Application of total glucosides of paeony in preparation of medicine for treating head and neck squamous cell carcinoma

Abstract

The invention discloses application of total glucosides of paeony in preparation of a medicine for treating head and neck squamous cell carcinoma, and belongs to the technical field of biological medicine. Aiming at the bottlenecks of high chemoradiotherapy toxicity, low immunotherapy response rate, easy drug resistance of targeted drugs and the like in the existing treatment of head and neck squamous cell carcinoma, the invention provides a preparation method of total glucosides of paeony and a preparation technology of a loaded drug of hydrogel obtained based on modified chitosan. The total glucosides of paeony can inhibit the proliferation rate, the clone forming ability and the in-vitro migration ability of SCC7, FADU and CAL27 head and neck squamous carcinoma cell lines in a dose-dependent manner, and obviously induce cell apoptosis. The multi-target action characteristic of the total glucosides of paeony is utilized, the head and neck squamous cell carcinoma resisting effect is achieved through the ways of adjusting the tumor microenvironment, enhancing immune surveillance and the like, the loaded medicine has good slow release performance and biological safety, a new efficient and low-toxicity strategy is provided for treatment of the head and neck squamous cell carcinoma, and important clinical application value is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of total glucosides of white paeony in preparing a drug for treating head and neck squamous cell carcinoma. Background Art

[0002] Head and neck squamous cell carcinoma is the sixth most common malignant tumor in the world, mainly occurring in the mucosal epithelial tissues of the head and neck, such as the oral cavity, pharynx, and larynx. The incidence of head and neck squamous cell carcinoma varies significantly in different regions and populations. Smoking, drinking, and human papillomavirus infection are its main risk factors. Although treatment strategies have gradually evolved from traditional combined sequential treatments of surgery, radiotherapy, and chemotherapy to a new comprehensive paradigm of targeted therapy, immune checkpoint inhibitors, and cell therapy in recent years, the improvement in patients' overall survival rate is still unsatisfactory. The five-year survival rate of patients with locally advanced head and neck squamous cell carcinoma still hovers around 50%, and more than half of the patients experience local recurrence or distant metastasis after receiving radical radiotherapy and chemotherapy, which has become a severe challenge for clinical treatment.

[0003] Current treatment strategies for head and neck squamous cell carcinoma (HNSCC) face multiple bottlenecks: First, damage to normal tissues by conventional radiotherapy and chemotherapy limits their dose intensity, resulting in poor therapeutic efficacy. Second, although immune checkpoint blockade therapy, represented by PD-1 inhibitors, offers hope for some patients, its response rate in platinum-resistant recurrent / metastatic HNSCC is very low. Third, while targeted therapies such as anti-EGFR monoclonal antibodies are incorporated into standard treatment, patient survival benefits are limited and drug resistance is inevitable. Given these challenges, exploring new, highly effective and low-toxic treatment strategies has become an urgent need in HNSCC research. Natural plant active ingredients are gaining increasing attention due to their multi-target action properties and favorable safety profile. Summary of the Invention

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

[0005] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are: A drug for treating head and neck squamous cell carcinoma, comprising total glucosides of white paeony and hydrogel, wherein the mass ratio of total glucosides of white paeony to hydrogel is 80-100 mg:10 g. Preferably, the hydrogel comprises modified chitosan and cellulose.

[0006] Preferably, the mass ratio of modified chitosan to cellulose is 3-7:5.

[0007] Preferably, the modifying agent in the modified chitosan includes epoxy acrolein diethyl acetal and oxiethyl methyl hexanoate.

[0008] Preferably, the mass ratio of epoxy acrolein diethyl acetal to chitosan is 1-4:2.

[0009] Preferably, the mass ratio of oxiranyl methyl hexanoate to chitosan is 1-4:2.

[0010] Epoxypropene diethyl acetal and glyoxalic acid methyl ester are used as modifiers to introduce aldehyde groups and ester groups. The two act together on the chitosan molecular chain and construct a functional group structure with both aldehyde groups and ester groups in its molecule by reacting with the active groups of chitosan. They form moderate cross-links with the aldehyde groups in aldehyde cellulose, which not only enhances the hydrophilicity of the hydrogel through polar groups, but also maintains the stability of the three-dimensional network structure through cross-linking, thereby balancing the swelling properties of the hydrogel and the drug release rate, providing a structural basis for the efficient loading and controlled release of total glucosides of white paeony.

[0011] Preferably, the total glucosides of white paeony are extracted by heating and refluxing white paeony in an extracting solution.

[0012] Preferably, the extract is a 65-75 v / v% ethanol aqueous solution.

[0013] Preferably, the mass volume ratio of white peony root to the extract is 300-800g:5L.

[0014] Preferably, the total glucosides of paeony are eluted and purified by a macroporous adsorption resin column.

[0015] Preferably, the elution solvent comprises deionized water and 45-55 v / v% ethanol aqueous solution.

[0016] Preferably, the amount of deionized water used is 1-4 BV.

[0017] Preferably, the amount of 45-55 v / v% ethanol aqueous solution is 6-8 BV.

[0018] Preferably, the total glucosides of white paeony are purified by precipitation with 2.5-3.5 wt % gelatin aqueous solution and ethanol.

[0019] Preferably, the volume ratio of ethanol to 2.5-3.5 wt% gelatin aqueous solution is 40-50:1.

[0020] Preferably, the medicament is for inhibiting the proliferation of head and neck squamous cell carcinoma cells.

[0021] Preferably, the head and neck squamous cell carcinoma cells comprise at least one of SCC7 cells, FADU cells and CAL27 cells.

[0022] Preferably, the drug is used for reducing the colony formation ability of the head and neck squamous cell carcinoma cells.

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

[0024] Preferably, the drug is used for inhibiting the in vitro migration ability of the head and neck squamous cell carcinoma cells.

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

[0026] Preferably, the drug is used for inducing the apoptosis of the head and neck squamous cell carcinoma cells.

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

[0028] More preferably, the hydrogel comprises modified chitosan and modified cellulose, the modifier of the modified cellulose comprises 3-trimethoxysilyl propyl acrylate, and the mass ratio of 3-trimethoxysilyl propyl acrylate to cellulose is 1-4:5. As the modifier of cellulose, 3-trimethoxysilyl propyl acrylate reacts with the hydroxyl in the molecular chain of cellulose to introduce silicon hydroxyl and acrylate groups, which significantly improves the water molecule binding capacity of cellulose. The modified cellulose and the modified chitosan form a more loose three-dimensional network structure, which reduces the resistance to drug diffusion and promotes the swelling of the hydrogel, thereby synergistically improving the release rate and release rate of total paeoniflorin, and providing structural support for the effective delivery of drugs in the treatment of head and neck squamous cell carcinoma.

[0029] The application also provides a preparation method of total paeoniflorin. The crushed paeonia suffruticosa is dispersed in an extraction liquid, and reflux extraction is performed 1-4 times at 75-85 DEG C for 1-4 hours each time. The extraction liquid is combined, the residue is filtered with filter paper, and the concentrated liquid is obtained by reducing pressure concentration. Deionized water is added to the concentrated liquid to obtain a diluted liquid. The diluted liquid is passed through a macroporous adsorption resin column at a flow rate of 3-5 BV / h, and the elution is performed with water phase at a flow rate until the effluent is colorless, and then the elution is performed with organic phase at a flow rate. The eluate is collected, concentrated under reduced pressure, and filtered. A 2.5-3.5 wt% gelatin aqueous solution is added to the filtrate, ethanol is added, and the mixture is statically placed for 20-25 hours. After filtration, the ethanol is recovered under reduced pressure, and the mixture is vacuum dried at 55-65 DEG C for 10-15 hours to obtain total paeoniflorin.

[0030] Preferably, the extracting solution is 65-75 v / v% ethanol aqueous solution, and the mass-volume ratio of white peony root to the extracting solution is 300-800 g:5 L.

[0031] Preferably, the mass-volume ratio of white peony root to the concentrated solution is 300-800 g:500 mL.

[0032] Preferably, the volume ratio of deionized water to the concentrated solution is 4-6 L:500 mL.

[0033] Preferably, the macroporous adsorption resin is HPD300.

[0034] Preferably, the water-phase elution solvent is deionized water, the elution flow rate of the water-phase elution solvent is 3-5 BV / h, and the amount of the water-phase elution solvent is 1-4 BV.

[0035] Preferably, the organic-phase elution solvent is 45-55 v / v% ethanol aqueous solution, the elution flow rate of the organic-phase elution solvent is 3-5 BV / h, and the amount of the organic-phase elution solvent is 6-8 BV.

[0036] Preferably, the volume ratio of the filtrate to 2.5-3.5 wt% gelatin aqueous solution is 5-15:1.

[0037] Preferably, the volume ratio of ethanol to 2.5-3.5 wt% gelatin aqueous solution is 40-50:1.

[0038] The application also provides a preparation method of modified chitosan, comprising: dispersing epoxypropylene aldehyde condensation diethyl alcohol and epoxyethyl methyl hexanoate in deionized water to obtain a modifier solution; dispersing chitosan in isopropyl alcohol at 55-65 °C, stirring for 1-4 h, adding the modifier solution, stirring and reacting at 75-85 °C for 6-10 h, adding the reaction liquid into acetone, collecting the precipitate by filtration, washing the precipitate with 80 v / v% isopropyl alcohol aqueous solution for 2-4 times, and vacuum drying at 45-55 °C for 10-15 h to obtain modified chitosan.

[0039] Preferably, the mass-volume ratio of epoxypropylene aldehyde condensation diethyl alcohol to deionized water is 1-4 mg:10 mL.

[0040] Preferably, the mass-volume ratio of epoxyethyl methyl hexanoate to deionized water is 1-4 mg:10 mL.

[0041] Preferably, the mass-volume ratio of chitosan to isopropyl alcohol is 1-4 mg:20 mL.

[0042] Preferably, the mass of the modifier solution is measured by the mass of epoxyethyl methyl hexanoate therein, and the mass ratio of epoxyethyl methyl hexanoate to chitosan is 1-4:2.

[0043] Preferably, the volume ratio of the reaction solution to acetone is 5-15:50.

[0044] The present invention also provides a method for preparing a hydrogel, comprising: Aldehyde cellulose is dispersed in a 0.3-0.5 wt% sodium hydroxide aqueous solution, modified chitosan is added, 0.6-0.8 mol / L glacial acetic acid solution is added, and the mixture is stirred for 5-15 minutes, sealed and allowed to stand for 10-15 hours, dialyzed in deionized water for 5-7 hours, and freeze-dried to obtain a hydrogel.

[0045] Preferably, the volume mass ratio of 0.3-0.5 wt % sodium hydroxide aqueous solution to aldehyde cellulose is 300-500 mL:5 g.

[0046] Preferably, the mass ratio of modified chitosan to aldehyde cellulose is 3-7:5.

[0047] Preferably, the volume mass ratio of 0.6-0.8 mol / L glacial acetic acid solution to aldehyde cellulose is 100-200 mL:5 g.

[0048] The present invention also provides a method for preparing a drug-loaded material, comprising: The total glucosides of white paeony were dispersed in deionized water, and the hydrogel was added and soaked at room temperature for 10-15 hours. The hydrogel was taken out and rinsed with deionized water to obtain the loaded drug.

[0049] Preferably, the mass volume ratio of total glucosides of white paeony to deionized water is 50-150 mg:10 mL.

[0050] Preferably, the mass ratio of total glucosides of white paeony to hydrogel is 80-100 mg:10 g.

[0051] The present invention uses total glucosides of paeony extracted and purified from white peony root, and modifies chitosan with epoxy acrolein diethyl acetal and epoxyethyl methyl hexanoate, and constructs a hydrogel loading system with cellulose to achieve efficient extraction and release of total glucosides of paeony root. Therefore, it has the following beneficial effects: the prepared drug for treating head and neck squamous cell carcinoma can inhibit the proliferation, cloning and migration of head and neck squamous cell carcinoma cells in a dose-dependent manner, significantly induce their apoptosis, has excellent biosafety, and can effectively solve the bottlenecks of high toxicity of chemotherapy and radiotherapy and low response rate of immunotherapy in existing treatments. Therefore, the present invention is a drug for treating head and neck cancer with high efficiency, low toxicity, and controllable delivery, which is suitable for the treatment of head and neck squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of the scanning electron microscope image of the hydrogel.

[0053] Figure 2Schematic diagram of the test results of the optimal concentration of total glucosides of paeony on SCC7 cells.

[0054] Figure 3 This is a schematic diagram of the test results of the optimal concentration of total glucosides of paeony on FADU cells.

[0055] Figure 4 Schematic diagram of the test results of the optimal concentration of total glucosides of paeony on CAL27 cells.

[0056] Figure 5 Schematic diagram of the test results of total glucosides of paeony on the proliferation ability of SCC7 cells.

[0057] Figure 6 Schematic diagram of the test results of total glucosides of paeony on the proliferation ability of FADU cells.

[0058] Figure 7 Schematic diagram of the test results of total glucosides of paeony on the proliferation ability of CAL27 cells.

[0059] Figure 8 Schematic diagram of the results of double fluorescence staining of CAL27 cells by total glucosides of white paeony using Hoechst and EdU-488.

[0060] Figure 9 Schematic diagram of the test results of total glucosides of paeony on the cell proliferation rate of CAL27 cells.

[0061] Figure 10 Schematic diagram of the results of double fluorescence staining of FADU cells by total glucosides of white paeony using Hoechst and EdU-488.

[0062] Figure 11 Schematic diagram of the test results of total glucosides of paeony on the cell proliferation rate of FADU cells.

[0063] Figure 12 Schematic diagram of the results of double fluorescence staining of SCC7 cells by Hoechst and EdU-488 by total glucosides of paeony.

[0064] Figure 13 Schematic diagram of the test results of total glucosides of paeony on the cell proliferation rate of SCC7 cells.

[0065] Figure 14 Schematic diagram of the effect of total glucosides of paeony on the clone-forming ability of CAL27 cells.

[0066] Figure 15 Schematic diagram of the test results of total glucosides of paeony on the clone formation rate of CAL27 cells.

[0067] Figure 16 Schematic diagram of the effect of total glucosides of paeony on the clone-forming ability of SCC7 cells.

[0068] Figure 17 Schematic diagram of the test results of total glucosides of paeony on the clone formation rate of SCC7 cells.

[0069] Figure 18 Schematic diagram of the effect of total glucosides of paeony on the clone-forming ability of FADU cells.

[0070] Figure 19 Schematic diagram of the test results of total glucosides of paeony on the clone formation rate of FADU cells.

[0071] Figure 20 Schematic diagram of the test results of the in vitro migration ability of total glucosides of paeony on head and neck squamous cell carcinoma cells.

[0072] Figure 21 Schematic diagram of the effect of total glucosides of paeony on the migration ability of SCC7 cells in vitro.

[0073] Figure 22 Schematic diagram of the effect of total glucosides of paeony on the migration ability of CAL27 cells in vitro.

[0074] Figure 23 Schematic diagram of the effect of total glucosides of paeony on the in vitro migration ability of FADU cells.

[0075] Figure 24 Schematic diagram of the effect of total glucosides of paeony on the migration ability of SCC7 cells in vitro.

[0076] Figure 25 Schematic diagram of the test results of total glucosides of paeony on the in vitro migration ability of SCC7 cells.

[0077] Figure 26 Schematic diagram of the effect of total glucosides of paeony on the in vitro migration ability of CAL27.

[0078] Figure 27 Schematic diagram of the test results of total glucosides of paeony on the in vitro migration ability of CAL27 cells.

[0079] Figure 28 Schematic diagram of the effect of total glucosides of paeony on the in vitro migration ability of FADU cells.

[0080] Figure 29 Schematic diagram of the test results of total glucosides of paeony on the in vitro migration ability of FADU cells.

[0081] Figure 30 This is a schematic diagram of the effect of the total glucosides of paeony control group on the apoptosis ability of SCC7 cells.

[0082] Figure 31 Schematic diagram of the effect of low-concentration total glucosides of white paeony on the apoptosis ability of SCC7 cells.

[0083] Figure 32 Schematic diagram of the effect of high-concentration total glucosides of white paeony on the apoptosis ability of SCC7 cells.

[0084] Figure 33 Schematic diagram of the test results of the apoptosis ability of total glucosides of paeony on SCC7 cells.

[0085] Figure 34 This is a schematic diagram of the effect of the total glucosides of white paeony control group on the apoptosis ability of CAL27.

[0086] Figure 35 This is a schematic diagram of the effect of the low-concentration group of total glucosides of white paeony on the apoptosis ability of CAL27.

[0087] Figure 36 This is a schematic diagram of the effect of high-concentration total glucosides of white paeony on the apoptosis ability of CAL27.

[0088] Figure 37 Schematic diagram of the test results of the apoptosis ability of total glucosides of paeony on CAL27 cells.

[0089] Figure 38 This is a schematic diagram of the effect of the total glucosides of white paeony control group on the apoptosis ability of FADU cells.

[0090] Figure 39 This is a schematic diagram of the effect of the low-concentration group of total glucosides of white paeony on the apoptosis ability of FADU cells.

[0091] Figure 40 This is a schematic diagram of the effect of high-concentration total glucosides of white paeony on the apoptosis ability of FADU cells.

[0092] Figure 41 Schematic diagram of the test results of the apoptosis ability of total glucosides of paeony on FADU cells. DETAILED DESCRIPTION

[0093] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0094] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0095] Example 1: Preparation of total glucosides of white paeony: Pulverize white paeony and disperse it in the extract, reflux extract twice at 80°C for 2 hours each time, combine the two extracts, filter the residue with filter paper, and concentrate under reduced pressure to obtain a concentrate; add deionized water to the concentrate to obtain a dilution; pass the dilution through an HPD300 macroporous adsorption resin column at a flow rate of 4BV / h, elute with an aqueous phase elution solvent at a flow rate until the effluent is colorless, elute with an organic phase elution solvent at a flow rate, collect the eluate, concentrate under reduced pressure, add 3wt% gelatin aqueous solution to the filtrate after filtration, add ethanol, let it stand for 24 hours, filter, recover the ethanol under reduced pressure, and then vacuum dry at 60°C for 12 hours to obtain total glucosides of white paeony. The extract is a 70v / v% ethanol aqueous solution, the mass volume ratio of white peony root to the extract is 500g:5L, the mass volume ratio of white peony root to the concentrate is 500g:500mL, and the volume ratio of deionized water to the concentrate is 5L:500mL; the aqueous phase elution solvent is deionized water, the elution flow rate of the aqueous phase elution solvent is 4BV / h, and the amount of the aqueous phase elution solvent is 2BV; the organic phase elution solvent is a 50v / v% ethanol aqueous solution, the elution flow rate of the organic phase elution solvent is 4BV / h, and the amount of the organic phase elution solvent is 7BV; the volume ratio of the filtrate to the 3wt% gelatin aqueous solution is 10:1, and the volume ratio of ethanol to the 3wt% gelatin aqueous solution is 44:1.

[0096] Modified chitosan was prepared by dispersing epoxypropanol diethyl acetal and oxiranyl methyl hexanoate in deionized water to obtain a modifier solution. Chitosan was then dispersed in isopropyl alcohol at 60°C and stirred for 2 hours. The modifier solution was then added and the reaction was stirred at 80°C for 8 hours. The reaction solution was then added to acetone, and the precipitate was collected by filtration. The precipitate was washed three times with an 80 v / v% isopropyl alcohol solution and dried under vacuum at 50°C for 12 hours to obtain the modified chitosan. The mass-to-volume ratio of epoxypropanol diethyl acetal to deionized water was 2 mg:10 mL, the mass-to-volume ratio of oxiranyl methyl hexanoate to deionized water was 2 mg:10 mL, and the mass-to-volume ratio of chitosan to isopropyl alcohol was 2 mg:20 mL. The mass of the modifier solution was based on the mass of oxiranyl methyl hexanoate in the solution, and the mass ratio of oxiranyl methyl hexanoate to chitosan was 2:2. The volume ratio of the reaction solution to acetone was 10:50.

[0097] Preparation of the hydrogel: Aldehyde cellulose was dispersed in a 0.4 wt% sodium hydroxide solution, modified chitosan was added, and 0.7 mol / L glacial acetic acid solution was added. After stirring for 10 minutes, the mixture was sealed and allowed to stand for 12 hours. The mixture was dialyzed in deionized water for 6 hours and freeze-dried to obtain the hydrogel. The volume-to-mass ratio of the 0.4 wt% sodium hydroxide solution to the aldehyde cellulose was 400 mL:5 g, the mass ratio of the modified chitosan to the aldehyde cellulose was 5:5, and the volume-to-mass ratio of the 0.7 mol / L glacial acetic acid solution to the aldehyde cellulose was 150 mL:5 g.

[0098] Preparation of drug-loaded solution: Disperse total glucosides of white paeony in deionized water, add hydrogel, and soak at room temperature for 12 hours. Remove the hydrogel and rinse with deionized water to obtain the drug-loaded solution. The mass-to-volume ratio of total glucosides of white paeony to deionized water is 100 mg:10 mL, and the mass ratio of total glucosides of white paeony to hydrogel is 100 mg:10 g.

[0099] Example 2: Compared with Example 1, the only difference between this example and Example 1 is the preparation of modified chitosan.

[0100] Modified chitosan was prepared by dispersing epoxypropanol diethyl acetal and oxiranyl methyl hexanoate in deionized water to obtain a modifier solution. Chitosan was then dispersed in isopropyl alcohol at 60°C and stirred for 2 hours. The modifier solution was then added and the reaction was stirred at 80°C for 8 hours. The reaction solution was then added to acetone, and the precipitate was collected by filtration. The precipitate was washed three times with an 80 v / v% isopropyl alcohol solution and dried under vacuum at 50°C for 12 hours to obtain the modified chitosan. The mass-to-volume ratio of epoxypropanol diethyl acetal to deionized water was 4 mg:10 mL, the mass-to-volume ratio of oxiranyl methyl hexanoate to deionized water was 2 mg:10 mL, and the mass-to-volume ratio of chitosan to isopropyl alcohol was 2 mg:20 mL. The mass of the modifier solution was based on the mass of oxiranyl methyl hexanoate in the solution, and the mass ratio of oxiranyl methyl hexanoate to chitosan was 2:2. The volume ratio of the reaction solution to acetone was 10:50.

[0101] Example 3: Compared with Example 1, the only difference between this example and Example 1 is the preparation of the hydrogel.

[0102] Preparation of hydrogel: cellulose was dispersed in anhydrous ethanol and stirred to obtain a cellulose dispersion; 3-trimethoxysilane acrylate was added to the cellulose dispersion, stirred to obtain a cellulose dispersion; concentrated hydrochloric acid was added, stirred and reacted at 60°C for 6 hours, filtered and the precipitate was collected, washed with anhydrous ethanol three times, and vacuum dried at 50°C for 12 hours to obtain modified cellulose; the modified cellulose was dispersed in a 0.4wt% sodium hydroxide aqueous solution, modified chitosan was added, 0.7mol / L glacial acetic acid solution was added, stirred for 10 minutes, sealed and allowed to stand for 12 hours, dialyzed in deionized water for 6 hours, and freeze-dried to obtain a hydrogel. In the cellulose dispersion, the volume mass ratio of anhydrous ethanol to cellulose is 100 mL:5 g; the mass ratio of 3-trimethoxysilane acrylate to cellulose is 1:5, the volume mass ratio of concentrated hydrochloric acid to cellulose is 0.5 mL:5 g, the volume mass ratio of 0.4 wt% sodium hydroxide aqueous solution to modified cellulose is 400 mL:5 g, the mass ratio of modified chitosan to modified cellulose is 5:5, and the volume mass ratio of 0.7 mol / L glacial acetic acid solution to modified cellulose is 150 mL:5 g.

[0103] Example 4: Compared with Example 1, the only difference between this example and Example 1 is the preparation of the hydrogel.

[0104] Preparation of hydrogel: cellulose was dispersed in anhydrous ethanol and stirred to obtain a cellulose dispersion; 3-trimethoxysilane acrylate was added to the cellulose dispersion, stirred to obtain a cellulose dispersion; concentrated hydrochloric acid was added, stirred and reacted at 60°C for 6 hours, filtered and the precipitate was collected, washed with anhydrous ethanol three times, and vacuum dried at 50°C for 12 hours to obtain modified cellulose; the modified cellulose was dispersed in a 0.4wt% sodium hydroxide aqueous solution, modified chitosan was added, 0.7mol / L glacial acetic acid solution was added, stirred for 10 minutes, sealed and allowed to stand for 12 hours, dialyzed in deionized water for 6 hours, and freeze-dried to obtain a hydrogel. In the cellulose dispersion, the volume mass ratio of anhydrous ethanol to cellulose is 100 mL:5 g; the mass ratio of 3-trimethoxysilane propyl acrylate to cellulose is 2:5, the volume mass ratio of concentrated hydrochloric acid to cellulose is 0.5 mL:5 g, the volume mass ratio of 0.4 wt% sodium hydroxide aqueous solution to modified cellulose is 400 mL:5 g, the mass ratio of modified chitosan to modified cellulose is 5:5, and the volume mass ratio of 0.7 mol / L glacial acetic acid solution to modified cellulose is 150 mL:5 g.

[0105] Example 5: Compared with Example 1, the only difference between this example is the preparation of the loaded drug.

[0106] Preparation of drug-loaded solution: Disperse total glucosides of white paeony in deionized water, add hydrogel, and soak at room temperature for 12 hours. Remove the hydrogel and rinse with deionized water to obtain the drug-loaded solution. The mass-to-volume ratio of total glucosides of white paeony to deionized water is 80 mg:10 mL, and the mass ratio of total glucosides of white paeony to hydrogel is 80 mg:10 g.

[0107] Example 6: Compared with Example 1, the only difference between this example is the preparation of the loaded drug.

[0108] Preparation of drug-loaded solution: Disperse total glucosides of white paeony in deionized water, add hydrogel, and soak at room temperature for 12 hours. Remove the hydrogel and rinse with deionized water to obtain the drug-loaded solution. The mass-to-volume ratio of total glucosides of white paeony to deionized water is 120 mg:10 mL, and the mass ratio of total glucosides of white paeony to hydrogel is 120 mg:10 g.

[0109] Example 7: Compared with Example 1, the only difference between this example is the preparation of the loaded drug.

[0110] Preparation of drug-loaded solution: Disperse total glucosides of white paeony in deionized water, add hydrogel, and soak at room temperature for 12 hours. Remove the hydrogel and rinse with deionized water to obtain the drug-loaded solution. The mass-to-volume ratio of total glucosides of white paeony to deionized water is 150 mg:10 mL, and the mass ratio of total glucosides of white paeony to hydrogel is 150 mg:10 g.

[0111] Comparative Example 1: This comparative example is different from Example 1 only in that epoxy acrolein diethyl acetal is not used in the preparation of the modified chitosan.

[0112] Comparative Example 2: This comparative example is different from Example 1 only in that oxiethyl hexanoate is not used in the preparation of the modified chitosan.

[0113] Comparative Example 3: This comparative example is different from Example 1 only in that epoxy acrolein diethyl acetal and epoxyethyl methyl hexanoate are not used in the preparation of the modified chitosan.

[0114] Comparative Example 4: Compared with Example 3, this comparative example differs only in the preparation of the hydrogel.

[0115] Preparation of hydrogel: cellulose was dispersed in anhydrous ethanol and stirred to obtain a cellulose dispersion; γ-methacryloxypropyltrimethoxysilane was added to the cellulose dispersion, stirred to obtain a cellulose dispersion; concentrated hydrochloric acid was added, stirred and reacted at 60°C for 6 hours, filtered and the precipitate was collected, washed with anhydrous ethanol three times, and vacuum dried at 50°C for 12 hours to obtain modified cellulose; the modified cellulose was dispersed in a 0.4wt% sodium hydroxide aqueous solution, modified chitosan was added, 0.7mol / L glacial acetic acid solution was added, stirred for 10 minutes, sealed and allowed to stand for 12 hours, dialyzed in deionized water for 6 hours, and freeze-dried to obtain a hydrogel. In the cellulose dispersion, the volume mass ratio of anhydrous ethanol to cellulose is 100 mL:5 g; the mass ratio of γ-methacryloxypropyltrimethoxysilane to cellulose is 1:5, the volume mass ratio of concentrated hydrochloric acid to cellulose is 0.5 mL:5 g, the volume mass ratio of 0.4 wt% sodium hydroxide aqueous solution to modified cellulose is 400 mL:5 g, the mass ratio of modified chitosan to modified cellulose is 5:5, and the volume mass ratio of 0.7 mol / L glacial acetic acid solution to modified cellulose is 150 mL:5 g.

[0116] Experimental Example 1: Microstructural characterization of hydrogels.

[0117] Test sample: hydrogel prepared in Example 1.

[0118] Test method: The hydrogel surface was gold-sprayed and observed using a scanning electron microscope at 25°C with an accelerating voltage set to 15kV.

[0119] The scanning electron microscope image of the hydrogel prepared by the present invention is as follows Figure 1 As shown, under a scanning electron microscope, it can be observed that it presents a three-dimensional porous network structure, the pore distribution is relatively uniform, the pore wall surface is relatively smooth, the network structure is tightly connected, and has good continuity, indicating that the hydrogel shown in the figure is successfully obtained.

[0120] Test Example 2: Swelling rate test of hydrogel.

[0121] Test samples: hydrogels prepared in Examples 1-4 and Comparative Examples 1-4.

[0122] Test method: The hydrogel sample was vacuum dried at 60°C to constant weight, and the initial dry mass m0 was accurately weighed. The dried hydrogel was added to 30 mL of pH 7.4 PBS buffer and allowed to stand at 37°C. After 2 hours, the hydrogel was taken out and the excess liquid on the surface was gently absorbed with filter paper. The sample was immediately weighed accurately, and the mass was m1. According to the formula, the swelling rate SR (%) = (m1-m0) / m0×100%, the swelling rate of the hydrogel was calculated, where m0 is the initial mass of the hydrogel and m1 is the mass of the hydrogel after swelling.

[0123] The swelling rate test results of the hydrogel prepared by the present invention are shown in Table 1.

[0124] Table 1 Hydrogel swelling rate test results

[0125] The modified chitosan in Example 1 is modified by epoxy acrolein diethanol acetal and epoxy ethyl methyl hexanoate. The two modifiers work synergistically to introduce appropriate aldehyde groups and ester groups into the chitosan molecular chain, forming moderate cross-linking with the aldehyde cellulose, and balancing hydrophilicity and structural stability; Example 2 increases the amount of epoxy acrolein diethanol acetal in the modified chitosan, enhances the interaction with water molecules, and the swelling rate is higher than that of Example 1; Example 3 uses 3-trimethoxysilane propyl acrylate to modify cellulose, the hydrophilicity of silanol and acrylate groups is significantly stronger than that of aldehyde groups, and the network combination of modified cellulose and modified chitosan is looser, and the swelling rate is further increased; Example 4 increases the amount of 3-trimethoxysilane propyl acrylate, which significantly increases the density of hydrophilic groups, and the steric hindrance of the acrylate group avoids excessive network density, and the swelling rate is the lowest. High; in the modified chitosan of Comparative Example 1, epoxy propylene glycol acetal was not used, and in the modified chitosan of Comparative Example 2, epoxy ethyl methyl hexanoate was not used. The cross-linking with the aldehyde cellulose was denser due to insufficient aldehyde groups, and the swelling rate was significantly lower than that of Example 1; in Comparative Example 3, epoxy propylene glycol acetal and epoxy ethyl methyl hexanoate were not used, and the reaction activity of the unmodified chitosan with the aldehyde cellulose was extremely low. The network structure had the lowest swelling rate due to insufficient cross-linking; in Comparative Example 4, γ-methacryloxypropyltrimethoxysilane was reacted with cellulose hydroxyl groups to improve the hydrophilicity of cellulose, maintain a certain network looseness, and enhance the swelling performance of the hydrogel. Therefore, the swelling rate was significantly higher than that of Comparative Examples 1-3, but the water molecule penetration and retention capacity of γ-methacryloxypropyltrimethoxysilane were lower than those of Examples 3-4 compared with 3-trimethoxysilane propyl acrylate.

[0126] Test Example 3: Release test of loaded drugs.

[0127] Test samples: loaded drugs prepared in various examples and comparative examples.

[0128] Test method: 10 g of loaded drug was added to 30 mL of pH 7.4 PBS buffer, sealed and shaken at 37°C and 100 rpm. 5 mL of release solution was drawn at 1, 2, 4, 8, 12, 24, 48 and 72 h, and 5 mL of fresh PBS buffer was added. The concentration C1 of total glucosides of white paeony in the release solution was determined by HPLC. The chromatographic column was a C18 column, 250 mm × 4.6 mm, 5 μm. The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution. The volume ratio of acetonitrile to 0.1% phosphoric acid aqueous solution was 15:85. The detection wavelength was 230 nm, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the injection volume was 20 μL. According to the formula, the release rate R (%) = (C1 × V1) / m0×100%, and the release rate of the loaded drug was calculated, where C1 is the concentration of total glucosides of paeony in the release solution, V1 is the volume of the release solution, and m0 is the mass of total glucosides of paeony in the initial loaded drug.

[0129] The 24h release test results of the loaded drug prepared by the present invention are shown in Table 2.

[0130] Table 2 24h release test results of loaded drugs

[0131] The hydrogel of Example 1 is prepared from aldehyde cellulose and modified chitosan, which form a moderately cross-linked three-dimensional porous network. The total glucosides of white paeony are released through diffusion through the pores. Affected by the moderately cross-linked network resistance and the hydrogen bond interaction between the drug and the carrier, the release rate is stable; in Example 2, the drug diffusion resistance is reduced, the release rate is accelerated, and the 24-hour release rate is higher than that of Example 1, effectively covering the critical window period for tumor treatment; in Example 3, cellulose is modified with 3-trimethoxysilane propyl acrylate, and the resulting hydrogel structure provides a smooth diffusion channel for the total glucosides of white paeony, so that the drug can be released quickly and fully; in Example 4, the amount of 3-trimethoxysilane propyl acrylate is increased, and the 24-hour release rate is further improved under the synergistic effect of high hydrophilicity and loose network; in Example 5, the amount of total glucosides of white paeony is reduced, and the drug does not reach the saturated loading capacity of the hydrogel, and the release rate is close to that of Example 1; in Example 6, the total glucosides of white paeony are increased in The dosage of the loaded drug is as follows: in Example 7, the loaded drug contains an excess of total glucosides of white paeony, the hydrogen bonding between total glucosides of white paeony and the hydrogel carrier is enhanced, and the network pores are partially blocked by drug molecules, resulting in increased release resistance, a decrease in release rate with increasing loading amount, and a decrease in 24h release rate compared with Example 1; in Comparative Example 1, chitosan is modified with only epoxyethyl methyl hexanoate, and in Comparative Example 2, chitosan is modified with only epoxypropionaldehyde diethyl acetal, resulting in large total glucosides diffusion resistance, slow release rate, and low cumulative release rate; in Comparative Example 3, no modifier is used, total glucosides of white paeony are difficult to be effectively released, the cumulative release rate is the lowest, and the demand for effective drug delivery in the treatment of head and neck squamous cell carcinoma cannot be met; in Comparative Example 4, the diffusion resistance of total glucosides of white paeony is increased by reacting γ-methacryloyloxypropyltrimethoxysilane with cellulose hydroxyl groups, the drug diffusion efficiency is decreased compared with Example 3, and the 24h release rate is lower than that of Example 3.

[0132] Test Example 4: Biosafety test of hydrogel.

[0133] Test samples: hydrogels prepared in Examples 1-4 and Comparative Examples 1-4.

[0134] Test method: After autoclaving, 1 g of hydrogel sample was added to 10 mL of RPMI1640 culture medium containing 10% fetal bovine serum and 1% double-antibody. The sample was incubated at 37°C for 24 h and sterilized by filtration with a 0.22 μm filter membrane to obtain the extract. L929 mouse fibroblasts in the logarithmic growth phase were taken and the concentration was adjusted to 5 × 10 4Cells were plated at 100 μL per well in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 h. The old culture medium in the wells was discarded, and 100 μL of hydrogel extract was added to the experimental group, 100 μL of RPMI1640 culture medium was added to the negative control group, and 100 μL of 0.64% phenol solution was added to the positive control group. Five replicate wells were set up for each group and cultured for another 24 h. 20 μL of 5 mg / mL MTT solution was added to each well. After incubation for 4 h, the supernatant was discarded, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min to fully dissolve the formazan crystals, and the absorbance was measured at 490 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 positive control group, and OD1 is the absorbance of the negative control group.

[0135] The biosafety test results of the hydrogel prepared by the present invention are shown in Table 3.

[0136] Table 3 Biosafety test results of hydrogels

[0137] The hydrogels prepared in the examples and comparative examples have extremely low toxicity to L92 cells, have excellent biosafety, and can be safely used in head and neck squamous cell carcinoma lesions to avoid local tissue irritation or toxic reactions.

[0138] Experimental Example 5: Test on the optimal concentration of total glucosides of white paeony on head and neck squamous cell carcinoma cells.

[0139] Test sample: total glucosides of paeony prepared in Example 1.

[0140] Test method: three cell lines SCC7, FADU and CAL27 purchased from Shengene were selected, wherein CAL27, FADU were cultured in DMEM medium containing 10v / v% fetal bovine serum and 1v / v% penicillin / streptomycin, and SCC7 was cultured in 1640 medium containing 10v / v% fetal bovine serum and 1v / v% penicillin / streptomycin, in a constant temperature incubator at 37°C containing 5% CO2; CCK-8 kit Vazyme A311 was used to detect cell proliferation activity, cells were inoculated in 96-well plates at an appropriate density, and after cell adhesion for 7h, fresh culture medium containing total paeoniflorin was replaced, and the same volume of solvent was added to the control group; detection was carried out at 0, 24, 48 and 72h after treatment, 10μL of CCK-8 solution was added to each well, mixed gently, incubated in the incubator for 2h, and the absorbance of each well was measured at 450nm using a microplate reader, according to the formula, cell survival rate R (%) = (OD2-OD0) / (OD1-OD0) x 100%, the cell survival rate was calculated, wherein OD2 is the absorbance of the experimental group, OD0 is the absorbance of the blank hole, and OD1 is the absorbance of the control group.

[0141] The results of the optimal concentration test of total paeoniflorin on SCC7 cells are shown in Table 1. Figure 2 The results of the optimal concentration test of total paeoniflorin on FADU cells are shown in Table 2. Figure 3 The results of the optimal concentration test of total paeoniflorin on CAL27 cells are shown in Table 3. Figure 4 The results of the optimal concentration test of total paeoniflorin on CAL27 cells are shown in Table 3.

[0142] Test example 6: Test of the proliferation ability of total paeoniflorin on head and neck squamous cell carcinoma cells.

[0143] Test sample: total paeoniflorin prepared in Example 1.

[0144] Test method: three cell lines SCC7, FADU and CAL27 purchased from Shang' an Biological were selected, wherein CAL27, FADU were cultured in DMEM medium containing 10v / v% fetal bovine serum and 1v / v% penicillin / streptomycin, and SCC7 was cultured in 1640 medium containing 10v / v% fetal bovine serum and 1v / v% penicillin / streptomycin in a constant temperature incubator at 37°C containing 5% CO2; CCK-8 kit Vazyme A311 was used to detect cell proliferation activity, and cells were inoculated in a 96-well plate at an appropriate density, and after cell adhesion for 7h, fresh medium containing total paeonol glycosides was replaced, and the same volume of solvent was added to the control group; wherein the low concentration group of total paeonol glycosides for SCC7 cells was 400μg / mL, and the high concentration group of total paeonol glycosides was 800μg / mL; the low concentration group of total paeonol glycosides for FADU cells was 500μg / mL, and the high concentration group of total paeonol glycosides was 1000μg / mL; the low concentration group of total paeonol glycosides for CAL27 cells was 800μg / mL, and the high concentration group of total paeonol glycosides was 1600μg / mL; detection was carried out at 0, 24, 48 and 72h after treatment respectively, 10μL of CCK-8 solution was added to each well, and the wells were mixed gently, incubated in the incubator for 2h in the dark, and the absorbance of each well was measured at 450nm using a microplate reader, according to the formula, cell survival rate R(%)=(OD2-OD0) / (OD1-OD0)×100%, the cell survival rate was calculated, wherein 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.

[0145] The results of the proliferation ability test of total paeonol glycosides on SCC7 cells are shown in Figure 5 The results of the proliferation ability test of total paeonol glycosides on FADU cells are shown in Figure 6 The results of the proliferation ability test of total paeonol glycosides on CAL27 cells are shown in Figure 7 When the concentration of total paeonol glycosides was low, the proliferation activity of total paeonol glycosides on Cal27, FADU and SCC7 three head and neck squamous cell carcinoma cell lines showed a significant inhibitory effect compared with the blank control group; the proliferation inhibition effect of high concentration of total paeonol glycosides on Cal27, FADU and SCC7 was more prominent.

[0146] Test example 7: Test of the proliferation rate of total paeonol glycosides on head and neck squamous cell carcinoma cells.

[0147] Test sample: total paeonol glycosides prepared in Example 1.

[0148] Test method: cell proliferation detection kit Click-iT™ Plus EdU Alexa Fluor™ 488 Imaging Kit, Thermo Fisher, C10637 was used to detect cell DNA synthesis activity, which directly reflected cell proliferation; cells were inoculated in a 96-well plate at a density of 1×10 5Cells / well were seeded in 24-well culture medium with crawling slides. After the cells adhered to the wall, the medium was replaced with fresh medium containing low and high concentrations of total glucosides of paeony. The control group was treated with an equal volume of solvent for 48 hours; the total glucosides of paeony low concentration group of SCC7 cells was 400 μg / mL, and the total glucosides of paeony high concentration group was 800 μg / mL; the total glucosides of paeony low concentration group of FADU cells was 500 μg / mL, and the total glucosides of paeony high concentration group was 1000 μg / mL; the total glucosides of paeony low concentration group of CAL27 cells was 800 μg / mL, and the total glucosides of paeony high concentration group was 1600 μg / mL; 2 hours before the end of the treatment, EdU working solution was prepared according to the instructions of the kit, and EdU working solution was added to the culture medium of each well. The cells were returned to the incubator and incubated for another 2 hours. After the EdU incubation was completed, the fixative was added and fixed at room temperature for 15 minutes, and 0.5% Triton was added. Permeabilize cells with X-100-PBS at room temperature for 20 minutes. Prepare the Click reaction mixture according to the kit instructions. Aspirate and discard the wash solution. Add enough Click reaction mixture to cover the cells to each well in the dark. Incubate at room temperature, wash, and counterstain the nuclei. Observe using a confocal microscope. Photograph five randomly selected fields per well. Quantitative analysis was performed using ImageJ. The EdU-positive cell percentage (%) per field was calculated as (number of EdU-positive cells) / (total number of cells) × 100%. Compare the EdU-positive percentages among the different treatment groups.

[0149] The results of double fluorescence staining of CAL27 cells by Hoechst and EdU-488 were shown in Figure 2. Figure 8 As shown in Figure 2, the results of the test on the cell proliferation rate of CAL27 cells by total glucosides of white paeony are as follows: Figure 9 As shown in the figure, the results of double fluorescence staining of Hoechst and EdU-488 on FADU cells by total glucosides of white paeony are shown in the figure. Figure 10 As shown in Figure 2, the results of the test on the cell proliferation rate of FADU cells by total glucosides of white paeony are as follows: Figure 11 As shown in the figure, the results of Hoechst and EdU-488 double fluorescence staining of SCC7 cells by total glucosides of white paeony are shown in the figure. Figure 12 As shown in Figure 2, the results of the test on the cell proliferation rate of SCC7 cells by total glucosides of paeony are as follows: Figure 13 As shown in Figure 3 . After treatment with total glucosides of white paeony, the EdU-positive cell rate in three head and neck squamous cell carcinoma cells, 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 further decreased, and was statistically different from the low-concentration group, suggesting that the drug inhibits cell proliferation in a concentration-dependent manner. Double fluorescence staining results showed no significant change in the total number of Hoechst-stained nuclei, excluding the possibility that the decrease in the total number was due to cytotoxicity, confirming that the decrease in EdU-positive rate was a direct result of suppressed proliferation activity.

[0150] Experimental Example 8: Test of the in vitro clone-forming ability of total glucosides of paeony on head and neck squamous cell carcinoma cells.

[0151] Test sample: total glucosides of paeony prepared in Example 1.

[0152] Test method: Three cell lines CAL27, FADU and SCC7 purchased from Sean Biotechnology were selected. CAL27 and FADU were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and SCC7 was cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, at 37°C in a constant temperature incubator with 5% CO2. The proliferation and adhesion survival ability of cells were evaluated by clone formation experiment. Single cell suspension was inoculated in 6-well plates at 500 μl and complete culture medium was added. After the cells adhered, the culture medium containing total glucosides of white paeony was replaced with fresh culture medium. The culture medium containing corresponding treatment was replaced every 3 days. After 10-14 days of culture, when clone formation was visible to the naked eye, the culture medium was discarded and the cells were fixed with 4% paraformaldehyde for 15 minutes and then stained with 0.1% crystal violet at room temperature for 20 minutes. EPSON The whole-well image was scanned by v850, and the number of clones was calculated using ImageJ. The clone formation rate was calculated according to the formula: R(%) = S1 / S0 × 100%, where S1 is the number of clones formed and S0 is the number of inoculated cells.

[0153] The plate staining results of the effect of total glucosides of paeony on the colony-forming ability of CAL27 cells are shown in Figure 2. Figure 14 As shown in Figure 2, the results of the test on the colony formation rate of CAL27 cells by total glucosides of white paeony are as follows: Figure 15 As shown; the plate staining results of the effect of total glucosides of paeony on the colony formation ability of SCC7 cells are shown Figure 16 As shown in Figure 2, the results of the test on the colony formation rate of SCC7 cells by total glucosides of paeony are as follows: Figure 17 As shown; the plate staining results of the effect of total glucosides of paeony on the clone formation ability of FADU cells are shown Figure 18 As shown in Figure 2, the results of the test on the clone formation rate of FADU cells by total glucosides of white paeony are as follows: Figure 19 After treatment with total glucosides of white paeony, the clone formation rates of CAL27, FADU, and SCC7 cells decreased significantly. The number of clones and the clone formation rate in the low-concentration group were lower than those in the control group. The clone formation rate in the high-concentration group was further reduced, and the clone size was smaller, indicating that the drug not only inhibited the number of clones but also reduced the clone proliferation ability.

[0154] Experimental Example 9: Testing the in vitro migration ability of total glucosides of paeony on head and neck squamous cell carcinoma cells.

[0155] Test sample: total glucosides of paeony prepared in Example 1.

[0156] Test method: Three cell lines, CAL27, FADU, and SCC7, purchased from Shane Biosciences, were selected. CAL27 and FADU were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, while SCC7 was cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a constant temperature incubator containing 5% CO2. The in vitro migration ability of the cells was assessed using a scratch wound test. The cells were plated at a density of 2×10 5 Cells / mL were inoculated into 6-well plates and cultured overnight to form a monolayer. A 200 μL sterile pipette tip was used to make vertical straight scratches on the bottom of the well plate. The cells were gently washed three times with PBS to remove detached cell debris. The culture medium was replaced with 1% FBS, and low-concentration and high-concentration groups of total glucosides of paeony were added. At 0 and 24 h, the scratch area was photographed at 100 times using an inverted phase contrast microscope, and the scratch width was measured using ImageJ software. Each experiment was repeated at least 3 times, and 3-5 fields of view were randomly selected from each well for measurement. The scratch healing rate R(%) was calculated according to the formula: (W0- W1) / W0×100%, where W1 is the scratch width at 24 h and W0 is the scratch width at 0 h.

[0157] The results of the in vitro migration test of total glucosides of paeony on head and neck squamous cell carcinoma cells are as follows Figure 20 The results of the effect of total glucosides of paeony on the migration ability of SCC7 cells in vitro are shown in Figure 21 The results of the effect of total glucosides of paeony on the migration ability of CAL27 cells in vitro are shown in Figure 22 The results of the effect of total glucosides of paeony on the migration ability of FADU cells in vitro are shown in Figure 23 After treatment with total glucosides of white paeony, the 24h scratch healing rate was significantly reduced. The healing rate of the low-concentration group was lower than that of the control group, and the decrease in the high-concentration group was more significant, indicating that the drug can inhibit the lateral migration ability of cells.

[0158] Experimental Example 10: Testing the in vitro migration ability of total glucosides of paeony on head and neck squamous cell carcinoma cells.

[0159] Test sample: total glucosides of paeony prepared in Example 1.

[0160] Test method: Three cell lines, CAL27, FADU and SCC7, purchased from Sean Biosciences, were selected. CAL27 and FADU were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and SCC7 was cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a constant temperature incubator containing 5% CO2. Cells were plated at a density of 5×10 4 / mL cells were resuspended in 200 μL serum-free medium and added to the upper chamber of the Transwell chamber, and 600 μL of 10% FBS complete medium was used; total glucosides of paeony were added to the culture medium in the upper chamber, and the chamber was incubated in a constant temperature incubator with 5% CO2 at 37°C; the low-concentration total glucosides group of SCC7 cells was 400 μg / mL, and the high-concentration total glucosides group was 800 μg / mL; the low-concentration total glucosides group of FADU cells was 500 μg / mL, and the high-concentration total glucosides group was 1000 μg / mL; the low-concentration total glucosides group of CAL27 cells was 800 μg / mL, and the high-concentration total glucosides group was 1600 μg / mL; after incubation, the non-migrated cells on the inner surface of the upper chamber membrane were gently wiped off with a cotton swab, and the cells that migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 20 minutes. Five fields of view were randomly selected and photographed under an inverted microscope at 200x magnification, and the number of migrated cells was counted using ImageJ.

[0161] The results of total glucosides of paeony on the scratching of SCC7 cells are as follows Figure 24 As shown in Figure 2, the results of the in vitro migration ability test of total glucosides of paeony on SCC7 cells are as follows: Figure 25 As shown; the results of total glucosides of white paeony on CAL27 cells scratch Figure 26 As shown in Figure 2, the results of the in vitro migration ability test of total glucosides of paeony on CAL27 cells are as follows: Figure 27 As shown; the results of total glucosides of white paeony on FADU cell scratches are shown as follows Figure 28 As shown in Figure 2, the results of the in vitro migration ability test of total glucosides of paeony on FADU cells are as follows: Figure 29 As shown in Figure 3, the number of cells that migrated to the membrane decreased with increasing drug concentration, and the number of migrated cells in the high-concentration group further decreased, confirming that the drug also has an inhibitory effect on the longitudinal invasion and migration of cells.

[0162] Experimental Example 11: Testing the apoptosis ability of total glucosides of paeony on head and neck squamous cell carcinoma cells.

[0163] Test sample: total glucosides of paeony prepared in Example 1.

[0164] Test method: Cells treated with total glucosides of white paeony for 48 hours were collected and operated according to the instructions of the Annexin V-FITC cell apoptosis detection kit BeyotimeC1062L. The samples were detected on an Attune NxT flow cytometer. FlowJo was used to analyze the data to distinguish between live cells, early apoptotic cells, late apoptotic / necrotic cells, and necrotic cells. According to the formula, the total apoptosis rate R (%) = R1 + R2, where R1 is the apoptosis rate of early apoptotic cells and R2 is the apoptosis rate of late apoptotic cells.

[0165] The flow cytometry results of the total glucosides of white paeony control group on apoptosis of SCC7 cells are shown in Figure 2. Figure 30 The flow scatter results of SCC7 cell apoptosis detection by low concentration group of total paeoniflorin are shown in Figure 6B. Figure 31 The flow scatter results of SCC7 cell apoptosis detection by high concentration group of total paeoniflorin are shown in Figure 6C. Figure 32 The apoptosis ability test results of SCC7 cells by total paeoniflorin are shown in Figure 6D. Figure 33 The flow scatter results of CAL27 cell apoptosis detection by control group of total paeoniflorin are shown in Figure 7B. Figure 34 The flow scatter results of CAL27 cell apoptosis detection by low concentration group of total paeoniflorin are shown in Figure 7C. Figure 35 The flow scatter results of CAL27 cell apoptosis detection by high concentration group of total paeoniflorin are shown in Figure 7D. Figure 36 The apoptosis ability test results of CAL27 cells by total paeoniflorin are shown in Figure 7E. Figure 37 The flow scatter results of FADU cell apoptosis detection by control group of total paeoniflorin are shown in Figure 8B. Figure 38 The flow scatter results of FADU cell apoptosis detection by low concentration group of total paeoniflorin are shown in Figure 8C. Figure 39 The flow scatter results of FADU cell apoptosis detection by high concentration group of total paeoniflorin are shown in Figure 8D. Figure 40 The apoptosis ability test results of FADU cells by total paeoniflorin are shown in Figure 8E. Figure 41 After treatment of total paeoniflorin, the total apoptosis rate of head and neck squamous cell carcinoma cells was significantly increased, the total apoptosis rate of the low concentration group was higher than that of the control group, and the apoptosis rate of the high concentration group was further increased; it is proved that total paeoniflorin can dose-dependently induce apoptosis of head and neck squamous cell carcinoma cells, and enhance cell death program by activating apoptosis pathway, which provides direct evidence for its anti-tumor effect.

[0166] The above-described embodiments and / or implementations are merely used to illustrate the preferred embodiments and / or implementations of the present application, and are not intended to limit the embodiments of the present application in any form. Any person skilled in the art can make some changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the present application, and such changes or modifications should be considered as substantially the same technology or embodiments as the present application.

[0167] The principles and implementations of the present application are described by using specific examples. The above description of the embodiments is only used to help understand the method and its core idea of the present application. The above description is only the preferred embodiments of the present application. It should be pointed out that due to the limitation of language expression, there are infinite specific structures. For ordinary skilled in the art, some improvements, refinements or changes can be made without departing from the principles of the present application. The above technical features can be combined in an appropriate manner. These improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solutions to other occasions without improvement, should be considered as the protection scope of the present application.

Claims

1. A drug for treating head and neck squamous cell carcinoma, characterized in that: The medicine comprises total glucosides of white paeony and hydrogel, and the mass ratio of the total glucosides of white paeony to the hydrogel is 80-100 mg:10 g.

2. A drug for treating head and neck squamous cell carcinoma according to claim 1, characterized in that: The hydrogel comprises modified chitosan and cellulose, and the mass ratio of the modified chitosan to the cellulose is 3-7:

5.

3. A drug for treating head and neck squamous cell carcinoma according to claim 2, characterized in that: The modifier in the modified chitosan includes epoxy acrolein diethanol acetal and hexanoic acid oxirane methyl ester, the mass ratio of epoxy acrolein diethanol acetal to chitosan is 1-4:2, and the mass ratio of hexanoic acid oxirane methyl ester to chitosan is 1-4:

2.

4. The drug for treating head and neck squamous cell carcinoma according to claim 1, wherein: The total glucosides of white paeony are extracted by heating and refluxing white paeony in an extracting solution, wherein the extracting solution is a 65-75 v / v% ethanol aqueous solution, and the mass volume ratio of the white paeony to the extracting solution is 300-800 g:5 L.

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

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

1.

7. The drug for treating head and neck squamous cell carcinoma according to claim 1, characterized in that: The drug is used for inhibiting the proliferation of head and neck squamous cell carcinoma cells, wherein the head and neck squamous cell carcinoma cells include at least one of SCC7 cells, FADU cells and CAL27 cells.

8. The drug for treating head and neck squamous cell carcinoma according to claim 1, characterized in that: The drug is used to reduce the clone-forming ability of head and neck squamous cell carcinoma cells, and the head and neck squamous cell carcinoma cells include at least one of SCC7 cells, FADU cells and CAL27 cells.

9. The drug for treating head and neck squamous cell carcinoma according to claim 1, characterized in that: The drug is used for inhibiting the in vitro migration ability of head and neck squamous cell carcinoma cells, wherein the head and neck squamous cell carcinoma cells include at least one of SCC7 cells, FADU cells and CAL27 cells.

10. The drug for treating head and neck squamous cell carcinoma according to claim 1, characterized in that: The drug is used to induce apoptosis of head and neck squamous cell carcinoma cells, and the head and neck squamous cell carcinoma cells include at least one of SCC7 cells, FADU cells and CAL27 cells.

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