A polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol, its preparation method and application

Abstract

This invention discloses a method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol. First, a kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex and a furfural-functionalized chitosan-mannose graft polymer are prepared separately. Then, the furfural-functionalized chitosan-mannose graft polymer and the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex undergo a cross-linking reaction to generate a hydrogel. This hydrogel is then mixed with rhubarb nanovesicles and incubated to obtain the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol. This invention also discloses the application of this polysaccharide hydrogel in the preparation of oral medications for treating ulcerative colitis. After oral administration, this polysaccharide hydrogel is not degraded in the gastrointestinal tract, exhibits dual targeting to the colon and macrophages, enabling precise drug delivery to the site of colonic inflammation. It also demonstrates good biocompatibility, low toxicity, and can significantly alleviate ulcerative colitis symptoms, reduce colonic atrophy, and mitigate colonic tissue damage.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis is a chronic, nonspecific inflammatory disease characterized by diarrhea, abdominal pain, bloody stools, and tenesmus. It commonly affects the rectal and colonic mucosa or submucosa, is prone to recurrence, and can potentially lead to cancer. It is listed by the World Health Organization as one of the most difficult-to-treat diseases. Currently, the main medications used clinically to treat ulcerative colitis include aminosalicylic acids, immunosuppressants, and glucocorticoids. These drugs offer significant short-term efficacy and high remission rates, but they also have drawbacks such as numerous adverse reactions, difficulty in achieving a cure, and strong toxic side effects with long-term use.

[0003] Oral administration is a highly compliant method of drug delivery for the treatment of ulcerative colitis, with advantages such as convenient administration, non-invasiveness, low infection rate, and low systemic toxicity. However, the design of oral drug delivery systems faces some limitations, such as low drug solubility and poor permeability, as well as enzymatic degradation of the gastrointestinal barrier and the mucus barrier.

[0004] Plant nanovesicles are membrane-bound vesicles with a lipid bilayer as their basic framework, carrying characteristic biological information molecules such as proteins, lipids, DNA, and miRNAs. Due to their natural composition, they have good biocompatibility and are not detected by the immune system. Researchers have demonstrated that the biological information contained in plant nanovesicles has a good therapeutic effect on ulcerative colitis. However, after oral administration, they can be destroyed by the harsh environment of the gastrointestinal tract, reducing their retention rate in the body and slowing down the therapeutic effect. Targeted delivery of plant nanovesicles using carriers can effectively solve the above problems. For example, encapsulating exosome-like nanovesicles with chitosan / β-glycerophosphate thermosensitive hydrogel can prolong their retention time in the body (Liu Feng, Zhang Yu, Wang Yanli, et al. Application of thermosensitive chitosan hydrogel encapsulation of exosomes in ischemic diseases [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(16):2479-2487).

[0005] Kaempferol is a flavonoid compound widely distributed in various plants. Studies have shown that it can improve the clinical symptoms of ulcerative colitis through multiple pathways, such as reducing the release of inflammatory factors and protecting the intestinal mucosal mechanical barrier. However, its extremely low solubility greatly affects its absorption and utilization in the human body, limiting its clinical application. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0007] Another object of the present invention is to provide the application of the above-mentioned polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol in the preparation of an oral medicament for treating ulcerative colitis.

[0008] Technical solution

[0009] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0010] (1) Hydroxypropyl-β-cyclodextrin and 3-maleimide propionic acid in a molar ratio of 1:5 were dissolved in anhydrous N,N-dimethylformamide and mixed evenly. Then, an anhydrous N,N-dimethylformamide solution of dicyclohexylcarbodiimide was added dropwise under stirring. After the reaction was carried out at -2℃, dimethylaminopyridine was added to continue the reaction. After the reaction was completed, the reaction solution was filtered and the filtrate was transferred to anhydrous diethyl ether. After standing treatment, the precipitate was dried to obtain 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0011] (2) Dissolve 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add kaempferol methanol solution dropwise under stirring to react, remove methanol by rotary evaporation under reduced pressure after reaction, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0012] (3) Dissolve the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain a kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve the furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. After standing for 20-30 hours, a polysaccharide hydrogel loaded with kaempferol is obtained.

[0013] The preparation method of furfural-functionalized chitosan-mannose is as follows: Chitosan-mannose graft polymer is dissolved in 2% glacial acetic acid aqueous solution, diluted with anhydrous methanol to obtain chitosan-mannose graft polymer solution. Under magnetic stirring, anhydrous methanol solution of furfural is added dropwise to chitosan-mannose graft polymer solution. The molar ratio of furfural to chitosan-mannose graft polymer is 3:1. After the reaction, methanol solution of sodium cyanoborohydride is added and the reaction is continued with stirring. The molar ratio of sodium cyanoborohydride to chitosan-mannose graft polymer is 4:1. After the reaction is completed, ammonia solution is added to adjust the pH to 11-12. Then the product is filtered and washed successively with deionized water, a mixture of water and ethanol and anhydrous ethanol. After filtration and drying, a light yellow crude product is obtained. After purification, furfural-functionalized chitosan-mannose graft polymer is obtained.

[0014] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles evenly, incubate at 4℃ for 20-30h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0015] In step (3), the preparation method of furfural-functionalized chitosan-mannose is as follows: Chitosan-mannose graft polymer is dissolved in 2% glacial acetic acid aqueous solution, diluted with anhydrous methanol to obtain chitosan-mannose graft polymer solution. Under magnetic stirring, anhydrous methanol solution of furfural is added dropwise to chitosan-mannose graft polymer solution. The molar ratio of furfural to chitosan-mannose graft polymer is 3:1. After the reaction, methanol solution of sodium cyanoborohydride is added and the reaction is continued with stirring. The molar ratio of sodium cyanoborohydride to chitosan-mannose graft polymer is 4:1. After the reaction is completed, ammonia solution is added to adjust the pH to 11-12. Then the product is filtered and washed successively with deionized water, a mixture of water and ethanol and anhydrous ethanol. After filtration and drying, a light yellow crude product is obtained. After purification, furfural-functionalized chitosan-mannose graft polymer is obtained.

[0016] Furthermore, in step (1), the molar ratio of dicyclohexylcarbodiimide to 3-maleimide propionic acid is (1.3-1.5):1, and the molar ratio of dimethylaminopyridine to 3-maleimide propionic acid is 0.05:1.

[0017] Furthermore, in step (2), the reaction temperature is 60°C and the time is 3-6 hours.

[0018] Furthermore, in step (2), the molar ratio of 3-maleimide-hydroxypropyl-β-cyclodextrin to kaempferol is 1:(3-5).

[0019] Furthermore, in step (3), the molar ratio of the 3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex to furfural-functionalized chitosan-mannose is 1:(2-5).

[0020] Further, in step (3), the preparation method of the chitosan-mannose graft polymer is as follows: mannose and chitosan with a molar ratio of 1:3 are dissolved in water, stirred evenly, and sodium cyanoborohydride is added. The molar ratio of sodium cyanoborohydride to mannose is (1.5-2):1. The mixture is stirred and reacted at room temperature for 40-50 hours. The obtained product is dialyzed in water and then freeze-dried to obtain the chitosan-mannose graft polymer.

[0021] Furthermore, in step (4), the mass ratio of the rhubarb nanovesicles to the polysaccharide hydrogel loaded with kaempferol is (10-120):1.

[0022] Further, in step (4), the preparation method of the rhubarb nanovesicles is as follows: Fresh rhubarb is chopped or rhubarb slices are soaked in 0.01M, pH 7.4 phosphate buffer and the juice is squeezed out. Then, the mixture is centrifuged at 3000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min in sequence. The supernatant is taken and centrifuged at 150000×g for 2 h. The precipitate is suspended in 0.01M, pH 7.4 phosphate buffer and then transferred to a gradient sucrose solution and centrifuged at 150000×g for 1 h. The yellow bands between the gradient sucrose solutions are collected. An equal amount of 0.01M, pH 7.4 phosphate buffer is added and centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

[0023] Take fresh rhubarb, chop it or use rhubarb slices, soak it in 0.01M, pH 7.4 phosphate buffer, squeeze out the juice, then centrifuge sequentially at 3000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. Take the supernatant and centrifuge at 150000×g for 2 h. Resuspend the precipitate in 0.01M, pH 7.4 phosphate buffer, then transfer it to a gradient sucrose solution and centrifuge at 150000×g for 1 h. Collect the yellow bands between the gradient sucrose solutions, add an equal volume of 0.01M, pH 7.4 phosphate buffer, and centrifuge again at 150000×g for 1 h to wash away the sucrose solution, obtaining rhubarb nanovesicles.

[0024] A polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol was prepared using the above method.

[0025] The above-mentioned polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol is used in the preparation of oral medications for the treatment of ulcerative colitis.

[0026] The beneficial effects of this invention are:

[0027] 1. The polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol of the present invention is prepared by cross-linking furfural-functionalized chitosan-mannose grafted polymer and 3-maleimide-hydroxypropyl-β-cyclodextrin. Chitosan is a natural polycationic polysaccharide that remains stable in the stomach and small intestine, and is degraded by specific enzymes produced by the colonic microbiota upon reaching the colon. Its strong positive charge can effectively adsorb negatively charged rhubarb nanovesicles, thereby improving the stability and retention rate of rhubarb nanovesicles in vivo after oral administration. Mannose is a monosaccharide that can be recognized and mediated by mannose receptors on the surface of macrophages. After being grafted with chitosan to form a polymer, it can specifically target macrophages. This is a novel strategy for targeted delivery of inflammatory drugs based on the macrophage recruitment effect at the site of inflammation. Hydroxypropyl-β-cyclodextrin, as the other main component of the polysaccharide hydrogel, utilizes its unique hydrophobic cavity structure to encapsulate the poorly soluble small molecule drug kaempferol, forming a host-guest inclusion complex, thereby effectively improving the solubility of the hydrophobic drug kaempferol.

[0028] 2. The polysaccharide hydrogel of the present invention has dual targeting properties to the colon and macrophages, enabling precise drug delivery to the site of colonic inflammation. It also has good biocompatibility and low toxicity, and can significantly relieve the symptoms of ulcerative colitis, alleviate colonic atrophy, and reduce colonic tissue damage.

[0029] 3. The polysaccharide hydrogel of the present invention is not degraded by the gastrointestinal tract after oral administration. It effectively accumulates in the colon and is degraded by glycosidases and other enzymes specific to the colon. Mannose is recognized by macrophages overexpressed in the inflamed areas of the colon, thereby breaking down the hydrogel structure and rapidly releasing rhubarb nanovesicles and kaempferol. This combination therapy enhances the therapeutic effect on ulcerative colitis and reduces toxic side effects. Attached Figure Description

[0030] Figure 1 Transmission electron microscopy image of rhubarb nanovesicles prepared in Example 3;

[0031] Figure 2 The infrared spectrum of the chitosan-mannose grafted polymer prepared in Example 3;

[0032] Figure 3 The infrared spectrum of 3-maleimide-hydroxypropyl-β-cyclodextrin prepared in Example 3;

[0033] Figure 4 The infrared spectrum of the furfural-functionalized chitosan-mannose grafted polymer prepared in Example 3;

[0034] Figure 5 The 1H NMR spectrum of 3-maleimide-hydroxypropyl-β-cyclodextrin prepared in Example 3;

[0035] Figure 6The 1H NMR spectrum of the furfural-functionalized chitosan-mannose graft polymer prepared in Example 3;

[0036] Figure 7 Differential calorimetry scan of the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex prepared in Example 3;

[0037] Figure 8 The HPLC chromatogram of the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex prepared in Example 3;

[0038] Figure 9 Scanning electron microscope (SEM) images of the polysaccharide hydrogel prepared in Example 3 and the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol;

[0039] Figure 10 The results of the in vitro degradation rate test of the polysaccharide hydrogel prepared in Example 3;

[0040] Figure 11 The in vitro release curve of the polysaccharide hydrogel loaded with kaempferol prepared in Example 3;

[0041] Figure 12 The results of cytotoxicity tests on RAW264.7 and CT26 cells at different concentrations of the polysaccharide hydrogel prepared in Example 3 are shown.

[0042] Figure 13 The colon length of mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 3;

[0043] Figure 14 HE staining image of mice after treatment with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 3;

[0044] Figure 15 The image shows the myeloperoxidase activity of mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 3.

[0045] Figure 16 The colon length of mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 1;

[0046] Figure 17 The image shows the myeloperoxidase activity of mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 4. Detailed Implementation

[0047] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0048] Example 1

[0049] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0050] (1) Take 10g of hydroxypropyl-β-cyclodextrin and 5.48g of 3-maleimide propionic acid and dissolve them in 200mL of anhydrous N,N-dimethylformamide. After mixing evenly, add 30mL of anhydrous N,N-dimethylformamide solution containing 8.71g of dicyclohexylcarbodiimide dropwise while stirring. After reacting at -2℃ for 1h, add 10mL of anhydrous N,N-dimethylformamide solution containing 0.19g of dimethylaminopyridine dropwise and continue the reaction. After the reaction is completed, filter the reaction solution and transfer the filtrate to anhydrous diethyl ether. After standing for half an hour, take the precipitate layer and dry it in a vacuum drying oven at 50℃ to obtain a pink powder, which is 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0051] (2) Dissolve 5.3g of 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add 0.2g of kaempferol in methanol solution dropwise while stirring, stir and react at 60℃ for 4h, remove methanol by rotary evaporation, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0052] (3) Dissolve 1g of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve 2.7g of furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. After standing at room temperature for 24h, kaempferol-loaded polysaccharide hydrogel is obtained.

[0053] The preparation method of the furfural-functionalized chitosan-mannose graft polymer:

[0054] 1) Dissolve 3.60g of mannose and 9.67g of chitosan in water, stir evenly, add 1.13g of sodium cyanoborohydride, stir and react at room temperature for 48h, dialyze the obtained product in water for 48h (MW: 3500), and then freeze dry to obtain chitosan-mannose graft polymer.

[0055] 2) Dissolve 3g of chitosan-mannose graft polymer in 100mL of 2% (w / v) glacial acetic acid aqueous solution, and dilute with 50mL of anhydrous methanol to obtain chitosan-mannose graft polymer solution. Dissolve 5.4g of furfural in anhydrous methanol and slowly add it dropwise to the chitosan-mannose graft polymer solution under magnetic stirring at 25℃. After stirring for 5h, add 60mL of methanol solution containing 3.06g of sodium cyanoborohydride and continue stirring for 1.5h. Then add 3mol / L ammonia solution to adjust the pH to 11-12. Filter the product and wash it successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. Redissolve the crude product and dialyze it in deionized water for 72h (MW: 3500) to remove unreacted furfural. Freeze-dry to obtain furfural-functionalized chitosan-mannose graft polymer.

[0056] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles at a mass ratio of 1:120, the mixture was incubated at 4°C for 24 h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0057] The preparation method of rhubarb nanovesicles is as follows: Rhubarb slices are soaked in 0.01M, pH 7.4 phosphate buffer for 48 hours, and the juice is squeezed out. The juice is centrifuged sequentially at 3000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. The supernatant is taken and subjected to ultracentrifugation at 150000×g (Optima L-100XP) for 2 h. The resulting precipitate is suspended in 1 mL of 0.01M, pH 7.4 phosphate buffer, and then transferred to sucrose solutions of four concentrations (8%, 30%, 45%, and 60%). The solutions are centrifuged at 150000×g for 1 h. The yellow bands between 30% and 45% sucrose concentrations are collected. An equal volume of 0.01M, pH 7.4 phosphate buffer is added, and the solution is washed away by centrifugation at 150000×g for 1 h to obtain rhubarb nanovesicles.

[0058] Example 2

[0059] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0060] (1) Take 5g of hydroxypropyl-β-cyclodextrin and 2.74g of 3-maleimide propionic acid and dissolve them in 100mL of anhydrous N,N-dimethylformamide. After mixing evenly, add 20mL of anhydrous N,N-dimethylformamide solution containing 4.35g of dicyclohexylcarbodiimide dropwise while stirring. After reacting at -2℃ for 1.5h, add 5mL of anhydrous N,N-dimethylformamide solution containing 0.09g of dimethylaminopyridine dropwise and continue the reaction. After the reaction is completed, filter the reaction solution and transfer the filtrate to anhydrous diethyl ether. After standing for half an hour, take the precipitate layer and dry it in a vacuum drying oven at 50℃ to obtain a pink powder, which is 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0061] (2) Dissolve 2.65g of 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add 0.1g of kaempferol methanol solution dropwise while stirring, stir and react at 60℃ for 3h, remove methanol by rotary evaporation, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0062] (3) Dissolve 1g of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve 3.6g of furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution evenly. After standing at room temperature for 24h, kaempferol-loaded polysaccharide hydrogel is obtained.

[0063] The preparation method of the furfural-functionalized chitosan-mannose graft polymer:

[0064] 1) Dissolve 3g of mannose and 8.06g of chitosan in water, stir evenly, add 1.26g of sodium cyanoborohydride, stir and react at room temperature for 48h, dialyze the obtained product in water for 48h (MW: 3500), and then freeze dry to obtain chitosan-mannose graft polymer.

[0065] 2) Dissolve 2g of chitosan-mannose graft polymer in 80mL of 2% (w / v) glacial acetic acid aqueous solution, and dilute with 30mL of anhydrous methanol to obtain a chitosan-mannose graft polymer solution. Dissolve 3.6g of furfural in anhydrous methanol and slowly add it dropwise to the chitosan-mannose graft polymer solution under magnetic stirring at 25℃. After stirring for 5h, add 50mL of methanol solution containing 2.04g of sodium cyanoborohydride and continue stirring for 1.5h. After the reaction is completed, add 3mol / L ammonia solution to adjust the pH to 11-12. Then filter the product and wash it successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. The crude product is purified by Soxhlet extraction with diethyl ether for 48h to remove unreacted furfural. After freeze drying, furfural-functionalized chitosan-mannose graft polymer is obtained.

[0066] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles at a mass ratio of 1:60, the mixture was incubated at 4°C for 24 h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0067] The preparation method of rhubarb nanovesicles is as follows: Fresh rhubarb is chopped and soaked in an equal volume of 0.01M, pH 7.4 phosphate buffer. The juice is then squeezed out and centrifuged sequentially at 3000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. The supernatant is then subjected to ultracentrifugation at 150000×g (Optima L-100XP) for 2 h. The resulting precipitate is suspended in 1 mL of 0.01M, pH 7.4 phosphate buffer and then transferred to sucrose solutions of four concentrations (8%, 30%, 45%, and 60%). The solutions are centrifuged at 150000×g for 1 h. Yellow bands between 30% and 45% sucrose concentrations are collected. An equal volume of 0.01M, pH 7.4 phosphate buffer is added, and the solutions are centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

[0068] Example 3

[0069] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0070] (1) Take 2.5g of hydroxypropyl-β-cyclodextrin and 1.37g of 3-maleimide propionic acid and dissolve them in 50mL of anhydrous N,N-dimethylformamide. After mixing evenly, add 20mL of anhydrous N,N-dimethylformamide solution containing 2.17g of dicyclohexylcarbodiimide dropwise while stirring. After reacting at -2℃ for 3h, add 3mL of anhydrous N,N-dimethylformamide solution containing 45mg of dimethylaminopyridine dropwise and continue the reaction. After the reaction is completed, filter the reaction solution and transfer the filtrate to anhydrous diethyl ether. After standing for half an hour, take the precipitate layer and dry it in a vacuum drying oven at 50℃ to obtain a pink powder, which is 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0071] (2) Dissolve 2g of 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add a methanol solution containing 80mg of kaempferol dropwise while stirring, stir and react at 60℃ for 4h, remove methanol by rotary evaporation, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0072] (3) Dissolve 500 mg of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve 1.35 g of furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. After standing at room temperature for 24 h, kaempferol-loaded polysaccharide hydrogel is obtained.

[0073] The preparation method of the furfural-functionalized chitosan-mannose graft polymer:

[0074] 1) Dissolve 2g of mannose and 5.37g of chitosan in water, stir evenly, add 626mg of sodium cyanoborohydride, stir and react at room temperature for 48h, dialyze the obtained product in water for 48h (MW: 3500), and then freeze dry to obtain chitosan-mannose graft polymer.

[0075] 2) Dissolve 1.5 g of chitosan-mannose graft polymer in 60 mL of 2% (w / v) glacial acetic acid aqueous solution, and dilute with 20 mL of anhydrous methanol to obtain a chitosan-mannose graft polymer solution. Dissolve 2.68 g of furfural in anhydrous methanol and slowly add it dropwise to the chitosan-mannose graft polymer solution under magnetic stirring at 25 °C. After stirring for 5 h, add 30 mL of methanol solution containing 1.52 g of sodium cyanoborohydride and continue stirring for 1.5 h. After the reaction is completed, add 3 mol / L ammonia solution to adjust the pH to 11-12. Then filter the product and wash it successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. Redissolve the crude product and dialyze it in deionized water for 72 h (MW: 3500) to remove unreacted furfural. Freeze-dry to obtain furfural-functionalized chitosan-mannose graft polymer.

[0076] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles at a mass ratio of 1:90, the mixture was incubated at 4°C for 24 h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0077] The preparation method of rhubarb nanovesicles is as follows: Rhubarb slices are soaked in 0.01M, pH 7.4 phosphate buffer for 48 hours, and the juice is squeezed out. The juice is centrifuged sequentially at 1000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. The supernatant is taken and subjected to ultracentrifugation at 150000×g (Optima L-100XP) for 2 h. The resulting precipitate is suspended in 1 mL of 0.01M, pH 7.4 phosphate buffer, and then transferred to sucrose solutions of four concentrations (8%, 30%, 45%, and 60%). The solutions are centrifuged at 150000×g for 1 h. The yellow bands between 30% and 45% sucrose concentrations are collected. An equal volume of 0.01M, pH 7.4 phosphate buffer is added, and the solutions are centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

[0078] Example 4

[0079] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0080] (1) Take 1.5g of hydroxypropyl-β-cyclodextrin and 0.82g of 3-maleimide propionic acid and dissolve them in 30mL of anhydrous N,N-dimethylformamide. After mixing evenly, add 15mL of anhydrous N,N-dimethylformamide solution containing 1.30g of dicyclohexylcarbodiimide dropwise while stirring. After reacting at -2℃ for 1h, add 2mL of anhydrous N,N-dimethylformamide solution containing 27mg of dimethylaminopyridine dropwise and continue the reaction for 10h. After the reaction is completed, filter the reaction solution and transfer the filtrate to anhydrous diethyl ether. After standing for half an hour, take the precipitate layer and dry it in a vacuum drying oven at 50℃ to obtain a pink powder, which is 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0081] (2) Dissolve 1g of 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add a methanol solution containing 60mg of kaempferol dropwise while stirring, stir and react at 60℃ for 5h, remove methanol by rotary evaporation, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0082] (3) Dissolve 500 mg of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve 1.8 g of furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. After standing at room temperature for 24 h, kaempferol-loaded polysaccharide hydrogel is obtained.

[0083] The preparation method of the furfural-functionalized chitosan-mannose graft polymer:

[0084] 1) Dissolve 1.8g of mannose and 4.84g of chitosan in water, stir evenly, add 563mg of sodium cyanoborohydride, stir and react at room temperature for 48h, dialyze the obtained product in water for 48h (MW: 3500), and then freeze dry to obtain chitosan-mannose graft polymer.

[0085] 2) Dissolve 1 g of chitosan-mannose graft polymer in 40 mL of 2% (w / v) glacial acetic acid aqueous solution, and dilute with 20 mL of anhydrous methanol to obtain a chitosan-mannose graft polymer solution. Dissolve 1.8 g of furfural in anhydrous methanol and slowly add it dropwise to the chitosan-mannose graft polymer solution under magnetic stirring at 25 °C. After stirring for 5 h, add 25 mL of methanol solution containing 1.02 g of sodium cyanoborohydride and continue stirring for 1.5 h. After the reaction is completed, add 3 mol / L ammonia solution to adjust the pH to 11-12. Then filter the product and wash it successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. Redissolve the crude product and dialyze it in deionized water for 72 h (MW: 3500) to remove unreacted furfural. Freeze-dry to obtain furfural-functionalized chitosan-mannose graft polymer.

[0086] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles at a mass ratio of 1:100, the mixture was incubated at 4°C for 24 h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0087] The preparation method of rhubarb nanovesicles is as follows: Rhubarb slices are soaked in 0.01M, pH 7.4 phosphate buffer for 48 hours, and the juice is squeezed out. The juice is centrifuged sequentially at 1000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. The supernatant is taken and subjected to ultracentrifugation at 150000×g (Optima L-100XP) for 2 h. The resulting precipitate is suspended in 1 mL of 0.01M, pH 7.4 phosphate buffer, and then transferred to sucrose solutions of four concentrations (8%, 30%, 45%, and 60%). The solutions are centrifuged at 150000×g for 1 h. The yellow bands between 30% and 45% sucrose concentrations are collected. An equal volume of 0.01M, pH 7.4 phosphate buffer is added, and the solutions are centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

[0088] Example 5

[0089] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0090] (1) Take 1g of hydroxypropyl-β-cyclodextrin and 548mg of 3-maleimide propionic acid and dissolve them in 20mL of anhydrous N,N-dimethylformamide. After mixing evenly, add 10mL of anhydrous N,N-dimethylformamide solution containing 871mg of dicyclohexylcarbodiimide dropwise while stirring. After reacting at -2℃ for 1h, add 1mL of anhydrous N,N-dimethylformamide solution containing 19mg of dimethylaminopyridine dropwise and continue the reaction for 10h. After the reaction is completed, filter the reaction solution and transfer the filtrate to anhydrous diethyl ether. After standing for half an hour, take the precipitate layer and dry it in a vacuum drying oven at 50℃ to obtain a pink powder, which is 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0091] (2) Dissolve 1g of 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add a methanol solution containing 60mg of kaempferol dropwise while stirring, stir and react at 60℃ for 4h, remove methanol by rotary evaporation, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0092] (3) Dissolve 250 mg of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve 675 mg of furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. After standing at room temperature for 24 h, kaempferol-loaded polysaccharide hydrogel is obtained.

[0093] The preparation method of the furfural-functionalized chitosan-mannose graft polymer:

[0094] 1) Dissolve 1g of mannose and 2.69g of chitosan in water, stir well, add 420mg of sodium cyanoborohydride, stir and react at room temperature for 48h, dialyze the obtained product in water for 48h (MW: 3500), and then freeze dry to obtain chitosan-mannose graft polymer.

[0095] 2) Dissolve 800 mg of chitosan-mannose graft polymer in 40 mL of 2% (w / v) glacial acetic acid aqueous solution, and dilute with 20 mL of anhydrous methanol to obtain a chitosan-mannose graft polymer solution. Dissolve 1.44 g of furfural in anhydrous methanol and slowly add it dropwise to the chitosan-mannose graft polymer solution under magnetic stirring at 25 °C. After stirring for 5 h, add 30 mL of methanol solution containing 816 mg of sodium cyanoborohydride and continue stirring for 1.5 h. After the reaction is completed, add 3 mol / L ammonia solution to adjust the pH to 11-12. Then filter the product and wash it successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. Redissolve the crude product and dialyze it in deionized water for 72 h (MW: 3500) to remove unreacted furfural. Freeze-dry to obtain furfural-functionalized chitosan-mannose graft polymer.

[0096] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles at a mass ratio of 1:80, the mixture was incubated at 4°C for 24 h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0097] The preparation method of rhubarb nanovesicles is as follows: Rhubarb slices are soaked in 0.01M, pH 7.4 phosphate buffer for 48 hours, and the juice is squeezed out. The juice is centrifuged sequentially at 1000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. The supernatant is taken and subjected to ultracentrifugation at 150000×g (Optima L-100XP) for 2 h. The resulting precipitate is suspended in 1 mL of 0.01M, pH 7.4 phosphate buffer, and then transferred to sucrose solutions of four concentrations (8%, 30%, 45%, and 60%). The solutions are centrifuged at 150000×g for 1 h. The yellow bands between 30% and 45% sucrose concentrations are collected. An equal volume of 0.01M, pH 7.4 phosphate buffer is added, and the solutions are centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

[0098] Example 6

[0099] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0100] (1) Dissolve 700 mg of hydroxypropyl-β-cyclodextrin and 383 mg of 3-maleimide propionic acid in 16 mL of anhydrous N,N-dimethylformamide. After mixing evenly, add 6 mL of anhydrous N,N-dimethylformamide solution containing 609 mg of dicyclohexylcarbodiimide dropwise while stirring. After reacting at -2℃ for 1 h, add 1 mL of anhydrous N,N-dimethylformamide solution containing 13 mg of dimethylaminopyridine dropwise and continue the reaction for 10 h. After the reaction is completed, filter the reaction solution and transfer the filtrate to anhydrous diethyl ether. After standing for half an hour, take the precipitate layer and dry it in a vacuum drying oven at 50℃ to obtain a pink powder, which is 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0101] (2) Dissolve 500 mg of 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add a methanol solution containing 20 mg of kaempferol dropwise while stirring, stir and react at 60 °C for 5 h, remove methanol by rotary evaporation, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0102] (3) Dissolve 500 mg of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve 1.8 g of furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. After standing at room temperature for 24 h, kaempferol-loaded polysaccharide hydrogel is obtained.

[0103] The preparation method of the furfural-functionalized chitosan-mannose graft polymer:

[0104] 1) Dissolve 0.8g of mannose and 2.15g of chitosan in water, stir evenly, add 250mg of sodium cyanoborohydride, stir and react at room temperature for 48h, dialyze the obtained product in water for 48h (MW: 3500), and then freeze dry to obtain chitosan-mannose graft polymer.

[0105] 2) Dissolve 500 mg of chitosan-mannose graft polymer in 30 mL of 2% (w / v) glacial acetic acid aqueous solution, and dilute with 20 mL of anhydrous methanol to obtain a chitosan-mannose graft polymer solution. Dissolve 0.9 g of furfural in anhydrous methanol and slowly add it dropwise to the chitosan-mannose graft polymer solution under magnetic stirring at 25 °C. After stirring for 5 h, add 30 mL of methanol solution containing 0.51 g of sodium cyanoborohydride and continue stirring for 1.5 h. After the reaction is completed, add 3 mol / L ammonia solution to adjust the pH to 11-12. Then filter the product and wash it successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. This crude product is purified by Soxhlet extraction with diethyl ether for 48 h to remove unreacted furfural. After freeze drying, furfural-functionalized chitosan-mannose graft polymer is obtained.

[0106] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles at a mass ratio of 1:30, the mixture was incubated at 4°C for 24 h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0107] The preparation method of rhubarb nanovesicles is as follows: Rhubarb slices are soaked in 0.01M, pH 7.4 phosphate buffer for 48 hours, and the juice is squeezed out. The juice is centrifuged sequentially at 1000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. The supernatant is taken and subjected to ultracentrifugation at 150000×g (Optima L-100XP) for 2 h. The resulting precipitate is suspended in 1 mL of 0.01M, pH 7.4 phosphate buffer, and then transferred to sucrose solutions of four concentrations (8%, 30%, 45%, and 60%). The solutions are centrifuged at 150000×g for 1 h. The yellow bands between 30% and 45% sucrose concentrations are collected. An equal volume of 0.01M, pH 7.4 phosphate buffer is added, and the solutions are centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

[0108] Example 7

[0109] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0110] (1) Dissolve 500 mg of hydroxypropyl-β-cyclodextrin and 274 mg of 3-maleimide propionic acid in 15 mL of anhydrous N,N-dimethylformamide. After mixing evenly, add 5 mL of anhydrous N,N-dimethylformamide solution containing 435 mg of dicyclohexylcarbodiimide dropwise while stirring. After reacting at -2℃ for 2 h, add 1 mL of anhydrous N,N-dimethylformamide solution containing 10 mg of dimethylaminopyridine dropwise and continue the reaction for 10 h. After the reaction is completed, filter the reaction solution and transfer the filtrate to anhydrous diethyl ether. After standing for half an hour, take the precipitate layer and dry it in a vacuum drying oven at 50℃ to obtain a pink powder, which is 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0111] (2) Dissolve 500 mg of 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add 30 mg of kaempferol in methanol solution dropwise while stirring, stir and react at 60 °C for 3 h, remove methanol by rotary evaporation, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0112] (3) Dissolve 250 mg of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve 0.9 g of furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. After standing at room temperature for 24 h, kaempferol-loaded polysaccharide hydrogel is obtained.

[0113] The preparation method of the furfural-functionalized chitosan-mannose graft polymer:

[0114] 1) Dissolve 0.5g of mannose and 1.34g of chitosan in water, stir evenly, add 209mg of sodium cyanoborohydride, stir and react at room temperature for 48h, dialyze the obtained product in water for 48h (MW: 3500), and then freeze dry to obtain chitosan-mannose graft polymer.

[0115] 2) Dissolve 500 mg of chitosan-mannose graft polymer in 30 mL of 2% (w / v) glacial acetic acid aqueous solution, and dilute with 20 mL of anhydrous methanol to obtain a chitosan-mannose graft polymer solution. Dissolve 0.9 g of furfural in anhydrous methanol and slowly add it dropwise to the chitosan-mannose graft polymer solution under magnetic stirring at 25 °C. After stirring for 5 h, add 20 mL of methanol solution containing 0.51 g of sodium cyanoborohydride and continue stirring for 1.5 h. After the reaction is completed, add 3 mol / L ammonia solution to adjust the pH to 11-12. Then filter the product and wash it successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. Redissolve the crude product and dialyze it in deionized water for 72 h (MW: 3500) to remove unreacted furfural. Freeze-dry to obtain furfural-functionalized chitosan-mannose graft polymer.

[0116] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles at a mass ratio of 1:60, the mixture was incubated at 4°C for 24 h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0117] The preparation method of rhubarb nanovesicles is as follows: Rhubarb slices are soaked in 0.01M, pH 7.4 phosphate buffer for 48 hours, and the juice is squeezed out. The juice is centrifuged sequentially at 1000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. The supernatant is taken and subjected to ultracentrifugation at 150000×g (Optima L-100XP) for 2 h. The resulting precipitate is suspended in 1 mL of 0.01M, pH 7.4 phosphate buffer, and then transferred to sucrose solutions of four concentrations (8%, 30%, 45%, and 60%). The solutions are centrifuged at 150000×g for 1 h. The yellow bands between 30% and 45% sucrose concentrations are collected. An equal volume of 0.01M, pH 7.4 phosphate buffer is added, and the solutions are centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

[0118] Example 8

[0119] A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol includes the following steps:

[0120] (1) Dissolve 300 mg of hydroxypropyl-β-cyclodextrin and 164 mg of 3-maleimide propionic acid in 12 mL of anhydrous N,N-dimethylformamide. After mixing evenly, add 3 mL of anhydrous N,N-dimethylformamide solution containing 243 mg of dicyclohexylcarbodiimide dropwise while stirring. After reacting at -2℃ for 1 h, add 1 mL of anhydrous N,N-dimethylformamide solution containing 6 mg of dimethylaminopyridine dropwise and continue the reaction for 10 h. After the reaction is completed, filter the reaction solution and transfer the filtrate to anhydrous diethyl ether. After standing for half an hour, take the precipitate layer and dry it in a vacuum drying oven at 50℃ to obtain a pink powder, which is 3-maleimide-hydroxypropyl-β-cyclodextrin.

[0121] (2) Dissolve 2g of 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add a methanol solution containing 67mg of kaempferol dropwise while stirring, stir and react at 60℃ for 4h, remove methanol by rotary evaporation, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex.

[0122] (3) Dissolve 100 mg of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve 470 mg of furfural-functionalized chitosan-mannose graft polymer in 2% glacial acetic acid aqueous solution, and then mix it with kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution evenly. After standing at room temperature for 24 h, kaempferol-loaded polysaccharide hydrogel is obtained.

[0123] The preparation method of the furfural-functionalized chitosan-mannose graft polymer:

[0124] 1) Dissolve 250 mg of mannose and 670 mg of chitosan in water, stir evenly, add 78 mg of sodium cyanoborohydride, stir and react at room temperature for 48 h, dialyze the obtained product in water for 48 h (MW: 3500), and then freeze dry to obtain chitosan-mannose graft polymer.

[0125] 2) Dissolve 300 mg of chitosan-mannose graft polymer in 30 mL of 2% (w / v) glacial acetic acid aqueous solution, and dilute with 20 mL of anhydrous methanol to obtain a chitosan-mannose graft polymer solution. Dissolve 540 mg of furfural in anhydrous methanol and slowly add it dropwise to the chitosan-mannose graft polymer solution under magnetic stirring at 25 °C. After stirring for 5 h, add 18 mL of methanol solution containing 306 mg of sodium cyanoborohydride and continue stirring for 1.5 h. After the reaction is completed, add 3 mol / L ammonia solution to adjust the pH to 11-12. Then filter the product and wash it successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. Redissolve the crude product and dialyze it in deionized water for 72 h (MW: 3500) to remove unreacted furfural. Freeze-dry to obtain furfural-functionalized chitosan-mannose graft polymer.

[0126] (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles at a mass ratio of 1:10, the mixture was incubated at 4°C for 24 h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol.

[0127] The preparation method of rhubarb nanovesicles is as follows: Fresh rhubarb is chopped and soaked in an equal volume of 0.01M, pH 7.4 phosphate buffer. The juice is then squeezed out and centrifuged sequentially at 3000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min. The supernatant is then subjected to ultracentrifugation at 150000×g (Optima L-100XP) for 2 h. The resulting precipitate is suspended in 1 mL of 0.01M, pH 7.4 phosphate buffer and then transferred to sucrose solutions of four concentrations (8%, 30%, 45%, and 60%). The solutions are centrifuged at 150000×g for 1 h. Yellow bands between 30% and 45% sucrose concentrations are collected. An equal volume of 0.01M, pH 7.4 phosphate buffer is added, and the solutions are centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

[0128] Taking Example 3 as an example, the following experiments further illustrate the application effects of the present invention:

[0129] 1. Morphological observation of rhubarb nanovesicles

[0130] One drop of the rhubarb nanovesicle solution prepared in Example 3 was taken with a pipette and dropped onto a copper grid. The grid was negatively stained with 3% glutaraldehyde solution, washed with purified water, and dried. The results were observed under a transmission electron microscope (Tecnai G2 Spirit Twin). See attached figure. Figure 1 .

[0131] Depend on Figure 1 It can be seen that rhubarb nanovesicles exhibit a distinct lipid bilayer structure.

[0132] 2. Rhubarb nanovesicle particle size and zeta potential

[0133] Rhubarb nanovesicles were taken, and their particle size, polydispersity index and zeta potential were determined using a nanoparticle size-zeta potential analyzer (Nicomp 380 / ZLS).

[0134] Test results: Zeta potential value is -5.11±0.48mV, average particle size is 264.8±2.80nm, and polydispersity index is 0.228±0.45.

[0135] 3. Identification and analysis of the structure of the reaction products

[0136] Fourier transform infrared spectroscopy (FTIR-8400s) and nuclear magnetic resonance spectroscopy (JNM-ECZ400s / L) were used to measure the infrared absorption spectra and proton nuclear magnetic resonance spectra of the chitosan-mannose grafted polymer, furfural-functionalized chitosan-mannose grafted polymer, and 3-maleimide-hydroxypropyl-β-cyclodextrin in Example 3, respectively.

[0137] Figure 2 The image shows the infrared spectrum of the chitosan-mannose grafted polymer prepared in Example 3, with the spectrum at 1579 cm⁻¹. -1 and 1421cm -1 The absorption peaks in the region represent the characteristic peaks of the two stretching vibrations of mannose; Figure 3 The infrared spectrum of 3-maleimide-hydroxypropyl-β-cyclodextrin prepared in Example 3 is shown, with the value at 1746 cm⁻¹. -1 and 1713cm -1 This indicates the presence of a dienophilic group; Figure 4 The infrared spectrum of the furfural-functionalized chitosan-mannose graft polymer prepared in Example 3 is shown below, with the 932 cm⁻¹ value being the highest. -1 and 883cm -1 The positions correspond to the unsaturated carbons (-C=C-) on the furan ring; Figure 5 The image shows the 1H NMR spectrum of 3-maleimide-hydroxypropyl-β-cyclodextrin prepared in Example 3. The peak at δ = 6.82 ppm is the proton peak of CH=CH on the maleimide ring. Figure 6 The figure shows the 1H NMR spectrum of the furfural-functionalized chitosan-mannose graft polymer prepared in Example 3. New peaks appear at δ = 7.62 ppm, 6.67 ppm and 6.51 ppm, which correspond to -O-CH=, -CH= and =CH- on the furan ring, respectively. Figure 2-6 This demonstrates the successful synthesis of chitosan-mannose branch polymers, furfural-functionalized chitosan-mannose, and 3-maleimide-modified hydroxypropyl-β-cyclodextrin.

[0138] 4. Physicochemical characterization of kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex

[0139] Five mg each of the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex prepared in Example 3, kaempferol, 3-maleimide propionic acid, hydroxypropyl-β-cyclodextrin, and a physical mixture of 3-maleimide propionic acid and hydroxypropyl-β-cyclodextrin were weighed and analyzed using a differential calorimeter (NY-DSC-101B). The results are as follows. Figure 7 As shown. By Figure 7 It can be seen that the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex has the same thermal absorption peak as 3-maleimide-hydroxypropyl-β-cyclodextrin, proving that kaempferol successfully formed an inclusion complex with 3-maleimide-hydroxypropyl-β-cyclodextrin, rather than a simple physical mixture.

[0140] The content of kaempferol included in the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex was determined and its inclusion rate was calculated using a methanol ultrasonic-high performance liquid chromatography (HPLC) method. 5 mg of the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex was dissolved in 0.5 mL of purified water, and then 0.5 mL of chromatographic methanol was added. The mixture was ultrasonicated for 30 min, passed through a 0.22 μm membrane, and the kaempferol content was detected using HPLC (1260 Infinity II). Kaempferol showed maximum absorption at 370 nm. The mobile phase was methanol-0.1% phosphoric acid aqueous solution (70:20, v / v). The elution time of kaempferol was 6.42 min. The inclusion rate was calculated using Formula 1.

[0141]

[0142] The HPLC chromatogram of kaempferol is shown in [reference needed]. Figure 8 Based on the measurement results, the inclusion rate was calculated to be 53.8 ± 0.33%.

[0143] 5. Observation of polysaccharide hydrogel morphology using scanning electron microscopy

[0144] The polysaccharide hydrogel prepared in Example 3 and the polysaccharide hydrogel samples co-loaded with rhubarb nanovesicles and kaempferol were subjected to ion sputtering gold sputtering, fixed on the sample stage and vacuumed, and scanned by scanning electron microscope (Teneo VS) with an accelerating voltage of 1KV.

[0145] Scanning electron microscope image (see) Figure 9 ,in, Figure 9 A is a polysaccharide hydrogel. Figure 9B is a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol. It can be seen that the polysaccharide hydrogel exhibits an irregular porous structure with a dense and smooth surface, while the porous mesh structure of the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol has a rough surface with obvious granules, proving that the rhubarb nanovesicles are loaded within the hydrogel mesh structure.

[0146] 6. In vitro degradation rate assessment

[0147] The polysaccharide hydrogels prepared in Example 3 were immersed in simulated gastric juice (pH 1.2 hydrochloric acid solution containing pepsin), simulated small intestinal juice (pH 6.8 phosphate buffer containing pancreatic enzyme), and simulated colonic juice (pH 7.4 phosphate buffer containing colonic enzyme extract), respectively, and placed in a constant temperature shaker (SHZ-82A) at 37°C with a shaking rate of 100 r / min. Oxygen was provided to simulate the human body environment to maintain enzyme activity. The gels were removed, weighed, and their morphology was observed at specified time intervals (0.5h, 1h, 2h, 4h, 8h, 12h, 24h). The degradation rate was calculated using the following formula:

[0148]

[0149] See results Figure 10 It can be seen that the polysaccharide hydrogel remains relatively stable in the stomach and small intestine, with degradation rates of 46.15% and 55.83% at 24 hours, respectively. However, it degrades rapidly in the colon, reaching 38.75% at 2 hours and 92.54% at 24 hours, indicating almost complete degradation.

[0150] 7. In vitro release experiment

[0151] The release behavior of polysaccharide hydrogels loaded with kaempferol was investigated using simulated gastric juice, simulated small intestinal juice, and simulated colonic juice as release media. The polysaccharide hydrogels were placed in different release media and subjected to isothermal shaking at 37℃ and 100 rpm. At different time points (0.25, 0.5, 1, 2, 4, 8, 12, and 24 h), 1 mL of the solution was taken as a sample solution, and an equal volume of fresh media was added simultaneously. The sample solutions were filtered through a 0.45 μm filter membrane, and the kaempferol content was determined by HPLC. The cumulative release rate was calculated, and in vitro release curves were plotted.

[0152] Figure 11 The in vitro release curve of the polysaccharide hydrogel loaded with kaempferol prepared in Example 3 shows that the release rate of kaempferol in phosphate buffer containing colon enzyme extract at pH 7.4 is much higher than that under other conditions, indicating that the polysaccharide hydrogel loaded with kaempferol has colon enzyme responsiveness.

[0153] 8. Cytotoxicity assay

[0154] The toxicity of polysaccharide hydrogels to RAW264.7 and CT26 cells (purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) was investigated using the MTT assay. Cells were inoculated at a concentration of 1×10⁻⁶ cells. 4 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured in a Thermo 3111 incubator for 24 hours until complete cell adhesion. The supernatant was removed, and sample solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL were added. After incubation for 24 hours, the supernatant was discarded, and the cells were washed twice with physiological saline. 100 μL MTT solution (0.5 mg / mL) was added, and the cells were incubated for another 4 hours. The MTT solution was then discarded, and 150 μL LDMSO solution was added. The cells were shaken for 10 minutes at 37°C and 100 rpm using a digital display shaker. The absorbance (OD) value was measured at 490 nm using a Varioskan LUX microplate reader, and cell viability was calculated using the following formula:

[0155]

[0156] The results are as follows Figure 12 As shown, when the polysaccharide hydrogel concentration was 400 μg / mL, the survival rates of RAW264.7 and CT26 cells were 85.24% and 90.6%, respectively, indicating that the polysaccharide hydrogel of the present invention has no obvious cytotoxicity when the concentration is less than 400 μg / mL.

[0157] 9. In vivo pharmacodynamic experiments against ulcerative colitis

[0158] Establishment of a mouse model of ulcerative colitis: Eighteen C57BL / 6 mice (provided by the Experimental Animal Center of Xuzhou Medical University) were used to establish a mouse model of ulcerative colitis by allowing them to drink 3% (w / v) sodium dextran sulfate (DSS) aqueous solution instead of drinking water for 5 days. The mice were then randomly divided into 6 treatment groups as follows: healthy control group, model control group, kaempferol group, polysaccharide hydrogel group, kaempferol-loaded polysaccharide hydrogel group, and polysaccharide hydrogel group co-loaded with rhubarb nanovesicles and kaempferol. The equivalent dose of kaempferol and rhubarb nanovesicles was 15 mg / kg. Administration was performed by gavage for 5 days. After administration, the mice were sacrificed, the colon was dissected and photographed, and 0.5 cm of colonic tissue was cut from near the rectum, fixed with 4% paraformaldehyde, prepared into paraffin blocks, stained with hematoxylin and eosin (HE), and observed and photographed under a microscope. A separate sample of colon tissue was taken for myeloperoxidase (MPO) activity detection. The assay was performed according to the instructions of Nanjing Jiancheng Biotechnology MPO reagent kit.

[0159] Figure 13The colon length of mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 3 was measured. It can be seen that the colon length of mice in the model control group was significantly shortened, while each treatment group could alleviate the colon shortening to some extent. Among them, the colon length of mice in the polysaccharide hydrogel group containing rhubarb nanovesicles and kaempferol was closest to that of the healthy control group. Figure 14 The images show HE staining of mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 3. Figure 14 A represents the healthy control group. Figure 14 B represents the model control group. Figure 14 C represents the kaempferol group. Figure 14 D represents the polysaccharide hydrogel group. Figure 14 E is a polysaccharide hydrogel loaded with kaempferol. Figure 14 F represents the polysaccharide hydrogel group co-loaded with rhubarb nanovesicles and kaempferol. It can be seen that the layered structure of the slices in the model control group was destroyed, with large areas of missing crypts, goblet cells and glands. In contrast, the polysaccharide hydrogel group co-loaded with rhubarb nanovesicles and kaempferol had clear crypt structure and goblet cells arranged tightly and regularly. Figure 15 The image shows the myeloperoxidase activity of mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 3. A represents the healthy control group, B the model control group, C the kaempferol group, D the polysaccharide hydrogel group, E the kaempferol-loaded polysaccharide hydrogel, and F the polysaccharide hydrogel group containing both rhubarb nanovesicles and kaempferol. It can be seen that the myeloperoxidase activity in the model control group was significantly enhanced compared to the healthy control group. Different drug groups showed a certain degree of reduction after treatment. The polysaccharide hydrogel group containing both rhubarb nanovesicles and kaempferol exhibited the strongest inhibitory effect on myeloperoxidase activity, and there were significant differences among the groups.

[0160] The in vivo anti-ulcerative colitis pharmacodynamic experiments were conducted on the polysaccharide hydrogels co-loaded with rhubarb nanovesicles and kaempferol prepared in Examples 1 and 4 using the same method. The experimental results are shown in [Figure 1]. Figure 16 and Figure 17 .

[0161] The colon length diagrams and MPO activity detection results for Examples 1 and 4 are shown below. Figure 16 , Figure 17 .

[0162] Figure 16 The colon length of mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 1 was measured. It can be seen that the results are the same as those of the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 3. The colon length of mice in the model control group was significantly shortened, while all treatment groups could alleviate the colon shortening to some extent. Among them, the colon length of mice in the polysaccharide hydrogel group containing rhubarb nanovesicles and kaempferol was closest to that of the healthy control group.

[0163] Figure 17 The image shows the myeloperoxidase activity in mice treated with the polysaccharide hydrogel containing rhubarb nanovesicles and kaempferol prepared in Example 4. A represents the healthy control group, B the model control group, C the kaempferol group, D the polysaccharide hydrogel group, E the polysaccharide hydrogel containing kaempferol, and F the polysaccharide hydrogel group containing both rhubarb nanovesicles and kaempferol. It can be seen that ulcerative colitis significantly enhances myeloperoxidase activity in colonic tissue. After treatment with different drugs, the activity of myeloperoxidase in the tissue decreased to some extent. The polysaccharide hydrogel group containing both rhubarb nanovesicles and kaempferol showed the strongest inhibitory effect on myeloperoxidase activity, and there were significant differences among the groups.

[0164] This invention utilizes the hydrophobic cavity structure of 3-maleimide-hydroxypropyl-β-cyclodextrin to encapsulate the poorly soluble small molecule drug kaempferol into a host-guest inclusion complex. Then, it undergoes a Diels-Alder cycloaddition reaction with furfural-functionalized chitosan-mannoside graft polymer to form a polysaccharide hydrogel. The strong positive charge of chitosan in the hydrogel adsorbs the negatively charged rhubarb nanovesicles, thereby preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol. The polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol prepared in this invention exhibits excellent therapeutic effects on ulcerative colitis, primarily due to three factors: First, the polysaccharide hydrogel effectively increases the retention rate of rhubarb nanovesicles in the colon, enhances intestinal absorption, improves the solubility and stability of poorly soluble small-molecule kaempferol, and fully leverages the anti-inflammatory effects of kaempferol. Second, after reaching the colon, the polysaccharide hydrogel, mediated by mannose, accumulates in macrophages overexpressed at the site of inflammation. Colonic enzymes degrade chitosan, the main carrier, thereby rapidly releasing rhubarb nanovesicles and kaempferol, effectively achieving targeted therapeutic effects. Third, chitosan itself, as the main carrier, possesses certain therapeutic effects on ulcerative colitis. Thus, the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol exerts a synergistic effect, maximizing the therapeutic efficacy against ulcerative colitis.

Claims

1. A method for preparing a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol, characterized in that, Includes the following steps: (1) Hydroxypropyl-β-cyclodextrin and 3-maleimide propionic acid in a molar ratio of 1:5 were dissolved in anhydrous N,N-dimethylformamide and mixed evenly. Then, an anhydrous N,N-dimethylformamide solution of dicyclohexylcarbodiimide was added dropwise under stirring. After the reaction was carried out at -2℃, dimethylaminopyridine was added to continue the reaction. After the reaction was completed, the reaction solution was filtered and the filtrate was transferred to anhydrous diethyl ether. After standing treatment, the precipitate was dried to obtain 3-maleimide-hydroxypropyl-β-cyclodextrin. (2) Dissolve 3-maleimide-hydroxypropyl-β-cyclodextrin in water, add kaempferol methanol solution dropwise under stirring to react, remove methanol by rotary evaporation under reduced pressure after reaction, and then freeze dry to obtain kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex. (3) Dissolve the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain a kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. Dissolve the furfural-functionalized chitosan-mannose graft polymer in a 2% (w / v) aqueous solution of glacial acetic acid. Then mix it with the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex solution. After standing for 20-30 h, a polysaccharide hydrogel loaded with kaempferol is obtained. The preparation method of the furfural-functionalized chitosan-mannose graft polymer is as follows: Chitosan-mannose graft polymer is dissolved in an aqueous solution of glacial acetic acid with a mass-volume concentration of 2%, and diluted with anhydrous methanol to obtain a chitosan-mannose graft polymer solution. Under magnetic stirring, anhydrous methanol solution of furfural is added dropwise to the chitosan-mannose graft polymer solution, with a molar ratio of furfural to chitosan-mannose graft polymer of 3:

1. After the reaction, methanol solution of sodium cyanoborohydride is added and the reaction is continued with stirring, with a molar ratio of sodium cyanoborohydride to chitosan-mannose graft polymer of 4:

1. After the reaction is completed, an aqueous ammonia solution is added to adjust the pH to 11-12. Then the product is filtered and washed successively with deionized water, a mixture of water and ethanol, and anhydrous ethanol. After filtration and drying, a pale yellow crude product is obtained. After purification, furfural-functionalized chitosan-mannose graft polymer is obtained. (4) After mixing the polysaccharide hydrogel loaded with kaempferol and rhubarb nanovesicles evenly, incubate at 4℃ for 20-30h to obtain a polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol. In step (3), the preparation method of the chitosan-mannose graft polymer is as follows: mannose and chitosan with a molar ratio of 1:3 are dissolved in water, stirred evenly, and sodium cyanoborohydride is added. The molar ratio of sodium cyanoborohydride to mannose is (1.5-2):

1. The mixture is stirred and reacted at room temperature for 40-50 hours. The obtained product is dialyzed in water and then freeze-dried to obtain the chitosan-mannose graft polymer. In step (4), the preparation method of rhubarb nanovesicles is as follows: Fresh rhubarb is chopped or processed, soaked in 0.01M, pH 7.4 phosphate buffer, and juice is squeezed out. Then, it is centrifuged at 3000×g for 20 min, 6000×g for 20 min, and 10000×g for 30 min in sequence. The supernatant is taken and centrifuged at 150000×g for 2 h. The precipitate is suspended in 0.01M, pH 7.4 phosphate buffer and then transferred to a gradient sucrose solution and centrifuged at 150000×g for 1 h. The yellow bands between the gradient sucrose solutions are collected. An equal amount of 0.01M, pH 7.4 phosphate buffer is added and centrifuged again at 150000×g for 1 h to wash away the sucrose solution, thus obtaining rhubarb nanovesicles.

2. The method for preparing the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol as described in claim 1, characterized in that, In step (1), the molar ratio of dicyclohexylcarbodiimide to 3-maleimide propionic acid is (1.3-1.5):1, and the molar ratio of dimethylaminopyridine to 3-maleimide propionic acid is 0.05:

1.

3. The method for preparing the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol as described in claim 1, characterized in that, In step (2), the reaction temperature is 60°C and the time is 3-6 hours.

4. The method for preparing the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol as described in claim 1, characterized in that, In step (2), the molar ratio of 3-maleimide-hydroxypropyl-β-cyclodextrin to kaempferol is 1:(3-5).

5. The method for preparing the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol as described in claim 1, characterized in that, In step (3), the molar ratio of the kaempferol-3-maleimide-hydroxypropyl-β-cyclodextrin inclusion complex to the furfural-functionalized chitosan-mannose graft polymer is 1:(2-5).

6. The method for preparing the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol as described in claim 1, characterized in that, In step (4), the mass ratio of rhubarb nanovesicles to polysaccharide hydrogel loaded with kaempferol is (10-120):

1.

7. A polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol prepared by the method described in any one of claims 1-6.

8. The use of the polysaccharide hydrogel co-loaded with rhubarb nanovesicles and kaempferol prepared by the method of any one of claims 1-6 in the preparation of an oral medicament for treating ulcerative colitis.

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