Application of total glucosides of paeony in the preparation of drugs for the treatment of head and neck squamous cell carcinoma

Abstract

This invention discloses the application of total paeoniflorin in the preparation of drugs for the treatment of head and neck squamous cell carcinoma, belonging to the field of biomedical technology. Addressing the bottlenecks in current treatments for head and neck squamous cell carcinoma, such as high toxicity of radiotherapy and chemotherapy, low response rates to immunotherapy, and easy resistance to targeted drugs, this invention provides a method for preparing total paeoniflorin and a drug-loaded preparation technology based on hydrogels obtained from modified chitosan. Total paeoniflorin can dose-dependently inhibit the proliferation rate, colony formation ability, and in vitro migration ability of SCC7, FADU, and CAL27 head and neck squamous cell carcinoma cell lines, and significantly induce apoptosis. This invention utilizes the multi-target effects of total paeoniflorin to exert anti-head and neck squamous cell carcinoma effects through modulation of the tumor microenvironment and enhancement of immune surveillance. Furthermore, the loaded drug exhibits good sustained-release properties and biosafety, providing a new, highly effective, and low-toxicity strategy for the treatment of head and neck squamous cell carcinoma, and has significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of total glucosides of paeony in the preparation of drugs for the treatment of head and neck squamous cell carcinoma. Background Technology

[0002] Squamous cell carcinoma of the head and neck is the sixth most common malignant tumor worldwide, primarily occurring in the mucosal epithelial tissues of the head and neck, including the oral cavity, pharynx, and larynx. The incidence of head and neck squamous cell carcinoma varies significantly across different regions and populations, with smoking, alcohol consumption, and human papillomavirus (HPV) infection being major risk factors. Although treatment strategies have evolved from the traditional combination of surgery, radiotherapy, and chemotherapy to a new comprehensive paradigm incorporating targeted therapy, immune checkpoint inhibitors, and cell therapy, the overall survival rate remains unsatisfactory. The five-year survival rate for patients with locally advanced head and neck squamous cell carcinoma hovers around 50%, with more than half experiencing local recurrence or distant metastasis after radical radiotherapy and chemotherapy, posing a significant challenge to clinical treatment.

[0003] Current treatment strategies for head and neck squamous cell carcinoma face multiple bottlenecks: First, the damage to normal tissues caused by traditional radiotherapy and chemotherapy limits the dosage intensity, leading to poor treatment outcomes; second, although immune checkpoint blockade therapies, represented by PD-1 inhibitors, offer hope to some patients, their response rate is very low in platinum-resistant recurrent / metastatic head and neck squamous cell carcinoma; third, while targeted therapies such as anti-EGFR monoclonal antibodies have been incorporated into the standard treatment system, patient survival benefits are limited and drug resistance is inevitable. Based on these challenges, exploring new, highly effective, and low-toxicity treatment strategies has become an urgent need in head and neck squamous cell carcinoma research, and natural plant active ingredients are increasingly attracting attention due to their multi-target effects and good safety profile. Summary of the Invention

[0004] The purpose of this invention is to provide an application of total paeoniflorin in the preparation of a therapeutic drug for head and neck squamous cell carcinoma, aiming to solve the bottlenecks in the existing treatment of head and neck squamous cell carcinoma, such as high toxicity of radiotherapy and chemotherapy, low response rate of immune checkpoint inhibitors, and easy drug resistance to targeted drugs. By utilizing the multi-target action characteristics of total paeoniflorin, in vitro experiments have confirmed that it can inhibit the proliferation of head and neck squamous cell carcinoma cells, reduce the ability of colony formation, block in vitro migration, and induce apoptosis. With its high efficiency and low toxicity, it provides a new strategy for the treatment of head and neck squamous cell carcinoma, improving the therapeutic effect and safety.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0006] A drug for treating head and neck squamous cell carcinoma, comprising total paeoniflorin and hydrogel, wherein the mass ratio of total paeoniflorin to hydrogel is 80-100 mg: 10 g.

[0007] Preferably, the hydrogel comprises modified chitosan and cellulose.

[0008] Preferably, the mass ratio of modified chitosan to cellulose is 3-7:5.

[0009] Preferably, the modifiers in the modified chitosan include epoxy acrolein diethanol and epoxy ethyl methyl hexanoate.

[0010] Preferably, the mass ratio of epoxy acrolein diethanolate to chitosan is 1-4:2.

[0011] Preferably, the mass ratio of epoxy ethyl methyl hexanoate to chitosan is 1-4:2.

[0012] Epoxy acrolein diacetate and epoxy ethyl methyl hexanoate are used as modifiers to introduce aldehyde and ester groups. The two work together on the chitosan molecular chain, and through the reaction with the active groups of chitosan, a functional group structure with both aldehyde and ester groups is constructed in the molecule. Moderate cross-linking is formed with the aldehyde groups in aldehyde cellulose, which enhances the hydrophilicity of the hydrogel through polar groups and maintains the stability of the three-dimensional network structure through cross-linking. This balances the swelling performance of the hydrogel and the drug release rate, providing a structural basis for the efficient loading and controlled release of total glucosides of paeony.

[0013] Preferably, the total glycosides of paeony are extracted by heating and refluxing paeony in an extract solution.

[0014] Preferably, the extract is a 65-75 v / v% aqueous ethanol solution.

[0015] Preferably, the mass-to-volume ratio of white peony root to extract is 300-800g:5L.

[0016] Preferably, the total glycosides of paeony are purified by elution using a macroporous adsorption resin column.

[0017] Preferably, the elution solvent comprises deionized water and a 45-55 v / v% aqueous solution of ethanol.

[0018] Preferably, the amount of deionized water used is 1-4 BV.

[0019] Preferably, the amount of 45-55 v / v% ethanol aqueous solution used is 6-8 BV.

[0020] Preferably, the total glucosides of paeony are purified by precipitation with ethanol in a 2.5-3.5 wt% aqueous solution of gelatin.

[0021] Preferably, the volume ratio of ethanol to 2.5-3.5 wt% gelatin aqueous solution is 40-50:1.

[0022] Preferably, the drug is used to inhibit the proliferation of squamous cell carcinoma cells in the head and neck.

[0023] Preferably, the head and neck squamous cell carcinoma cells include at least one of SCC7 cells, FADU cells, and CAL27 cells.

[0024] Preferably, the drug is used to reduce the clonogenic ability of head and neck squamous cell carcinoma cells.

[0025] Preferably, the head and neck squamous cell carcinoma cells include at least one of SCC7 cells, FADU cells, and CAL27 cells.

[0026] Preferably, the drug is used to inhibit the in vitro migration ability of head and neck squamous cell carcinoma cells.

[0027] Preferably, the head and neck squamous cell carcinoma cells include at least one of SCC7 cells, FADU cells, and CAL27 cells.

[0028] Preferably, the drug is used to induce apoptosis in head and neck squamous cell carcinoma cells.

[0029] Preferably, the head and neck squamous cell carcinoma cells include at least one of SCC7 cells, FADU cells, and CAL27 cells.

[0030] More preferably, the hydrogel comprises modified chitosan and modified cellulose, wherein the modifier of the modified cellulose includes 3-trimethoxysilane propylene acrylate, and the mass ratio of 3-trimethoxysilane propylene acrylate to cellulose is 1-4:5. As a cellulose modifier, 3-trimethoxysilane propylene acrylate reacts with the hydroxyl groups in the cellulose molecular chain to introduce silanol groups and acrylate groups, significantly enhancing the water molecule binding capacity of cellulose. This allows the modified cellulose and modified chitosan to form a looser three-dimensional network structure, reducing resistance to drug diffusion, promoting hydrogel swelling, and thus synergistically improving the release rate and release percentage of total paeoniflorin, providing structural support for the effective delivery of drugs in the treatment of head and neck squamous cell carcinoma.

[0031] This invention also provides a method for preparing total paeoniflorin, comprising:

[0032] After pulverizing the white peony root, disperse it in the extract and reflux extract 1-4 times at 75-85℃, 1-4 hours each time. Combine the extracts, filter the residue with filter paper, and concentrate under reduced pressure to obtain a concentrated solution. Add deionized water to the concentrated solution to obtain a diluted solution. Pass the diluted solution through a macroporous adsorption resin column at a flow rate of 3-5 BV / h, elute with the aqueous phase solvent at a flow rate of [missing value] until the eluent is colorless, elute with the organic phase solvent at a flow rate of [missing value], collect the eluent, concentrate under reduced pressure, filter, add 2.5-3.5 wt% gelatin aqueous solution to the filtrate, add ethanol, let stand for 20-25 hours, filter, recover the ethanol under reduced pressure, and vacuum dry at 55-65℃ for 10-15 hours to obtain total peony glycosides.

[0033] Preferably, the extract is a 65-75% v / v ethanol aqueous solution, and the mass-volume ratio of white peony root to extract is 300-800 g: 5 L.

[0034] Preferably, the mass-to-volume ratio of white peony root to concentrated liquid is 300-800g:500mL.

[0035] Preferably, the volume ratio of deionized water to concentrate is 4-6L:500mL.

[0036] Preferably, the macroporous adsorption resin is HPD300.

[0037] Preferably, the aqueous elution solvent is deionized water, the elution flow rate of the aqueous elution solvent is 3-5 BV / h, and the amount of aqueous elution solvent used is 1-4 BV.

[0038] Preferably, the organic phase elution solvent is a 45-55 v / v% aqueous ethanol solution, the elution flow rate of the organic phase elution solvent is 3-5 BV / h, and the amount of organic phase elution solvent used is 6-8 BV.

[0039] Preferably, the volume ratio of the filtrate to the 2.5-3.5 wt% gelatin aqueous solution is 5-15:1.

[0040] Preferably, the volume ratio of ethanol to 2.5-3.5 wt% gelatin aqueous solution is 40-50:1.

[0041] This invention also provides a method for preparing modified chitosan, comprising:

[0042] Epoxy acrolein diethanolate and epoxy ethyl methyl hexanoate were dispersed in deionized water to obtain a modifier solution. Chitosan was dispersed in isopropanol at 55-65℃ and stirred for 1-4 hours. The modifier solution was added and the mixture was stirred at 75-85℃ for 6-10 hours. The reaction solution was added to acetone, the precipitate was collected by filtration, washed 2-4 times with 80 v / v% isopropanol aqueous solution, and vacuum dried at 45-55℃ for 10-15 hours to obtain modified chitosan.

[0043] Preferably, the mass-to-volume ratio of epoxy acrolein diethanol condensate to deionized water is 1-4 mg:10 mL.

[0044] Preferably, the mass-to-volume ratio of epoxy ethyl methyl hexanoate to deionized water is 1-4 mg: 10 mL.

[0045] Preferably, the mass-to-volume ratio of chitosan to isopropanol is 1-4 mg: 20 mL.

[0046] Preferably, the mass of the modifier solution is measured by the mass of hexanoic acid epoxy ethyl methyl ester, and the mass ratio of hexanoic acid epoxy ethyl methyl ester to chitosan is 1-4:2.

[0047] Preferably, the volume ratio of the reaction solution to acetone is 5-15:50.

[0048] This invention also provides a method for preparing a hydrogel, comprising:

[0049] Aldehyde cellulose was dispersed in a 0.3-0.5 wt% sodium hydroxide aqueous solution, modified chitosan was added, and a 0.6-0.8 mol / L glacial acetic acid solution was added. After stirring for 5-15 min, the mixture was sealed and allowed to stand for 10-15 h. After dialyzing in deionized water for 5-7 h, the mixture was lyophilized to obtain a hydrogel.

[0050] Preferably, the volume-to-mass ratio of 0.3-0.5 wt% sodium hydroxide aqueous solution to aldehyde cellulose is 300-500 mL: 5 g.

[0051] Preferably, the mass ratio of modified chitosan to aldehyde cellulose is 3-7:5.

[0052] Preferably, the volume-to-mass ratio of 0.6-0.8 mol / L glacial acetic acid solution to aldehyde cellulose is 100-200 mL: 5 g.

[0053] The present invention also provides a method for preparing a drug-loaded substance, comprising:

[0054] Total glucosides of paeony were dispersed in deionized water, and hydrogel was added. The mixture was soaked at room temperature for 10-15 hours. The hydrogel was then removed and rinsed with deionized water to obtain the drug-loaded product.

[0055] Preferably, the mass-to-volume ratio of total paeoniflorin to deionized water is 50-150 mg: 10 mL.

[0056] Preferably, the mass ratio of total paeoniflorin to hydrogel is 80-100 mg: 10 g.

[0057] This invention utilizes total paeoniflorin extracted and purified from Paeonia lactiflora, and modifies chitosan with acrolein diethanoloxide and hexanoic acid ethyl methyl ester to construct a hydrogel loading system with cellulose, achieving highly efficient extraction and release of total paeoniflorin. Therefore, it has the following beneficial effects: the prepared head and neck squamous cell carcinoma treatment drug can dose-dependently inhibit the proliferation, colony formation, and migration of head and neck squamous cell carcinoma cells, significantly induce apoptosis, and exhibits excellent biosafety, effectively overcoming the bottlenecks of high toxicity of radiotherapy and chemotherapy and low response rates of immunotherapy in existing treatments. Therefore, this invention is a highly effective, low-toxicity, and controllable delivery head and neck squamous cell carcinoma treatment drug suitable for the treatment of head and neck squamous cell carcinoma. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of a scanning electron microscope image of a hydrogel.

[0059] Figure 2 This is a schematic diagram showing the optimal concentration of total paeoniflorin for SCC7 cells.

[0060] Figure 3 This is a schematic diagram showing the optimal concentration of total paeoniflorin for FADU cells.

[0061] Figure 4 This is a schematic diagram showing the optimal concentration of total paeoniflorin in CAL27 cells.

[0062] Figure 5 This is a schematic diagram showing the results of the test on the proliferation ability of SCC7 cells by total glucosides of paeony.

[0063] Figure 6 This is a schematic diagram showing the results of the test on the proliferation ability of FADU cells by total glucosides of paeony.

[0064] Figure 7 This is a schematic diagram showing the results of the test on the proliferation ability of CAL27 cells by total glucosides of paeony.

[0065] Figure 8 This is a schematic diagram showing the results of Hoechst and EdU-488 dual fluorescence staining of CAL27 cells with total paeoniflorin.

[0066] Figure 9 This is a schematic diagram showing the cell proliferation rate test results of total paeoniflorin on CAL27 cells.

[0067] Figure 10 This is a schematic diagram showing the results of Hoechst and EdU-488 dual fluorescence staining of FADU cells with total paeoniflorin.

[0068] Figure 11 This is a schematic diagram showing the results of the test on the cell proliferation rate of FADU cells by total glucosides of paeony.

[0069] Figure 12 This is a schematic diagram showing the results of Hoechst and EdU-488 dual fluorescence staining of SCC7 cells with total paeoniflorin.

[0070] Figure 13 This is a schematic diagram showing the cell proliferation rate test results of total paeoniflorin in SCC7 cells.

[0071] Figure 14 This is a schematic diagram showing the effect of total paeoniflorin on the clonogenic ability of CAL27 cells.

[0072] Figure 15 This is a schematic diagram showing the results of the clonogenicity test of total paeoniflorin in CAL27 cells.

[0073] Figure 16 This is a schematic diagram showing the effect of total paeoniflorin on the colony-forming ability of SCC7 cells.

[0074] Figure 17 This is a schematic diagram showing the results of the colony formation rate test of total paeoniflorin in SCC7 cells.

[0075] Figure 18 This is a schematic diagram showing the effect of total glucosides of paeony on the clonogenic ability of FADU cells.

[0076] Figure 19 This is a schematic diagram showing the results of the clonogenicity test of total paeoniflorin in FADU cells.

[0077] Figure 20 This is a schematic diagram showing the results of the in vitro migration ability test of total paeoniflorin on head and neck squamous cell carcinoma cells.

[0078] Figure 21 This is a schematic diagram showing the effect of total glucosides of paeony on the in vitro migration ability of SCC7 cells.

[0079] Figure 22 This is a schematic diagram showing the effect of total paeoniflorin on the in vitro migration ability of CAL27 cells.

[0080] Figure 23 This is a schematic diagram showing the effect of total glucosides of paeony on the in vitro migration ability of FADU cells.

[0081] Figure 24 This is a schematic diagram showing the effect of total glucosides of paeony on the in vitro migration ability of SCC7 cells.

[0082] Figure 25 This is a schematic diagram showing the results of the in vitro migration ability test of total paeoniflorin on SCC7 cells.

[0083] Figure 26 This is a schematic diagram showing the effect of total paeoniflorin on the in vitro migration ability of CAL27.

[0084] Figure 27 This is a schematic diagram showing the results of the in vitro migration ability test of total paeoniflorin on CAL27 cells.

[0085] Figure 28 This is a schematic diagram showing the effect of total glucosides of paeony on the in vitro migration ability of FADU cells.

[0086] Figure 29 This is a schematic diagram showing the results of the in vitro migration ability test of total paeony glycosides on FADU cells.

[0087] Figure 30 This is a schematic diagram showing the effect of total paeoniflorin control group on the apoptosis ability of SCC7 cells.

[0088] Figure 31 This is a schematic diagram showing the effect of low concentrations of total paeoniflorin on the apoptosis capacity of SCC7 cells.

[0089] Figure 32 This is a schematic diagram showing the effect of high concentrations of total paeoniflorin on the apoptosis capacity of SCC7 cells.

[0090] Figure 33 This is a schematic diagram showing the results of the test on the apoptosis ability of total paeony glycosides in SCC7 cells.

[0091] Figure 34 This is a schematic diagram showing the effect of total paeoniflorin control group on the apoptosis ability of CAL27 cells.

[0092] Figure 35 This is a schematic diagram showing the effect of low concentrations of total paeoniflorin on the apoptosis ability of CAL27 cells.

[0093] Figure 36 This is a schematic diagram showing the effect of high concentrations of total paeoniflorin on the apoptosis ability of CAL27 cells.

[0094] Figure 37 This is a schematic diagram showing the results of the test on the apoptosis ability of total paeony glycosides in CAL27 cells.

[0095] Figure 38 This is a schematic diagram showing the effect of total paeoniflorin control group on the apoptosis ability of FADU cells.

[0096] Figure 39 This is a schematic diagram showing the effect of low concentrations of total paeoniflorin on the apoptosis capacity of FADU cells.

[0097] Figure 40 This is a schematic diagram showing the effect of high concentrations of total paeoniflorin on the apoptosis capacity of FADU cells.

[0098] Figure 41 This is a schematic diagram showing the results of the test on the apoptosis ability of total paeony glycosides in FADU cells. Detailed Implementation

[0099] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0100] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0101] Example 1:

[0102] Preparation of total glycosides of paeony: Pulverized paeony was dispersed in the extract and refluxed twice at 80℃ for 2 hours each time. The two extracts were combined, the residue was filtered through filter paper, and the extract was concentrated under reduced pressure to obtain a concentrated solution. Deionized water was added to the concentrated solution to obtain a diluted solution. The diluted solution was passed through an HPD300 macroporous adsorption resin column at a flow rate of 4 BV / h. The aqueous phase elution solvent was used to elute at a flow rate of until the eluent was colorless. The organic phase elution solvent was used to elute at a flow rate of . The eluent was collected, concentrated under reduced pressure, filtered, and 3 wt% gelatin aqueous solution was added to the filtrate. Ethanol was added, and the solution was allowed to stand for 24 hours before filtration. The ethanol was recovered under reduced pressure, and the solution was vacuum dried at 60℃ for 12 hours to obtain total glycosides of paeony. The extract was a 70 v / v% ethanol aqueous solution. The mass-to-volume ratio of white peony root to extract was 500 g: 5 L, the mass-to-volume ratio of white peony root to concentrate was 500 g: 500 mL, and the volume ratio of deionized water to concentrate was 5 L: 500 mL. The aqueous phase elution solvent was deionized water, the elution flow rate was 4 BV / h, and the volume of aqueous phase elution solvent used was 2 BV. The organic phase elution solvent was a 50 v / v% ethanol aqueous solution, the elution flow rate was 4 BV / h, and the volume of organic phase elution solvent used was 7 BV. The volume ratio of filtrate to 3 wt% gelatin aqueous solution was 10:1, and the volume ratio of ethanol to 3 wt% gelatin aqueous solution was 44:1.

[0103] Preparation of modified chitosan: Epoxy acrolein diacetate and epoxy ethyl methyl hexanoate were dispersed in deionized water to obtain a modifier solution. Chitosan was dispersed in isopropanol at 60°C and stirred for 2 hours. The modifier solution was then added, and the mixture was stirred at 80°C for 8 hours. The reaction solution was added to acetone, the precipitate was collected by filtration, washed three times with 80 v / v% isopropanol aqueous solution, and vacuum dried at 50°C for 12 hours to obtain modified chitosan. The mass-to-volume ratio of epoxy acrolein diacetate to deionized water was 2 mg:10 mL, the mass-to-volume ratio of epoxy ethyl methyl hexanoate to deionized water was 2 mg:10 mL, and the mass-to-volume ratio of chitosan to isopropanol was 2 mg:20 mL. The mass of the modifier solution was measured by the mass of epoxy ethyl methyl hexanoate, and the mass ratio of epoxy ethyl methyl hexanoate to chitosan was 2:2. The volume ratio of the reaction solution to acetone was 10:50.

[0104] Preparation of hydrogel: Aldehyde cellulose was dispersed in a 0.4 wt% sodium hydroxide aqueous solution, modified chitosan was added, and a 0.7 mol / L glacial acetic acid solution was added. After stirring for 10 min, the mixture was sealed and allowed to stand for 12 h. Dialysis was performed in deionized water for 6 h, and the hydrogel was obtained by lyophilization. The volume-to-mass ratio of 0.4 wt% sodium hydroxide aqueous solution to aldehyde cellulose was 400 mL:5 g, the mass ratio of modified chitosan to aldehyde cellulose was 5:5, and the volume-to-mass ratio of 0.7 mol / L glacial acetic acid solution to aldehyde cellulose was 150 mL:5 g.

[0105] Preparation of the loaded drug: Total paeoniflorin was dispersed in deionized water, hydrogel was added, and the mixture was soaked at room temperature for 12 hours. The hydrogel was then removed and rinsed with deionized water to obtain the loaded drug. The mass-to-volume ratio of total paeoniflorin to deionized water was 100 mg: 10 mL, and the mass ratio of total paeoniflorin to hydrogel was 100 mg: 10 g.

[0106] Example 2: The only difference between this example and Example 1 is the preparation of the modified chitosan.

[0107] Preparation of modified chitosan: Epoxy acrolein diacetate and epoxy ethyl methyl hexanoate were dispersed in deionized water to obtain a modifier solution. Chitosan was dispersed in isopropanol at 60°C and stirred for 2 hours. The modifier solution was then added, and the mixture was stirred at 80°C for 8 hours. The reaction solution was added to acetone, the precipitate was collected by filtration, washed three times with 80 v / v% isopropanol aqueous solution, and vacuum dried at 50°C for 12 hours to obtain modified chitosan. The mass-to-volume ratio of epoxy acrolein diacetate to deionized water was 4 mg:10 mL, the mass-to-volume ratio of epoxy ethyl methyl hexanoate to deionized water was 2 mg:10 mL, and the mass-to-volume ratio of chitosan to isopropanol was 2 mg:20 mL. The mass of the modifier solution was measured by the mass of epoxy ethyl methyl hexanoate, and the mass ratio of epoxy ethyl methyl hexanoate to chitosan was 2:2. The volume ratio of the reaction solution to acetone was 10:50.

[0108] Example 3: The only difference between this example and Example 1 is the preparation of the hydrogel.

[0109] Preparation of hydrogel: Cellulose was dispersed in anhydrous ethanol and stirred until homogeneous to obtain a cellulose dispersion; 3-trimethoxysilane propylene acrylate was added to the cellulose dispersion and stirred until homogeneous; concentrated hydrochloric acid was added and the mixture was stirred at 60℃ for 6 h; the precipitate was collected after filtration, washed three times with anhydrous ethanol, and vacuum dried at 50℃ for 12 h to obtain modified cellulose; the modified cellulose was dispersed in a 0.4 wt% sodium hydroxide aqueous solution, modified chitosan was added, and 0.7 mol / L glacial acetic acid solution was added; the mixture was stirred for 10 min, sealed and allowed to stand for 12 h; dialyzed in deionized water for 6 h; and lyophilized to obtain a hydrogel. In the cellulose dispersion, the volume-to-mass ratio of anhydrous ethanol to cellulose was 100 mL: 5 g; the mass ratio of 3-trimethoxysilane propylene acrylate to cellulose was 1: 5; the volume-to-mass ratio of concentrated hydrochloric acid to cellulose was 0.5 mL: 5 g; the volume-to-mass ratio of 0.4 wt% sodium hydroxide aqueous solution to modified cellulose was 400 mL: 5 g; the mass ratio of modified chitosan to modified cellulose was 5: 5; and the volume-to-mass ratio of 0.7 mol / L glacial acetic acid solution to modified cellulose was 150 mL: 5 g.

[0110] Example 4: The only difference between this example and Example 1 is the preparation of the hydrogel.

[0111] Preparation of hydrogel: Cellulose was dispersed in anhydrous ethanol and stirred until homogeneous to obtain a cellulose dispersion; 3-trimethoxysilane propylene acrylate was added to the cellulose dispersion and stirred until homogeneous; concentrated hydrochloric acid was added and the mixture was stirred at 60℃ for 6 h; the precipitate was collected after filtration, washed three times with anhydrous ethanol, and vacuum dried at 50℃ for 12 h to obtain modified cellulose; the modified cellulose was dispersed in a 0.4 wt% sodium hydroxide aqueous solution, modified chitosan was added, and 0.7 mol / L glacial acetic acid solution was added; the mixture was stirred for 10 min, sealed and allowed to stand for 12 h; dialyzed in deionized water for 6 h; and lyophilized to obtain a hydrogel. In the cellulose dispersion, the volume-to-mass ratio of anhydrous ethanol to cellulose was 100 mL: 5 g; the mass ratio of 3-trimethoxysilane propylene acrylate to cellulose was 2: 5; the volume-to-mass ratio of concentrated hydrochloric acid to cellulose was 0.5 mL: 5 g; the volume-to-mass ratio of 0.4 wt% sodium hydroxide aqueous solution to modified cellulose was 400 mL: 5 g; the mass ratio of modified chitosan to modified cellulose was 5: 5; and the volume-to-mass ratio of 0.7 mol / L glacial acetic acid solution to modified cellulose was 150 mL: 5 g.

[0112] Example 5: The only difference between this example and Example 1 is the preparation of the loaded drug.

[0113] Preparation of the loaded drug: Total paeoniflorin was dispersed in deionized water, hydrogel was added, and the mixture was soaked at room temperature for 12 hours. The hydrogel was then removed and rinsed with deionized water to obtain the loaded drug. The mass-to-volume ratio of total paeoniflorin to deionized water was 80 mg:10 mL, and the mass ratio of total paeoniflorin to hydrogel was 80 mg:10 g.

[0114] Example 6: The only difference between this example and Example 1 is the preparation of the loaded drug.

[0115] Preparation of the loaded drug: Total paeoniflorin was dispersed in deionized water, hydrogel was added, and the mixture was soaked at room temperature for 12 hours. The hydrogel was then removed and rinsed with deionized water to obtain the loaded drug. The mass-to-volume ratio of total paeoniflorin to deionized water was 120 mg:10 mL, and the mass ratio of total paeoniflorin to hydrogel was 120 mg:10 g.

[0116] Example 7: The only difference between this example and Example 1 is the preparation of the loaded drug.

[0117] Preparation of the loaded drug: Total paeoniflorin was dispersed in deionized water, hydrogel was added, and the mixture was soaked at room temperature for 12 hours. The hydrogel was then removed and rinsed with deionized water to obtain the loaded drug. The mass-to-volume ratio of total paeoniflorin to deionized water was 150 mg:10 mL, and the mass ratio of total paeoniflorin to hydrogel was 150 mg:10 g.

[0118] Comparative Example 1: The only difference between this comparative example and Example 1 is that epoxy acrolein diethanol was not used in the preparation of the modified chitosan.

[0119] Comparative Example 2: The only difference between this comparative example and Example 1 is that epoxy ethyl hexanoate was not used in the preparation of the modified chitosan.

[0120] Comparative Example 3: The only difference between this comparative example and Example 1 is that epoxy acrolein diethanolate and epoxy ethyl hexanoate were not used in the preparation of the modified chitosan.

[0121] Comparative Example 4: The only difference between this comparative example and Example 3 is the preparation of the hydrogel.

[0122] Preparation of hydrogel: Cellulose was dispersed in anhydrous ethanol and stirred until homogeneous to obtain a cellulose dispersion; γ-methacryloxypropyltrimethoxysilane was added to the cellulose dispersion and stirred until homogeneous; concentrated hydrochloric acid was added and the mixture was stirred at 60℃ for 6 h; the precipitate was collected after filtration, washed three times with anhydrous ethanol, and vacuum dried at 50℃ for 12 h to obtain modified cellulose; the modified cellulose was dispersed in a 0.4 wt% sodium hydroxide aqueous solution, modified chitosan was added, and 0.7 mol / L glacial acetic acid solution was added; the mixture was stirred for 10 min, sealed and allowed to stand for 12 h; dialyzed in deionized water for 6 h; and lyophilized to obtain a hydrogel. In the cellulose dispersion, the volume-to-mass ratio of anhydrous ethanol to cellulose was 100 mL: 5 g; the mass ratio of γ-methacryloyloxypropyltrimethoxysilane to cellulose was 1: 5; the volume-to-mass ratio of concentrated hydrochloric acid to cellulose was 0.5 mL: 5 g; the volume-to-mass ratio of 0.4 wt% sodium hydroxide aqueous solution to modified cellulose was 400 mL: 5 g; the mass ratio of modified chitosan to modified cellulose was 5: 5; and the volume-to-mass ratio of 0.7 mol / L glacial acetic acid solution to modified cellulose was 150 mL: 5 g.

[0123] Experimental Example 1: Microstructure characterization of hydrogels.

[0124] Test sample: Hydrogel prepared in Example 1.

[0125] Test method: The surface of the hydrogel was sputtered with gold and observed under a scanning electron microscope at 25°C with an accelerating voltage of 15kV.

[0126] Scanning electron microscope image of the hydrogel prepared in this invention is shown below. Figure 1 As shown, a three-dimensional porous network structure can be observed under a scanning electron microscope. The pores are relatively uniformly distributed, the pore walls are relatively smooth, and the network structure is tightly connected and has good continuity, indicating that the hydrogel shown in the figure was successfully obtained.

[0127] Experimental Example 2: Swelling rate test of hydrogel.

[0128] Test samples: Hydrogels prepared in Examples 1-4 and Comparative Examples 1-4.

[0129] Test method: The hydrogel sample was vacuum dried to constant weight at 60℃. The initial dry mass m0 was accurately weighed. The dried hydrogel was added to 30mL of PBS buffer at pH 7.4 and allowed to stand at 37℃ for 2 hours. After that, the hydrogel was taken out and the excess liquid on the surface was gently blotted with filter paper. The mass was immediately and accurately weighed as m1. The swelling rate SR (%) was calculated according to the formula (m1-m0) / m0×100%, where m0 is the initial mass of the hydrogel and m1 is the mass of the hydrogel after swelling.

[0130] The swelling rate test results of the hydrogel prepared in this invention are shown in Table 1.

[0131] Table 1. Results of swelling ratio test of hydrogels

[0132]

[0133] In Example 1, the modified chitosan was co-modified with epoxy acrolein diacetate and epoxy ethyl methyl hexanoate. The two modifiers worked synergistically to introduce appropriate amounts of aldehyde and ester groups into the chitosan molecular chain, forming moderate crosslinks with aldehyde cellulose, achieving a balance between hydrophilicity and structural stability. In Example 2, the amount of epoxy acrolein diacetate in the modified chitosan was increased, enhancing the interaction with water molecules, resulting in a higher swelling rate than in Example 1. In Example 3, cellulose was modified with 3-trimethoxysilane propylene acrylate. The hydrophilicity of the silanol groups and acrylate groups was significantly stronger than that of the aldehyde groups, and the network bonding between the modified cellulose and modified chitosan was looser, further increasing the swelling rate. In Example 4, increasing the amount of 3-trimethoxysilane propylene acrylate significantly increased the density of hydrophilic groups, and the steric hindrance of the acrylate groups prevented excessive network density, resulting in the highest swelling rate. The modified chitosan in Comparative Example 1 did not use epoxy acrolein diacetate, and the modified chitosan in Comparative Example 2 did not use epoxy ethyl methyl hexanoate. Due to the insufficient aldehyde groups, the crosslinking with aldehyde cellulose was denser, and the swelling rate was significantly lower than that in Example 1. Comparative Example 3 did not use epoxy acrolein diacetate and epoxy ethyl methyl hexanoate. The unmodified chitosan had extremely low reactivity with aldehyde cellulose, and the network structure had the lowest swelling rate due to insufficient crosslinking. Comparative Example 4 improved the hydrophilicity of cellulose by reacting γ-methacryloxypropyltrimethoxysilane with the hydroxyl groups of cellulose, maintained a certain degree of network looseness, and improved the swelling performance of the hydrogel. Therefore, the swelling rate was significantly higher than that of Comparative Examples 1-3. However, compared with 3-trimethoxysilane propylene acrylate, γ-methacryloxypropyltrimethoxysilane had lower water molecule penetration and retention capacity than Examples 3-4.

[0134] Experimental Example 3: Drug Release Test.

[0135] Test samples: Drug-loaded samples prepared in each example and comparative example.

[0136] Test method: 10g of the drug-loaded compound was added to 30mL of pH 7.4 PBS buffer, sealed, and shaken at 37℃ and 100rpm. At time points of 1, 2, 4, 8, 12, 24, 48, and 72h, 5mL of the release solution was collected, and 5mL of fresh PBS buffer was added simultaneously. The concentration (C1) of total paeoniflorin in the release solution was determined by HPLC. The chromatographic column was a C18 column, 250mm × 4.6mm, 5μm. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, with a volume ratio of acetonitrile to 0.1% phosphoric acid aqueous solution of 15:85. The detection wavelength was 230nm, the flow rate was 1.0mL / min, the column temperature was 30℃, and the injection volume was 20μL. The release rate R(%) was calculated using the formula: R(%) = (C1 × V1) / V1. The release rate of the loaded drug is calculated by m0×100%, where C1 is the concentration of total paeoniflorin in the released solution, V1 is the volume of the released solution, and m0 is the mass of total paeoniflorin in the initial loaded drug.

[0137] The 24-hour release test results of the loaded drug prepared in this invention are shown in Table 2.

[0138] Table 2 Results of 24-hour drug release test

[0139]

[0140] Example 1: The hydrogel was prepared from aldehyde cellulose and modified chitosan, forming a moderately cross-linked three-dimensional porous network. Total paeoniflorin was released through diffusion through the pores. The release rate was stable due to the moderate cross-linking network resistance and hydrogen bonding between the drug and the carrier. Example 2: Drug diffusion resistance was reduced, and the release rate was accelerated. The 24-hour release rate was higher than in Example 1, effectively covering the critical window period for tumor treatment. Example 3: 3-Trimethoxysilane propylene acrylate was used to modify cellulose. The resulting hydrogel structure provided a smooth diffusion channel for total paeoniflorin, allowing for rapid and sufficient drug release. Example 4: Increasing the amount of 3-trimethoxysilane propylene acrylate further improved the 24-hour release rate through the synergistic effect of high hydrophilicity and a loose network. Example 5: Reducing the amount of total paeoniflorin resulted in a release rate close to that of Example 1, even though the drug did not reach the hydrogel's saturation loading. Example 6: Increasing the amount of total paeoniflorin in… Regarding the dosage of the loaded drug, in Example 7, the total glucosides of paeony were in excess. The hydrogen bonding between the total glucosides of paeony and the hydrogel carrier was enhanced, and the network pores were partially blocked by drug molecules, resulting in increased release resistance and a decrease in release rate with increasing loading. The 24-hour release rate was lower than that of Example 1. In Comparative Example 1, only hexanoic acid epoxy ethyl methyl ester was used to modify chitosan, and in Comparative Example 2, only epoxy acrolein diethanolamide was used to modify chitosan. The diffusion resistance of total glucosides of paeony was high, the release rate was slow, and the cumulative release rate was low. In Comparative Example 3, no modifier was used, and the total glucosides of paeony were difficult to release effectively, resulting in the lowest cumulative release rate, which could not meet the requirements for effective drug delivery in the treatment of head and neck squamous cell carcinoma. In Comparative Example 4, the diffusion resistance of total glucosides of paeony was increased by reacting γ-methacryloxypropyltrimethoxysilane with the hydroxyl groups of cellulose. The drug diffusion efficiency was lower than that of Example 3, and the 24-hour release rate was lower than that of Example 3.

[0141] Experimental Example 4: Biosafety Test of Hydrogels.

[0142] Test samples: Hydrogels prepared in Examples 1-4 and Comparative Examples 1-4.

[0143] Test method: After autoclaving 1g of hydrogel sample, add it to 10mL of RPMI 1640 culture medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. Extract at 37℃ for 24h, then filter through a 0.22μm membrane for sterilization to obtain the extract. Take L929 mouse fibroblasts in logarithmic growth phase and adjust the concentration to 5×10⁻⁶. 4Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The old culture medium was discarded. 100 μL of hydrogel extract was added to each experimental group, 100 μL of RPMI 1640 medium to the negative control group, and 100 μL of 0.64% phenol solution to the positive control group. Five replicates were made for each group, and the cells were incubated for another 24 h. 20 μL of 5 mg / mL MTT solution was added to each well, and after incubation for 4 h, the supernatant was discarded. 150 μL of DMSO was added to each well, and the mixture was shaken for 10 min to fully dissolve the formazan crystals. The absorbance was measured at 490 nm using a microplate reader. Cell viability R(%) was calculated using the formula: R(%) = (OD2 - OD0) / (OD1 - OD0) × 100%, where OD2 is the absorbance of the experimental group, OD0 is the absorbance of the positive control group, and OD1 is the absorbance of the negative control group.

[0144] The biosafety test results of the hydrogel prepared in this invention are shown in Table 3.

[0145] Table 3. Biosafety test results of hydrogels

[0146]

[0147] The hydrogels prepared in the examples and comparative examples showed extremely low toxicity to L92 cells and excellent biocompatibility, making them safe for use at head and neck squamous cell carcinoma lesions, avoiding local tissue irritation or toxic reactions.

[0148] Experimental Example 5: Optimal concentration test of total glucosides of paeony against head and neck squamous cell carcinoma cells.

[0149] Test sample: Total paeoniflorin prepared in Example 1.

[0150] Assay methods: Three cell lines purchased from Shangen Biotechnology, namely SCC7, FADU, and CAL27, were used. CAL27 and FADU were cultured in DMEM medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin / streptomycin, while SCC7 was cultured in 1640 medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin / streptomycin. Cell proliferation was detected using the CCK-8 assay kit (Vazyme A311). Cells were seeded at an appropriate density in 96-well plates and allowed to adhere for 7 hours. Afterwards, the culture medium was replaced with fresh medium containing total paeoniflorin, and the control group was added with an equal volume of solvent. The results were measured at 0, 24, 48 and 72 h after treatment. 10 μL of CCK-8 solution was added to each well, gently mixed, and incubated in the dark for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. The cell viability R (%) was calculated according to the formula (OD2-OD0) / (OD1-OD0)×100%, where OD2 is the absorbance of the experimental group, OD0 is the absorbance of the blank well, and OD1 is the absorbance of the control group.

[0151] The optimal concentration of total paeoniflorin in SCC7 cells was determined as follows: Figure 2 As shown, the half-maximal inhibitory concentration (IC50) of total paeoniflorin against SCC7 cells was 802 μg / mL; the optimal concentration of total paeoniflorin against FADU cells was determined as follows. Figure 3 As shown, the half-maximal inhibitory concentration (IC50) of total paeoniflorin against FADU cells was 1145 μg / mL; the optimal concentration of total paeoniflorin against CAL27 cells was determined as follows. Figure 4 As shown, the half-maximal inhibitory concentration (IC50) of total paeoniflorin in CAL27 cells was 1650 μg / mL.

[0152] Experimental Example 6: Test on the proliferative capacity of total paeoniflorin in head and neck squamous cell carcinoma cells.

[0153] Test sample: Total paeoniflorin prepared in Example 1.

[0154] Test methods: Three cell lines purchased from Shangen Biotechnology, namely SCC7, FADU, and CAL27, were used. CAL27 and FADU were cultured in DMEM medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin / streptomycin, while SCC7 was cultured in 1640 medium containing 10 v / v% fetal bovine serum and 1 v / v% penicillin / streptomycin. All cells were cultured at 37°C in a 5% CO2 incubator. Cell proliferation was detected using the CCK-8 assay kit (Vazyme A311). Cells were seeded at an appropriate density in 96-well plates. After 7 hours of cell adhesion, the medium was replaced with fresh medium containing total paeoniflorin. The control group received an equal volume of solvent. The low concentration of total paeoniflorin in SCC7 cells was 400 μg / mL, and the high concentration was 800 μg / mL. The total paeoniflorin concentration (TPS) of FADU cells was 500 μg / mL for the low-concentration group and 1000 μg / mL for the high-concentration group. For CAL27 cells, the total paeoniflorin concentration (TPS) was 800 μg / mL for the low-concentration group and 1600 μg / mL for the high-concentration group. Detection was performed at 0, 24, 48, and 72 h after treatment. 10 μL of CCK-8 solution was added to each well, gently mixed, and incubated in the dark for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability R(%) was calculated using the formula: R(%) = (OD2 - OD0) / (OD1 - OD0) × 100%, where OD2 is the absorbance of the experimental group, OD0 is the absorbance of the blank well, and OD1 is the absorbance of the control group.

[0155] The results of the test on the proliferation ability of SCC7 cells by total paeoniflorin are as follows: Figure 5 As shown in the figure, the results of the test on the proliferation ability of FADU cells by total paeoniflorin are as follows: Figure 6 As shown in the figure, the results of the test on the proliferation ability of CAL27 cells by total paeoniflorin are as follows. Figure 7 As shown, at low concentrations, total paeoniflorin significantly inhibited the proliferation of three head and neck squamous cell carcinoma cell lines (Cal27, FADU, and SCC7) compared to the blank control group; the inhibitory effect of high concentrations of total paeoniflorin on the proliferation of Cal27, FADU, and SCC7 was even more pronounced.

[0156] Experimental Example 7: Test on the proliferation rate of head and neck squamous cell carcinoma cells by total glucosides of paeony.

[0157] Test sample: Total paeoniflorin prepared in Example 1.

[0158] Test method: The Click-iT™ Plus EdU Alexa Fluor™ 488 Imaging Kit (Thermo Fisher, C10637) was used to detect cellular DNA synthesis activity, directly reflecting cell proliferation. Cells were cultured at 1×10⁻⁶ cells / year. 5Cells were seeded per well in 24 mL of medium containing a climbing slide. After cell adhesion, the medium was replaced with fresh medium containing low and high concentrations of total paeoniflorin (TP). The control group was treated with an equal volume of solvent for 48 h. Specifically, for SCC7 cells, the low-concentration TP group was 400 μg / mL, and the high-concentration TP group was 800 μg / mL; for FADU cells, the low-concentration TP group was 500 μg / mL, and the high-concentration TP group was 1000 μg / mL; for CAL27 cells, the low-concentration TP group was 800 μg / mL, and the high-concentration TP group was 1600 μg / mL. Two hours before the end of treatment, EdU working solution was prepared according to the kit instructions and added to each well of the medium. The cells were then returned to the incubator for another 2 h of incubation. After EdU incubation, fixative was added at room temperature for 15 min, followed by the addition of 0.5% Triton [solution / concentration]. X-100 was dissolved in PBS at room temperature for 20 min. Click reaction mixture was prepared according to the kit instructions. Washing buffer was discarded, and sufficient Click reaction mixture to cover cells was added to each well. The cells were incubated in the dark at room temperature, followed by washing and nuclear counterstaining. Confocal microscopy was used for observation, and five fields of view were randomly selected from each well for photographing. Quantitative analysis was performed using ImageJ, calculating the EdU-positive cell rate per field (%) = (Number of EdU-positive cells) / (Total number of cells) × 100%. Differences in EdU positivity rates among different treatment groups were compared.

[0159] The results of Hoechst and EdU-488 dual fluorescence staining of total paeoniflorin on CAL27 cells are as follows: Figure 8 As shown, the results of the test on the cell proliferation rate of CAL27 cells by total paeoniflorin are as follows. Figure 9 As shown; the results of Hoechst and EdU-488 dual fluorescence staining of total paeony glucosides on FADU cells are as follows. Figure 10 As shown, the results of the test on the cell proliferation rate of FADU cells by total paeoniflorin are as follows. Figure 11 As shown; the results of Hoechst and EdU-488 dual fluorescence staining of SCC7 cells by total paeoniflorin are as follows. Figure 12 As shown, the results of the test on the cell proliferation rate of SCC7 cells by total paeoniflorin are as follows. Figure 13 As shown, after treatment with total paeoniflorin, the EdU-positive cell rate of three types of head and neck squamous cell carcinoma (CAL27, FADU, and SCC7) decreased significantly with increasing drug concentration, indicating a significant reduction in DNA synthesis activity. The EdU-positive rate in the high-concentration group was further reduced, and there was a statistically significant difference compared with the low-concentration group, suggesting that the drug's inhibition of cell proliferation is concentration-dependent. Dual fluorescence staining results showed no significant change in the total number of cell nuclei stained with Hoechst, ruling out a reduction in the total number of nuclei due to cytotoxicity, confirming that the decrease in the EdU-positive rate was a direct result of inhibited proliferation activity.

[0160] Experimental Example 8: In vitro clonogenic ability test of total paeoniflorin on head and neck squamous cell carcinoma cells.

[0161] Test sample: Total paeoniflorin prepared in Example 1.

[0162] Test Methods: Three cell lines purchased from Shangen Biotechnology, namely CAL27, FADU, and SCC7, were used. CAL27 and FADU were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, while SCC7 was cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. All cells were cultured at 37°C in a 5% CO2 incubator. Cell proliferation and adherence were assessed using a colony formation assay. Single-cell suspensions were seeded at 500 cells / well in 6-well plates, and complete culture medium was added. After cell adherence, the medium was replaced with fresh medium containing total paeoniflorin. The medium was changed every 3 days, and after 10-14 days, when colony formation was visible to the naked eye, the medium was discarded. Cells were fixed with 4% paraformaldehyde for 15 min and then stained with 0.1% crystal violet at room temperature for 20 min. EPSON was used as the assay medium. The v850 scanned the whole-well image and used ImageJ to calculate the number of clones. According to the formula, the clone formation rate R(%) = S1 / S0 × 100%, the clone formation rate was calculated, where S1 is the number of clones formed and S0 is the number of seeded cells.

[0163] The plate staining results showed the effect of total glucosides of paeony on the colony-forming ability of CAL27 cells. Figure 14 As shown, the results of the clonogenicity test of total paeoniflorin on CAL27 cells are as follows: Figure 15 As shown; the plate staining results of the effect of total paeoniflorin on the colony-forming ability of SCC7 cells are as follows. Figure 16 As shown, the results of the clonogenicity test of total paeoniflorin on SCC7 cells are as follows: Figure 17 As shown; the plate staining results of the effect of total paeoniflorin on the colony-forming ability of FADU cells are as follows. Figure 18 As shown, the results of the clonogenicity test of total paeoniflorin on FADU cells are as follows: Figure 19 As shown, after treatment with total paeoniflorin, the colony formation rate of CAL27, FADU, and SCC7 cells decreased significantly. The number of clones and the colony formation rate in the low-concentration group were reduced compared with the control group, and the colony formation rate in the high-concentration group was further reduced, and the clone volume was smaller, indicating that the drug not only inhibited the number of clones, but also reduced the clone proliferation capacity.

[0164] Experimental Example 9: In vitro migration ability test of total paeony glucosides against head and neck squamous cell carcinoma cells.

[0165] Test sample: Total paeoniflorin prepared in Example 1.

[0166] Test methods: Three cell lines purchased from Shangen Biotechnology, namely CAL27, FADU, and SCC7, were used. CAL27 and FADU were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, while SCC7 was cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. All cells were cultured at 37°C in a 5% CO2 incubator. The in vitro migration ability of the cells was assessed using a scratch assay. Cells were sputtered at a rate of 2 × 10⁶ cells / year. 5 Cells were seeded at 1000 μL / mL in 6-well plates and cultured overnight until a monolayer was formed. A straight scratch was made vertically at the bottom of the well using a 200 μL sterile pipette tip. The cells were gently washed three times with PBS to remove detached cell debris. The medium was then replaced with 1% FBS, and low- and high-concentration total paeoniflorin glucosides were added. At 0 and 24 h, the scratched areas were photographed at 100x magnification using an inverted phase-contrast microscope. The scratch width was measured using ImageJ software. Each experiment was repeated at least three times, and 3-5 fields of view were randomly selected from each well for measurement. The scratch healing rate R(%) was calculated using the formula: (W0 - W1) / W0 × 100%, where W1 is the scratch width at 24 h and W0 is the scratch width at 0 h.

[0167] The results of the in vitro migration ability test of total glucosides of paeony against head and neck squamous cell carcinoma cells are as follows: Figure 20 As shown, the effects of total paeoniflorin on the in vitro migration ability of SCC7 cells are as follows: Figure 21 As shown, the effect of total paeoniflorin on the in vitro migration ability of CAL27 cells is as follows: Figure 22 As shown, the effect of total paeoniflorin on the in vitro migration ability of FADU cells is as follows: Figure 23 As shown, treatment with total paeoniflorin significantly reduced the wound healing rate after 24 hours. The healing rate was lower in the low-concentration group than in the control group, and the decrease was more significant in the high-concentration group, indicating that the drug can inhibit the lateral migration ability of cells.

[0168] Experimental Example 10: In vitro migration ability test of total glucosides of paeony against head and neck squamous cell carcinoma cells.

[0169] Test sample: Total paeoniflorin prepared in Example 1.

[0170] Test methods: Three cell lines purchased from Shangen Biotechnology, namely CAL27, FADU, and SCC7, were used. CAL27 and FADU were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, while SCC7 was cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. All cultures were incubated at 37°C in a constant temperature incubator containing 5% CO2. Cells were then cultured at a density of 5 × 10⁶ cells / year. 4Cells were resuspended in 200 μL of serum-free medium at a concentration of / mL. The upper chamber of the Transwell chamber was added, and 600 μL of 10% [amount of culture medium] was added to the lower chamber. Complete culture medium for FBS was prepared. Total paeoniflorin was added to the culture medium in the upper chamber, and the chamber was incubated in a 37°C incubator containing 5% CO2. The concentration of total paeoniflorin in SCC7 cells was 400 μg / mL for the low concentration group and 800 μg / mL for the high concentration group. The concentration of total paeoniflorin in FADU cells was 500 μg / mL for the low concentration group and 1000 μg / mL for the high concentration group. The concentration of total paeoniflorin in CAL27 cells was 800 μg / mL for the low concentration group and 1600 μg / mL for the high concentration group. After incubation, the unmigrated cells on the inner surface of the upper chamber membrane were gently wiped away with a cotton swab. Cells that migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 20 min. Five fields of view were randomly selected and photographed under an inverted microscope at 200x magnification, and the number of migrating cells was counted using ImageJ.

[0171] The results of total paeoniflorin on SCC7 cell scratch inhibition are as follows: Figure 24 As shown in the figure, the results of the in vitro migration ability test of total paeoniflorin on SCC7 cells are as follows: Figure 25 As shown; the results of total paeoniflorin on CAL27 cell scratch therapy are as follows. Figure 26 As shown in the figure, the results of the in vitro migration ability test of total paeoniflorin on CAL27 cells are as follows: Figure 27 As shown; the results of total paeoniflorin on FADU cell scratch therapy are as follows. Figure 28 As shown in the figure, the results of the in vitro migration ability test of total paeoniflorin on FADU cells are as follows: Figure 29 As shown, the number of cells migrating to the subcellular region decreased with increasing drug concentration, and the number of migrating cells further decreased in the high-concentration group, confirming that the drug also inhibits the longitudinal invasion and migration of cells.

[0172] Experimental Example 11: Test on the apoptosis-inducing ability of total paeoniflorin in head and neck squamous cell carcinoma cells.

[0173] Test sample: Total paeoniflorin prepared in Example 1.

[0174] Test method: Cells treated with total paeoniflorin for 48 h were collected. The Annexin V-FITC apoptosis detection kit Beyotime C1062L was used according to the instructions. The samples were analyzed by Attune NxT flow cytometer. FlowJo was used to analyze the data and distinguish between live cells, early apoptotic cells, late apoptotic / necrotic cells and necrotic cells. The total apoptosis rate R (%) was calculated according to the formula R1 + R2, where R1 is the apoptosis rate of early apoptotic cells and R2 is the apoptosis rate of late apoptotic cells.

[0175] The flow cytometry scatter plot results of the total paeoniflorin control group for SCC7 cell apoptosis detection are as follows: Figure 30 As shown in the figure, the flow cytometry scatter plot results of the low concentration group of total paeony glycosides on the apoptosis detection of SCC7 cells are as follows. Figure 31 As shown, the flow cytometry scatter plot results of the high concentration group of total paeony glycosides on the apoptosis detection of SCC7 cells are as follows. Figure 32 As shown in the figure, the results of the test on the apoptosis ability of total paeony glycosides in SCC7 cells are as follows. Figure 33 As shown; the flow cytometry scatter plot results of the total paeoniflorin control group for CAL27 cell apoptosis detection are as follows. Figure 34 As shown, the flow cytometry scatter plot results of the low concentration group of total paeoniflorin in CAL27 cells for apoptosis detection are as follows: Figure 35 As shown, the flow cytometry scatter plot results of the high concentration group of total paeoniflorin in CAL27 cells for apoptosis detection are as follows: Figure 36 As shown in the figure, the results of the test on the apoptosis ability of total paeoniflorin in CAL27 cells are as follows. Figure 37 As shown; the flow cytometry scatter plot results of FADU cell apoptosis detection in the total paeoniflorin control group are as follows. Figure 38 As shown, the flow cytometry scatter plot results of the low concentration group of total paeony glucosides on FADU cell apoptosis detection are as follows: Figure 39 As shown, the flow cytometry scatter plot results of the high concentration group of total paeony glucosides on FADU cell apoptosis detection are as follows: Figure 40 As shown in the figure, the results of the test on the apoptosis ability of total paeony glycosides in FADU cells are as follows. Figure 41 As shown, treatment with total paeoniflorin significantly increased the total apoptosis rate of head and neck squamous cell carcinoma cells. The total apoptosis rate in the low-concentration group was higher than that in the control group, and the apoptosis rate in the high-concentration group was further increased. This indicates that total paeoniflorin can dose-dependently induce apoptosis in head and neck squamous cell carcinoma cells, enhance cell death programs by activating apoptosis pathways, and provide direct evidence for its anti-tumor effect.

[0176] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0177] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A drug for treating head and neck squamous cell carcinoma, characterized in that, The drug comprises total paeoniflorin and hydrogel, wherein the mass ratio of total paeoniflorin to hydrogel is 80-100 mg:10 g. The hydrogel comprises modified chitosan and modified cellulose. The modifier in the modified chitosan comprises epoxy acrolein diacetate and epoxy ethyl methyl hexanoate, wherein the mass ratio of epoxy acrolein diacetate to chitosan is 1-4:2, and the mass ratio of epoxy ethyl methyl hexanoate to chitosan is 1-4:

2. The modifier in the modified cellulose comprises propyl 3-trimethoxysilane acrylate, wherein the mass ratio of propyl 3-trimethoxysilane acrylate to cellulose is 1-4:

5. The total paeoniflorin is extracted by heating and refluxing paeoniflorin in an extractant, wherein the extractant is a 65-75 v / v% ethanol aqueous solution, and the mass-volume ratio of paeoniflorin to the extractant is 300-800 g:5 L.

2. The drug for treating head and neck squamous cell carcinoma according to claim 1, characterized in that: The total glucosides of paeony were purified by elution using a macroporous adsorption resin column. The elution solvent included deionized water and a 45-55 v / v% ethanol aqueous solution, with the amount of deionized water being 1-4 BV and the amount of the 45-55 v / v% ethanol aqueous solution being 6-8 BV.

3. The drug for treating head and neck squamous cell carcinoma according to claim 1, characterized in that: The total glycosides of paeony were purified by precipitation with 2.5-3.5 wt% gelatin aqueous solution and ethanol, wherein the volume ratio of ethanol to 2.5-3.5 wt% gelatin aqueous solution was 40-50:

1.

4. The use of the head and neck squamous cell carcinoma treatment drug according to any one of claims 1-3 in the preparation of a drug for treating head and neck squamous cell carcinoma, characterized in that: The drug targets head and neck squamous cell carcinoma cells, which include at least one of SCC7 cells, FADU cells, and CAL27 cells.

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