffron petal polysaccharide APPCS-1a and positive control drug PFA, different concentrations (10, 50, 100 μg / ml) of saffron petal polysaccharide APPCS-1a or positive control drug PFA (200 μg / ml) were added to the cell culture medium 2 h (-2 h) before HCMV infection, and then HCMV was inoculated (MOI=0.5). The changes in cell morphology, viral protein expression, and viral DNA copy number were observed daily in the groups treated with saffron petal polysaccharide APPCS-1a (10, 50, 100 μg / ml) and PFA (200 μg / ml).

[0028] Example 4: Effect of saffron petal polysaccharide APPCS-1a pretreatment on WI-38 cells in HCMV-infected cytopathic effects After WI-38 cells were inoculated with HCMV (MOI=0.5), a typical cytopathic effect was observed. Figure 3 HCMV-infected cells swelled, with increased intercellular spaces and large pores in the cell pores. Pretreatment with different concentrations of saffron petal polysaccharide APPCS-1a (10, 50, 100 μg / ml) significantly alleviated cytopathic effects. The protective effect of saffron petal polysaccharide APPCS-1a on infected cells was comparable to that of the positive control phosphonoformic acid (PFA, 200 μg / ml), with only very weak cytopathic effects. This result indicates that saffron petal polysaccharide APPCS-1a has significant anti-HCMV infection ability.

[0029] Example 5: Effect of saffron petal polysaccharide APPCS-1a pretreatment on HCMV viral protein expression The effect of saffron petal polysaccharide APPCS-1a treatment on the expression of immediate early proteins IE1 / 2 and p52 in HCMV was verified by Western blot experiment, aiming to determine the effect of saffron petal polysaccharide APPCS-1a on HCMV infection. The experiment included six groups: a negative control group (-), an HCMV infection group (+), a positive drug PFA treatment group (PFA), and three saffron petal polysaccharide APPCS-1a pretreatment groups (10, 50, and 100 μg / ml). Cell culture, drug treatment, and HCMV infection were performed as described in Example 3 above. HCMV was inoculated at 0.5 MOI. Five days after infection, samples were collected and subjected to SDS-PAGE electrophoresis, membrane transfer, and blocking at room temperature using standard methods. Then, specific antibodies for IE1 / 2 and p52 were added and incubated overnight at 4°C. The next day, HRP-labeled goat anti-mouse secondary antibody was added and incubated at room temperature for 1 hour. Finally, chemiluminescence was used to detect protein expression levels. The results showed that saffron petal polysaccharide APPCS-1a could significantly inhibit the expression of HCMV immediate early proteins IE1 / 2 and p52. Figure 4 This result indicates that saffron petal polysaccharide APPCS-1a has a clear anti-HCMV activity at the protein level, and is consistent with... Figure 3 The observed cytopathic effects were consistent with the results.

[0030] Example 6: Effect of saffron petal polysaccharide APPCS-1a pretreatment on HCMV DNA copy number in host cells Cell culture, saffron petal polysaccharide APPCS-1a treatment, and HCMV inoculation were performed as described in Example 3 above. Three experimental groups were set with saffron petal polysaccharide APPCS-1a concentrations (25, 50, and 100 μg / ml). HCMV was inoculated at an MOI of 0.5. Samples were collected 3 days post-HCMV infection (3 dpi). Viral DNA was extracted using the QIAamp DNA Mini Kit; specific methods were described in the kit's instructions. 10 ng of total DNA was used for qPCR experiments with 2×Universal SYBR GreenFast qPCR Mix. The primers used are shown in the table below. 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′- GGTTTC TGGCTCGTGGATGTCG-3′ 5′- CACACAACACCGTCGTCCGATTAC-3′ -forward-reverse 5′-CTGTTGCTGTAGCCAAATTCGT-3′5′-ACCCACTCCTCCACCTTTGAC-3′ Amplification conditions: 95℃ for 5 min, (95℃ for 5 sec, 60℃ for 30 sec) × 40 cycles, for 2... -△△Ct The method was used to calculate the DNA copy number of samples infected with HCMV alone (HCMV only) as a control. The results are as follows: Figure 5As shown, concentrations of 25 μg / ml, 50 μg / ml, and 100 μg / ml can all immediately reduce HCMV. UL123 , UL44 and UL32 DNA copy number. Compared with the group inoculated with HCMV alone, saffron petal polysaccharide APPCS-1a can suppress the DNA copy number of three genes to about 30%.

[0031] 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. Application of saffron petal polysaccharide in the preparation of anti-HCMV infection drugs.

2. The application as described in claim 1, characterized in that... The term "anti-HCMV infection" refers to the action against HCMV immediate early proteins IE1 / 2, p52, and the immediate early gene. UL123 Early genes UL44 and late genes UL32 It inhibits the DNA copy number.

3. The application as described in claim 1, characterized in that... The extraction method of saffron petal polysaccharide includes the following steps: Fresh saffron petals are dried, ground into powder, and filtered after adding 95% ethanol. The residue is collected and dried for later use. Then, 8% NaOH solution is added, and the mixture is heated in a water bath at 80℃ and stirred with a magnetic stirrer for 2 hours. Extraction is performed twice, and the extract is immediately neutralized to neutral after extraction. The mixture is centrifuged at 4000 rpm for 10 minutes, and the supernatant is compressed under reduced pressure and dialyzed in a 3500 Da dialysis bag for 3 days to obtain the polysaccharide product. A small amount of KCl is added to the concentrated polysaccharide to promote polysaccharide aggregation, followed by precipitation with anhydrous ethanol. After filtration, the polysaccharide is dried to obtain crude polysaccharide. The crude polysaccharide is separated using a DEAE-52 cellulose column (5.5×50cm) and continuously eluted with deionized water, 0.1, 0.3, and 0.5M NaCl. Finally, it is purified using an S-100 gel column. The eluents are combined and dialyzed continuously in a 500 Da dialysis bag at 4℃ for 3 days. After concentration, the polysaccharide is dried using a vacuum freeze dryer. The obtained polysaccharide is designated APPCS-1a.

4. The application as described in claim 1, characterized in that... The pharmaceutical preparation also includes pharmaceutically acceptable carriers, diluents, and excipients.

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

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