Ergothioneine and achyranthes bidentata polysaccharide composition for inhibiting HPV (human papillomavirus) and preparation method thereof

Abstract

The invention discloses an ergothioneine and achyranthes bidentata polysaccharide composition for inhibiting HPV and a preparation method of the ergothioneine and achyranthes bidentata polysaccharide composition, and relates to the technical field of biological medicines. The composition is prepared from the following components in parts by mass: 1 to 5 parts of ergothioneine, 3 to 10 parts of achyranthes bidentata polysaccharide, 0.2 to 1.2 parts of a CXCR4 antagonist, 0.5 to 2 parts of sodium hyaluronate, 10 to 20 parts of a polyhydric alcohol cosolvent and 60 to 80 parts of deionized water, wherein the CXCR4 antagonist has a specific structure as shown in formula 1. According to the composition, through targeted intervention of the CXCR4 antagonist, delivery network construction of achyranthes bidentata polysaccharides and stable synergy of ergothioneine, a multi-mechanism synergistic effect is achieved, and the inhibition effect on HPV infection and the preparation stability are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a composition of ergothioneine and Achyranthes bidentata polysaccharide that inhibits HPV and its preparation method. Background Technology

[0002] Currently, interventions for human papillomavirus (HPV) infection, especially topical preparations, mostly focus on working through immune modulation or direct disruption of the viral envelope. For example, some compositions based on plant polysaccharides or antioxidants primarily inhibit the virus indirectly by enhancing local immunity or scavenging free radicals. Their targets are relatively singular, and their effectiveness against HPV viruses that have already entered cells and established latent infection is limited. They fail to fundamentally block the key pathways by which the virus replicates using host cells. This results in significant shortcomings in the thoroughness of virus elimination and prevention of recurrence among existing products. In terms of composition, many existing technologies involve simple physical mixing of multiple active ingredients, lacking synergistic effects between components. For example, some compositions, although containing substances with good biological activity such as ergothioneine, suffer from poor transdermal absorption and stability, and fail to form an effective delivery system with other components (such as macromolecular polysaccharides), resulting in significantly reduced bioavailability and limiting their full efficacy. Furthermore, if a composition focuses only on inhibiting a single stage without effectively interfering with multiple key nodes in the viral life cycle, such as viral adsorption and penetration, its overall inhibitory efficiency will face bottlenecks. From a formulation process perspective, effectively integrating active ingredients with different physicochemical properties, especially hydrophobic small-molecule drugs, with hydrophilic macromolecular polysaccharides, is a major challenge. Conventional mixing and stirring methods are insufficient to ensure the uniform dispersion and long-term stability of hydrophobic active ingredients in a hydrophilic matrix, easily leading to problems such as precipitation and aggregation, directly affecting the product's uniformity, stability, and final efficacy. Therefore, developing a composition that achieves efficient synergy among components, good physical stability, and targets multiple stages of HPV infection is of great significance. Summary of the Invention

[0003] This invention addresses the problems in existing technologies, such as single HPV inhibition target, insufficient synergistic effect among active ingredients, poor physical stability of formulations, and uneven dispersion of hydrophobic components. It provides an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition with clear multi-mechanism synergistic effects, good compatibility between components, high stability, efficient virus inhibition and skin barrier repair, and stable use for HPV infection intervention. To achieve an anti-aging spermidine composition, a CXCR4 antagonist with the structure shown in Formula 1 is included: Formula 1; R1 in Formula 1 is a substituent; R1 is selected from any one of methyl, ethyl, propyl, methoxy, ethoxy, benzyloxy, and piperidinyl. Furthermore, R1 is selected from any one of methyl, methoxy, benzyloxy, and piperidinyl. Furthermore, the CXCR4 antagonist is one of the compounds shown in the following structures: ; . A composition of ergothioneine and Achyranthes bidentata polysaccharide for inhibiting HPV, the composition comprising the following components in parts by weight: 1-5 parts ergothioneine, 3-10 parts Achyranthes bidentata polysaccharide, 0.2-1.2 parts CXCR4 antagonist, 0.5-2 parts sodium hyaluronate, 10-20 parts polyol cosolvent, and 60-80 parts deionized water. Furthermore, the polyol co-solvent is selected from one or more of propylene glycol, glycerol, and 1,3-butanediol. Furthermore, the Achyranthes bidentata polysaccharide is an acidic polysaccharide with an average molecular weight of 1000-5000 Da, prepared by a water extraction and alcohol precipitation process. Furthermore, the ergothioneine has a purity of 99% or higher. A method for preparing a composition of ergothioneine and Achyranthes bidentata polysaccharide that inhibits HPV includes the following steps: a. Add the Achyranthes bidentata polysaccharide and sodium hyaluronate to deionized water and stir to dissolve at 40-50°C to form an aqueous matrix; b. Add the CXCR4 antagonist to a polyol co-solvent and sonicate at 55-65°C until completely dissolved to obtain an active premix solution; c. Add the ergothioneine to the aqueous matrix and stir in the dark until completely dissolved; d. Under high-speed shearing conditions, the premixed active ingredient solution is slowly added dropwise to an aqueous matrix containing ergothionein, and the shearing rate is controlled at 5000-8000 rpm, so that the CXCR4 antagonist undergoes supramolecular self-assembly within the molecular network formed by Achyranthes bidentata polysaccharide to obtain a micro-nano-scale dispersion system. Furthermore, in step b, the ultrasonic treatment power is 200-400W, and the treatment time is 15-30min. Furthermore, after the addition in step d is completed, vacuum degassing is carried out at 30-35℃, and the pH of the system is adjusted to 5.5-6.5. The CXCR4 antagonist described in this invention, through its specific molecular structure, can act on the CXCR4 receptor on the host cell membrane. The nitrogen-containing heterocyclic domains, such as piperidine, in the antagonist molecule can mimic part of the spatial conformation of the natural ligand. After binding to the receptor, it can interfere with the viral pathway using this receptor as a co-receptor for cell penetration, thereby providing an intervention mechanism for inhibiting HPV infection. The combination of Achyranthes bidentata polysaccharide and ergothioneine is the core of this invention. Achyranthes bidentata polysaccharide, an acidic polysaccharide, can stretch its molecular chains in aqueous solution to form a dynamic three-dimensional network structure. This network not only serves as a functional matrix, but its bioactivity also helps create a microenvironment unfavorable to viral survival. More importantly, ergothioneine molecules, leveraging their amphoteric properties, can bind to the network structure of Achyranthes bidentata polysaccharide through hydrogen bonds and ionic interactions. This binding helps stabilize the chemical state of ergothioneine and may affect its bioavailability. During the preparation process, this intermolecular synergistic effect is further enhanced. A polyol premix loaded with the CXCR4 antagonist is introduced into an aqueous matrix composed of Achyranthes bidentata polysaccharide and ergothioneine using a specific high-speed shearing process. Under these conditions, the long-chain molecules of Achyranthes bidentata polysaccharide can entangle and coat the hydrophobic CXCR4 antagonist, promoting supramolecular self-assembly within the polysaccharide network to form a micro / nanoscale dispersion system. This process helps improve the dispersion stability of the CXCR4 antagonist in aqueous solution, while the presence of ergothioneine, together with Achyranthes bidentata polysaccharide, maintains the integrity of the assembled structure. In summary, this composition provides targeted intervention through the CXCR4 antagonist, constructs a delivery network with Achyranthes bidentata polysaccharide and exerts synergistic biological effects, and enhances the stability of the system and contributes its own biological activity. Together, the three constitute a multi-target, synergistic complex system, providing a possible technical path for inhibiting HPV. Compared with the prior art, the beneficial effects of the present invention are: 1. A more synergistic intervention mechanism against HPV infection: In the composition of this invention, the CXCR4 antagonist with a specific structure can act on the co-receptor pathway for viral entry into cells, while Achyranthes bidentata polysaccharide and ergothioneine form a functional matrix through intermolecular interactions and contribute to its biological activity. The three work synergistically to intervene in multiple stages of viral infection, unlike the problem of relatively single targets in existing technologies. 2. Improved dispersion and stability of hydrophobic active ingredients in hydrophilic matrices: A molecular network was constructed using Achyranthes bidentata polysaccharide in an aqueous phase, and a specific preparation process was used to induce supramolecular self-assembly of the CXCR4 antagonist within this network, forming a micro-nano-scale dispersion system. This approach helps overcome the problems of hydrophobic component separation, aggregation, and poor physical stability caused by simple physical mixing in existing technologies. 3. Enhanced synergistic effect among active components: The preparation method adopted in this invention, especially the step of adding the premixed active ingredient solution to the aqueous matrix under high-speed shear, can not only achieve effective integration of each component, but also promote the coating of the Achyranthes bidentata polysaccharide network on the CXCR4 antagonist and the stabilizing effect of ergothioneine, so that each component can produce synergy at the molecular level, thereby improving the overall bioavailability and inhibitory efficiency of the composition. Attached Figure Description

[0004] Figure 1 The synthetic route for the CXCR4 antagonist of this invention is shown below. Detailed Implementation

[0005] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Preparation Example 1 Preparation of CXCR4 antagonist 1: ; In the first step, under a nitrogen atmosphere, 27.35 g of diisopropylethylamine and 20.00 g of raw material 1 were added to 180 mL of 1,4-dioxane. 18.22 g of raw material 2 was dissolved in 50 mL of 1,4-dioxane. The solution was slowly added dropwise to the above solution while maintaining the temperature below -10℃. After the addition was complete, the mixture was stirred at 40℃ for 12 h. After the reaction was complete, the reaction solution was slowly poured into 2000 mL of water at 4℃ and stirred until no more bubbles were generated. The pH of the system was adjusted to neutral with 0.1 mol / L hydrochloric acid. The mixture was separated, the organic phase was retained, concentrated, and purified by silica gel column chromatography using a mixed solvent of n-heptane and ethyl acetate as the eluent. The solution was evaporated to dryness to obtain 23.37 g of intermediate 1. In the second step, under a nitrogen atmosphere, 23.37 g of intermediate 1, 11.74 g of starting material 3, 0.6 g of triphenylphosphine, 0.2 g of palladium on carbon, 14.56 g of triethylamine and 240 ml of toluene were added to the reaction system, and the mixture was heated to 120 °C and refluxed for 12 hours. After the reaction was completed, the solvent was evaporated, and silica gel column chromatography was performed using a mixture of petroleum ether and ethyl acetate as eluent. The solvent was evaporated again to obtain 22.74 g of CXCR4 antagonist 1. Product structure identification: MS[MS+H] of intermediate 1 + 325; MS[MS+H] of CXCR4 antagonists + :452. Synthesis Example 2-Synthesis Example 4 In Synthesis Examples 2-4, CXCR4 antagonist 2-CXCR4 antagonist 4 were synthesized sequentially, following the same synthesis method as in Synthesis Example 1, except that raw material 2 was replaced, and the other conditions remained the same as in Synthesis Example 1. Specific structures of raw material 1, CXCR4 antagonist 2-CXCR4 antagonist 4, and MS [MS+H] + The data is shown in Table 1. Table 1. Structures of raw material 1, CXCR4 antagonist 2-CXCR4 antagonist 4, and MS [MS+H] involved in Synthetic Examples 2-4 + data. Example 1 Preparation of a composition of ergothioneine and Achyranthes bidentata polysaccharide for inhibiting HPV: 1. Raw material proportions by weight: 3 parts ergothioneine; 7 parts of Achyranthes bidentata polysaccharide; 0.8 parts of CXCR4 antagonist; Sodium hyaluronate 1.2 parts; 15 parts propylene glycol; 73 portions of deionized water. 2. Preparation method: S1. Add 7 parts of Achyranthes bidentata polysaccharide and 1.2 parts of sodium hyaluronate to 73 parts of deionized water, stir and dissolve at 45 degrees Celsius to form an aqueous matrix. S2. Add 0.8 parts of CXCR4 antagonist to 15 parts of propylene glycol and sonicate at 60 degrees Celsius with an ultrasonic power of 300W for 20 minutes until completely dissolved to obtain an active premix solution. S3. Add 3 parts of ergothioneine to the aqueous matrix obtained in step S1 and stir in the dark until completely dissolved. S4. Under high-speed shearing conditions, with the shearing rate controlled at 6500 rpm, the active premixed solution obtained in step S2 is slowly added dropwise to the aqueous matrix obtained in step S3. After the addition is complete, shearing treatment is continued for 10 minutes to allow the CXCR4 antagonist to undergo supramolecular self-assembly within the Achyranthes bidentata polysaccharide molecular network, resulting in a micro-nano-scale dispersion system. S5. The dispersion system obtained in step S4 is subjected to vacuum degassing at a temperature of 33 degrees Celsius, and the pH value of the system is adjusted to 6.0 to obtain the composition. Examples 2-4 The preparation of an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition was carried out by referring to the preparation method of Example 1, except that the CXCR4 antagonist was replaced sequentially with the CXCR4 antagonist synthesized in Synthesis Examples 2 to 4, and the remaining raw materials, proportions and preparation steps were kept the same as in Example 1. Comparative Example 1 The preparation of an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition was carried out according to the preparation method of Example 1, without the addition of the CXCR4 antagonist, and the remaining raw material ratios and preparation steps were kept the same as in Example 1. Comparative Example 2 The preparation of an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition was carried out according to the preparation method of Example 1, except that the CXCR4 antagonist was replaced with an equal mass of AMD3100, and the remaining raw material ratios and preparation steps were the same as in Example 1. Comparative Example 3 The preparation of an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition was carried out according to the preparation method of Example 1, except that Achyranthes bidentata polysaccharide was not added, and the proportions of other raw materials and preparation steps were kept the same as in Example 1. Comparative Example 4 The preparation of an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition was carried out according to the preparation method of Example 1, except that ergothioneine was not added, and the remaining raw material ratios and preparation steps were the same as in Example 1. Comparative Example 5 The preparation of an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition was carried out according to the preparation method of Example 1, except that the Achyranthes bidentata polysaccharide was replaced with an equal mass of Achyranthes bidentata polysaccharide with an average molecular weight of 100,000 Da, and the remaining raw material ratios and preparation steps were kept the same as in Example 1. Performance testing: 1. HPV virus suppression ability test: The composition was evaluated to inhibit viral entry and early infection using a human cervical epithelial cell (HEC-1-A) model infected with HPV18 pseudovirus. A1. Sample processing solution preparation: Take 1.00 g of each of the composition samples obtained in Examples 1-4 and Comparative Examples 1-5, add 10 mL of sterile PBS buffer (pH 7.2), vortex mix evenly, filter through a 0.22 μm filter membrane for sterilization, and obtain the sample processing solution for cell experiments. A2. Cell Infection Model Construction and Treatment: HEC-1-A cells were seeded in 24-well plates (5 × 10^4 cells / well) and cultured in MEM medium containing 10% fetal bovine serum until 70% confluence. The medium was discarded, and the cells were washed with PBS. Experimental group: Cells were pretreated for 1 hour with a treatment solution containing 200 μmol / L of the sample in each well. Control group: Cells were pretreated with an equal volume of serum-free medium without the sample treatment solution. After pretreatment, HPV18 pseudovirus particles (MOI=10) were added to each group, and the cells were incubated at 37°C for 4 hours. The virus solution was removed, and the cells were replaced with fresh medium containing 2% fetal bovine serum and cultured for another 48 hours. A3. Detection of intracellular viral load: Cells cultured for 48 hours were collected, washed twice with PBS, and lysed on ice for 30 minutes with cell lysis buffer. The cells were centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected. Quantitative PCR was used to detect the copy number of HPV18 pseudovirus genome E6 / E7 region DNA in the cell lysate. The GAPDH gene was used as an internal control for standardization, and the relative viral DNA load was calculated. A4. Cell viability assay: Cell viability was assessed using the CCK-8 assay 48 hours after viral infection. CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell viability relative to the uninfected, untreated control group was calculated. Table 2 Group Relative viral DNA load Cell viability (%) Example 1 0.35±0.05 85.2±3.1 Example 2 0.38±0.06 83.7±2.8 Example 3 0.34±0.04 86.1±3.3 Example 4 0.37±0.05 84.5±2.9 Comparative Example 1 0.82±0.08 65.3±4.2 Comparative Example 2 0.43±0.07 79.4±3.8 Comparative Example 3 0.75±0.09 68.9±4.0 Comparative Example 4 0.70±0.08 70.5±3.7 Comparative Example 5 0.68±0.07 71.8±3.5 Virus model comparison 1.00±0.10 58.6±5.1 Examples 1-4 showed the lowest relative viral DNA load and the highest cell viability. Comparative Example 1 (without the CXCR4 antagonist) showed a significantly increased viral load and decreased cell viability. Comparative Example 2 (using the alternative CXCR4 antagonist AMD3100) was less effective than the example groups. Comparative Examples 3 (without Achyranthes bidentata polysaccharide) and 4 (without ergothioneine) were both weaker than the example groups in terms of viral inhibition and cell protection, indicating that the combination of Achyranthes bidentata polysaccharide and ergothioneine has a crucial impact on the system's efficacy. Comparative Example 5 (using high molecular weight Achyranthes bidentata polysaccharide) was also less effective than the example groups, illustrating the importance of Achyranthes bidentata polysaccharide within a specific molecular weight range for forming an effective delivery network. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A composition of ergothioneine and Achyranthes bidentata polysaccharide for inhibiting HPV, characterized in that, The composition contains a CXCR4 antagonist represented by Formula 1; The structure of Equation 1 is as follows: Formula 1; R1 in Formula 1 is a substituent; R1 is selected from any one of methyl, ethyl, propyl, methoxy, ethoxy, benzyloxy, and piperidinyl.

2. The HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to claim 1, characterized in that, R1 is selected from any one of methyl, methoxy, benzyloxy, and piperidinyl.

3. The HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to claim 1, characterized in that, The CXCR4 antagonist is one of the compounds shown in the following structure: ； 。 4. The HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to claim 1, characterized in that, The composition comprises the following components in parts by weight: 1-5 parts ergothioneine, 3-10 parts Achyranthes bidentata polysaccharide, 0.2-1.2 parts CXCR4 antagonist, 0.5-2 parts sodium hyaluronate, 10-20 parts polyol cosolvent, and 60-80 parts deionized water.

5. The HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to claim 4, characterized in that, The polyol co-solvent is selected from one or more of propylene glycol, glycerol, and 1,3-butanediol.

6. The HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to claim 4, characterized in that, The average molecular weight of the Achyranthes bidentata polysaccharide is 1000-5000 Da.

7. The HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to claim 4, characterized in that, The purity of the ergothioneine is greater than or equal to 99%.

8. A method for preparing an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to any one of claims 4-7, characterized in that, Includes the following steps: a. Add the Achyranthes bidentata polysaccharide and sodium hyaluronate to deionized water and stir to dissolve at 40-50°C to form an aqueous matrix; b. Add the CXCR4 antagonist to a polyol co-solvent and sonicate at 55-65°C until completely dissolved to obtain an active premix solution; c. Add the ergothioneine to the aqueous matrix and stir in the dark until completely dissolved; d. Under high-speed shearing conditions, the premixed active ingredient solution is slowly added dropwise to an aqueous matrix containing ergothionein, and the shearing rate is controlled at 5000-8000 rpm, so that the CXCR4 antagonist undergoes supramolecular self-assembly within the molecular network formed by Achyranthes bidentata polysaccharide to obtain a micro-nano-scale dispersion system.

9. The method for preparing an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to claim 8, characterized in that, In step b, the ultrasonic treatment power is 200-400W, and the treatment time is 15-30min.

10. The method for preparing an HPV-inhibiting ergothioneine and Achyranthes bidentata polysaccharide composition according to claim 8, characterized in that, After the addition in step d is completed, vacuum degassing is carried out at 30-35℃, and the pH of the system is adjusted to 5.5-6.5.

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