Hydrogel microspheres as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of hydrogel, and discloses hydrogel microspheres as well as a preparation method and application thereof. According to the invention, epigallocatechin gallate and vitamin E are self-assembled into hydrophobic polyphenol-based micelles, the hydrophobic polyphenol-based micelles take EGCG as a core, a stable structure is formed through non-covalent interaction (such as hydrophobic interaction, hydrogen bond and pi-pi accumulation), and the stability and functionality of the hydrophobic core are enhanced; the preparation method comprises the following steps: preparing a hydrophobic structure, mixing the hydrophobic structure with hydrophilic sodium alginate for gelling to finally obtain the hydrogel microsphere with a unique amphiphilic heterostructure, and the hydrogel microsphere can stably exist in a water environment, actively captures dietary fat, improves the capture capability on the dietary fat, and improves the dietary fat capture efficiency. The stability of the hydrogel microspheres in gastrointestinal tracts is improved.

Description

Technical Field

[0001] The invention relates to the technical field of hydrogels, and in particular to hydrogel microspheres and a preparation method and application thereof. Background Art

[0002] Preventing the intestinal absorption of dietary fat is the main determinant of treating obesity. In the small intestine, dietary fat forms micelles after bile acid-mediated emulsification and lipase-driven hydrolysis, which are transported to the blood through intestinal epithelial cells for systemic circulation, among which the absorption of dietary fat at the emulsification interface is crucial. In view of these physiological facts, a series of treatments have been proposed to prevent intestinal absorption of dietary fat and curb obesity, and the most widely used are lifestyle interventions and medical therapies, such as dietary modification, anti-obesity drugs, and bariatric surgery. However, these methods are limited by low individual compliance and multiple side effects. Therefore, biomaterials have an encouraging prospect in solving the problem of obesity due to their high efficiency, versatility and biocompatibility.

[0003] Currently reported biomaterials that target dietary fat intestinal absorption mainly include ionic liquids, sucrose salts, and dietary fiber. Ionic liquids and sucrose salts reduce the absorption of dietary fat by forming a barrier between intestinal cells and dietary fat, but undigested fat remains in the intestine, leading to physiological problems such as steatorrhea. Dietary fiber (resistant starch and nanocellulose) can reduce such intestinal side effects. Based on its unique macromolecular complex structure, it can effectively reduce the intestinal absorption of dietary fat. However, due to its non-intelligent response structure, it lacks the ability to dynamically target and capture dietary fat. Summary of the invention

[0004] To solve the problems in the background technology, the present invention provides a hydrogel microsphere and a preparation method and application thereof. The hydrogel microsphere provided by the present invention is a heterogeneous hydrogel, which improves the stability of the hydrogel microsphere in the intestine and can dynamically capture dietary fat.

[0005] To achieve the above object, the first technical solution adopted by the present invention is: A method for preparing hydrogel microspheres, the method comprising: Epigallocatechin gallate and vitamin E were self-assembled in an ethanol solution to form hydrophobic polyphenol-based micelles; The hydrophobic polyphenol-based micelles are uniformly mixed with a sodium alginate solution to form a pre-gel; The pregel was cross-linked with calcium ions, washed, and freeze-dried.

[0006] Preferably, the mass ratio of epigallocatechin gallate, vitamin E and sodium alginate is 1:(0.7-0.9):(0.6-1.2).

[0007] Preferably, the mass concentration of the sodium alginate is 1.5-3%.

[0008] Preferably, the freeze drying is freeze drying at -80 °C for 36-48 h.

[0009] Preferably, the calcium ion concentration is 1.5-2%.

[0010] The second technical solution adopted by the present invention is: The hydrogel microspheres are obtained according to the preparation method of the first technical solution.

[0011] The third technical solution adopted by the present invention is: The second technical solution is the use of hydrogel microspheres in the preparation of drugs for preventing intestinal absorption of dietary fat.

[0012] The fourth technical solution adopted by the present invention is: The second technical solution is the use of hydrogel microspheres in the preparation of drugs for treating obesity.

[0013] Compared with the prior art, the present invention has the following effects: The present invention self-assembles epigallocatechin gallate and vitamin E into hydrophobic polyphenol-based micelles, wherein the hydrophobic polyphenol-based micelles take EGCG as the core and form a stable structure through non-covalent interactions (such as hydrophobic interaction, hydrogen bonding, and π-π stacking), thereby enhancing the stability and functionality of the hydrophobic core; and then the hydrophobic structure is mixed with hydrophilic sodium alginate to form a gel, and the finally obtained hydrogel microspheres have unique amphiphilic heterogeneous structure hydrogel microspheres, which can stably exist in an aqueous environment, actively capture dietary fat, improve the ability to capture dietary fat, and improve the stability of the hydrogel microspheres in the gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the demulsification effect of hydrogel microspheres with different ratios in Example 1, a is the light transmittance of the emulsion; b is the weight difference of the hydrogel microspheres before and after liposuction; Figure 2 a is the UV spectrum of the hydrogel microspheres in Example 1, b is the dynamic light scattering particle size, and c is the transmission electron microscope image of the hydrophobic cavity; Figure 3 The amphiphilic properties of the hydrogel microspheres prepared in Example 2, a is the macroscopic morphology of the hydrogel microspheres before and after freeze-drying, b is the amphiphilic interface of the hydrogel microspheres photographed by a fluorescence microscope, and c is the amphiphilicity result of the contact angle test of the hydrogel microspheres; Figure 4The structure characterization of the hydrogel microspheres prepared in Example 2, a is the Fourier infrared spectrum of the hydrogel microspheres, b is the elemental analysis diagram, c and d are thermogravimetric analysis diagrams; Figure 5 is the biocompatibility of the hydrogel prepared in Example 2, a is cytotoxicity, b is cell viability; Figure 6 is the gastrointestinal stability of the hydrogel microspheres prepared in Example 2, a is the EGCG / V in the supernatant after the microspheres were immersed in simulated gastrointestinal fluid E a is the content, b is the weight difference of the microspheres after being soaked in simulated gastrointestinal fluid, and c is the structural difference of the microspheres after being soaked in simulated gastrointestinal fluid; Figure 7 is the absorption capacity of the hydrogel microspheres prepared in Example 2 for dietary fat, a is a digital photo and a fluorescence microscope image of soybean oil, b is the weight difference of the hydrogel microspheres before and after liposuction, and c is the absorption rate of the hydrogel for all dietary lipids; Figure 8 a is the fluorescence imaging of the gastrointestinal tract of rats in Example 3, b is the content of triglycerides and free fatty acids in the blood of rats; Fig. 9 This is a statistical diagram of food intake of each group of animals in Example 4; Fig.10 The application effect of the hydrogel microspheres in the management of obesity in Example 4, a is a graph showing the weight changes of rats in different treatment groups after feeding for 30 days, b is a graph showing the morphology of rats in different treatment groups after feeding for 30 days, and c is a graph showing the weights of the liver and adipose tissue of rats in different treatment groups after feeding for 30 days; Fig.11 The in vitro elimination capacity of the hydrogel microspheres for dietary fat in Example 4, a is the feces discharged by rats in each group; b is the content of triglyceride, cholesterol, free fatty acid and bile acid in the feces of each group from left to right; Fig.12 is the particle size of different micelles in the comparative example. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments.It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.Unspecified conditions in the embodiments are carried out according to the conditions of normal conditions or manufacturer recommendations.Reagents used or instruments that do not specify manufacturers are conventional products that can be purchased and obtained commercially.

[0016] The first embodiment of the present invention provides a method for preparing hydrogel microspheres, which comprises: Epigallocatechin gallate and vitamin E were self-assembled in an ethanol solution to form hydrophobic polyphenol-based micelles; The hydrophobic polyphenol-based micelles are uniformly mixed with a sodium alginate solution to form a pre-gel; The pregel was cross-linked with calcium ions, washed, and freeze-dried.

[0017] The embodiment of the present invention self-assembles epigallocatechin gallate (EGCG) and vitamin E into hydrophobic polyphenol-based micelles, wherein the hydrophobic polyphenol-based micelles have EGCG as the core and form a stable structure through non-covalent interactions (such as hydrophobic interaction, hydrogen bonding, and π-π stacking), thereby enhancing the stability and functionality of the hydrophobic core; the hydrophobic structure is then mixed with hydrophilic sodium alginate to form a gel, and the finally obtained hydrogel microspheres have unique amphiphilic heterostructure hydrogel microspheres, which cleverly combine hydrophobic and hydrophilic characteristics on the same level to form an integrated heterogeneous network, so that the hydrogel microspheres can stably exist in an aqueous environment, actively capture dietary fat, improve the ability to capture dietary fat, and improve the stability of the hydrogel microspheres in the gastrointestinal tract.

[0018] It should be noted that the simple EGCG component can only break the emulsion, and its binding force with dietary fat is insufficient, so that dietary fat is not easily retained in the EGCG microspheres. During the implementation of the present invention, it was found that only EGCG, a polyphenolic substance, can self-assemble with vitamin E to obtain cavity hydrophobic polyphenol-based micelles, which enhances the binding force with dietary fat, firmly locks dietary fat inside the microspheres, and enhances the functionality of the hydrophobic core. The hydrophobic polyphenol-based micelles are then filled in the dietary matrix to construct amphiphilic heterostructure hydrogel microspheres.

[0019] It should also be noted that the heterogeneous structure hydrogel obtained by the present invention specifically refers to the amphiphilic interface structure formed, which is composed of different materials or different structures of the same material. These heterogeneous regions have different structures at the microscopic or macroscopic level, resulting in different mechanical properties, water absorption performance or biological activity; these heterogeneous hydrogels can have layered structures, encapsulated particles or other types of composite structures at the same time. However, the heterogeneous hydrogels in the prior art are mixtures containing different components, which are just compared with the heterogeneous concept proposed by homogeneous hydrogels: homogeneous hydrogels refer to hydrogels composed of a single type of polymer network, whose chemical and physical properties are uniformly distributed throughout the system; in homogeneous hydrogels, polymer chains and cross-linking points are uniformly distributed on a macroscopic scale; this means that the same structure and composition will be found in any part of the hydrogel, whether observed from a microscopic or macroscopic perspective; heterogeneous hydrogels in the prior art are usually composed of different composite components for their functions, and are mostly heterogeneous multi-level structures.

[0020] The ethanol solution is used to dissolve vitamin E and EGCG. The amount of ethanol solution used is sufficient to dissolve vitamin E and EGCG, and after sodium alginate is added, the sodium alginate gel does not agglomerate. Those skilled in the art can select a suitable amount of ethanol according to actual conditions.

[0021] In some preferred embodiments, the mass ratio of epigallocatechin gallate, vitamin E and sodium alginate is 1:(0.7-0.9):(0.6-1.2).

[0022] In some preferred embodiments, the mass concentration of sodium alginate is 1.5-3%.

[0023] There is no need to specifically limit the freeze-drying conditions, and those skilled in the art can adjust them according to the desired product performance. For example, in some preferred embodiments, the freeze-drying is freeze-drying at -80 ° C for 48 h.

[0024] In some preferred embodiments, in order to further increase the mechanical properties of the hydrogel, calcium salt may be added during the preparation of the pregel. After the pregel is cross-linked with calcium ions, hydrochloric acid is added to release the Ca in the calcium salt. 2+ , cross-linked with the hydrogel inside the microspheres, improving the gelation inside the microspheres. Wherein, the calcium salt can be calcium sodium ethylenediaminetetraacetate.

[0025] The second embodiment of the present invention provides the hydrogel microspheres obtained according to the first embodiment.

[0026] The hydrogel microspheres according to the embodiment of the present invention are hydrogels with an amphiphilic heterogeneous structure, which can enhance the adsorption capacity of dietary fat and achieve efficient capture of dietary fat.

[0027] The third embodiment of the present invention provides the use of the hydrogel microspheres of the second embodiment in preparing a drug for preventing intestinal absorption of dietary fat.

[0028] The hydrogel microspheres according to the embodiments of the present invention have a dual role in preventing the intestinal absorption of dietary fat: first, the hydrophilic area on its surface can contact with the emulsified fat droplets to effectively capture them, laying the foundation for their subsequent fat-locking behavior; second, the hydrophobic structure in the hydrogel microspheres can interact with the oils and fats released by demulsification, firmly locking the oils and fats in the spheres, and excreting them from the body without causing adverse reactions such as steatorrhea. Driven by the dual effects of the interfacial activity of EGCG and the non-absorbability of the sodium alginate dietary matrix, the hydrogel microspheres can maintain their structural integrity in the gastrointestinal environment and safely wrap dietary fats inside the microspheres until they are naturally excreted from the body. Therefore, the hydrogel microspheres according to the embodiments of the present invention can be used to prepare drugs that prevent the intestinal absorption of dietary fats.

[0029] The fourth embodiment of the present invention provides use of the hydrogel microspheres of the second embodiment in preparing a drug for treating obesity.

[0030] The hydrogel microspheres of the embodiments of the present invention achieve the treatment of obesity by preventing the intestinal absorption of dietary fat, effectively avoiding the common discomfort reactions in traditional anti-obesity biomaterial intervention measures, such as diarrhea, nausea and flatulence, and show great potential in obesity management. Therefore, the hydrogel microspheres of the embodiments of the present invention can be used to prepare drugs for the treatment of obesity.

[0031] In order to make the technical solution of the present invention clearer, the preparation and performance of hydrogel microspheres are described below through multiple specific examples.

[0032] Example 1 1.1 Preparation of hydrogel microspheres First, epigallocatechin gallate (EGCG) and vitamin E (Vitamin, V E ) self-assembled in an ethanol solution to form hydrophobic polyphenol-based micelles Fat lock. Then, the hydrophobic polyphenol-based micelles were added dropwise to a 2% sodium alginate solution and stirred to form a uniform pre-gel. 2+ Hydrogel microspheres formed by ionic crosslinking. Finally, the microspheres were rinsed with distilled water several times to remove residual ions and freeze-dried at −80 °C for 48 h to obtain amphiphilic hydrogel microspheres (Polyphenol-based fat trap, PFAT).

[0033] 1.2 Study the effect of different raw material dosages on the effect of hydrogel microspheres According to the method in 1.1, prepare sodium alginate:EGCG:V E The mass ratios of 0:0:0, 1:0:0, 1:1:0, 6:10:7, 6:10:9, 4:5:4, 12:10:9, 12:10:7 were used to prepare hydrogel microspheres, and then the transmittance changes before and after the emulsion treatment were detected by UV spectrophotometer to explore the effect of demulsification and liposuction. The ability of hydrogel microspheres to absorb fat from emulsified fat was measured to test the absorbability of hydrogel microspheres to dietary fat.

[0034] The specific method is as follows: soybean oil is used as a fat model, and it is mixed with artificial bile acid and simulated intestinal fluid in a ratio of 1:2:99, and stirred at 2000 rpm for 1 hour to prepare emulsified fat. Then, the hydrogel microspheres are placed in the emulsified fat at 37°C and 100 rpm for 2 hours. The transmittance (T) of the emulsified fat before and after treatment is observed by an ultraviolet spectrophotometer (UV), and the fat content in the emulsified fat is observed by a microscope. The liposorption ability of the hydrogel microspheres is evaluated by the weight difference of the hydrogel microspheres.

[0035] The results are as follows Figure 1 As shown, a is the transmittance of the emulsion, and the transmittances are 27.05±0.73, 32.03±0.59, 42.10±1.43, 49.60±1.66, 55.35±1.42, 89.15±1.20, 77.60±0.71, 63.93±1.26 from left to right, in units of %; b is the weight difference of the hydrogel microspheres before and after liposuction, and the weight differences are 0.149±0.027, 0.298±0.017, 0.210±0.017, 0.277±0.029, 0.347±0.021, 0.246±0.014, 0.190±0.034 from left to right, in units of g.

[0036] For the above sodium alginate:EGCG:V E The structure of the hydrogel microspheres prepared with a mass ratio of 4:5:4 was tested. Figure 2 As shown, Figure 2 a is the UV spectrum obtained by UV spectrophotometer, b is the dynamic light scattering particle size obtained by dynamic scattering instrument, and c is the transmission electron microscope image of hydrophobic cavity. The particle size of EGCG in Figure b is 379.83±11.77 nm, V E The particle size is 171.23±7.72nm, and the Fat lock particle size is 574±32.54nm. Figure 2 It can be seen that the hydrogel microspheres prepared in the present invention have hydrophobic cavities.

[0037] Example 2 Preparation and performance testing of hydrogel microspheres 2.1 Preparation of hydrogel microspheres 1 g EGCG and 0.8 g vitamin E were dissolved in 3 mL anhydrous ethanol solution and shaken at 200 rpm for 30 min to form hydrophobic polyphenol-based micelles. Subsequently, 2% w / v sodium alginate solution and 2% w / v sodium calcium ethylenediaminetetraacetic acid solution were magnetically stirred at a volume ratio of 4:1 to form a uniform mixed solution. Next, the hydrophobic polyphenol-based micelles were added dropwise to the mixed solution to form a uniform pre-gel, which was then stirred by a constant flow pump and a thin hose (3 cm 3) Add the pregel dropwise to 100 mL of 2% w / v calcium chloride (CaCl 2 ) aqueous solution and stirred gently at 300 rpm. The hydrogel microsphere intermediates formed in CaCl 2 The intermediate was then immersed in a 0.1 mol / L hydrochloric acid (HCl) solution for 1 h to allow EDTA-Ca to release Ca 2+ Finally, the pregel inside the cross-linked microspheres was rinsed with distilled water several times to remove residual ions, and the resulting hydrogel microspheres PFAT were collected by freeze drying at −80 °C for 48 h.

[0038] The contact angle of the hydrogel microspheres PFAT was measured, and the amphiphilic interface was photographed using a fluorescence microscope. Figure 3 The results showed that the prepared hydrogel microspheres were amphiphilic, demonstrating the potential for demulsification and liposuction. Figure 3 Figure a shows the macroscopic morphology of hydrogel microspheres before freeze-drying (Fresh PFAT) and after freeze-drying (Dried PFAT). Figure b shows the amphiphilic interface of hydrogel microspheres photographed by fluorescence microscopy: pink is sodium alginate matrix-hydrophilic; green is hydrophobic cavity-hydrophobic; bright field image; red and green coexistence graphics merge. Figure c shows the amphiphilicity results of the contact angle test of the hydrogel microspheres, with a water contact angle of 17.5° and an oil contact angle of 51.1°.

[0039] 2.2 Preparation and characterization of hydrogel microspheres The hydrogel microspheres were analyzed by Fourier transform infrared spectroscopy (FTIR), organic element analyzer (EA), and thermogravimetric analyzer (TGA). The specific method is as follows: the hydrogel microspheres were fully mixed with potassium bromide (KBr) at a ratio of 1:100 w / w, and then pressed into transparent sheets. -1 The FTIR of the samples was scanned over the wavelength range. After 5 mg of hydrogel microspheres were thoroughly ground, the TGA curves were tested in the temperature range of 30-900 °C. The test conditions were nitrogen environment, flow rate of 40 mL / min, and heating rate of 10 °C / min. To quantify the content of carbon (C) and hydrogen (H), the samples were completely burned in an oxidation tube at 1150 °C in a pure oxygen environment. To analyze the oxygen (O) content, the hydrogel microspheres were heated in a hydrogen (H) tube at 1150 °C. 2 ) and helium (He) mixed gas flow, and the concentrations of C, H and O were determined by thermal conductivity detection.

[0040] The results showed that polyphenol cavity micelles were successfully filled in dietary matrix to construct hydrogel microspheres, e.g. Figure 4 shown.

[0041] Figure 4 Figure a is the Fourier transform infrared spectrum of the hydrogel microspheres, which is used to show the composition of the microspheres. Each component has a specific peak. The final microspheres contain sodium alginate / EGCG / V E The characteristic peaks of , indicating that the hydrogel microspheres were successfully prepared. b is an elemental analysis diagram, PFFT is the microspheres prepared using pure sodium alginate in 2.1, and PFAT is the hydrogel microspheres prepared in 2.1. The change in the element ratio shows that the hydrophobic polyphenol-based micelles are successfully filled in the sodium alginate matrix, that is, the hydrogel microspheres are successfully prepared. c and d are thermogravimetric analysis diagrams, analyzing the temperature change of the microspheres PFFT prepared with pure sodium alginate and the microspheres PFAT prepared with sodium alginate + EGCG micelles. The results show that the hydrophobic polyphenol-based micelles are successfully filled in the sodium alginate matrix, that is, the hydrogel microspheres are successfully prepared.

[0042] 2.3 Biosafety assessment of hydrogel microspheres NIH3T3 fibroblasts were used as the research object, and the biosafety of hydrogel microspheres was evaluated by cell activity and cell proliferation. The cytotoxicity of the hydrogel microspheres prepared in 2.1 was evaluated by the cell counting kit-8 (CCK-8) assay. The effect of hydrogel microspheres on cell proliferation was evaluated by the Annexin V-FITC apoptosis detection kit and confocal laser scanning microscopy (CLSM).

[0043] The results are as follows Figure 5 As shown, Figure a is cell live and dead fluorescence staining, green represents live cells, red represents dead cells, PBS is used as the control group, and Fat locks are polyphenol-based micelles; the results show that there is no visible difference in red and green colors compared with the control, qualitatively indicating that the cytotoxicity of the hydrogel prepared in the present application is negligible; Figure b is the CCK-8 test result for cell viability, which quantitatively analyzes the safety of the material, wherein the Alginate matrix is ​​sodium alginate, and the cell viability values ​​from left to right are 100.00±2.26, 103.71±3.17, 97.18±1.60, and 98.18±2.05, respectively. The results show that there is no significant difference in the values ​​between the treatment group and the control group, indicating that the hydrogel microspheres prepared in the present application have no significant effect on cell viability.

[0044] 2.4 Gastrointestinal stability testing of hydrogel microspheres 50 mg of the hydrogel microspheres prepared in 2.1 were immersed in simulated saliva (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) for 10 min, 2 h and 2 h, respectively, at 37 °C and 200 rpm for shaking reaction; after the reaction, the hydrogel microspheres were separated and 1 mL of SSF, SGF and SIF were filtered with a 0.22 μm filter membrane, respectively, and then analyzed.

[0045] EGCG and V in simulated liquid were analyzed by high performance liquid chromatography (HPLC). E Quantitative determination: HPLC analysis was performed using a CAPCELL PAK HPLC column (C18, 250 mm × 4.6 mm, 5 μm) equipped with a UV detector; the chromatographic conditions were set at an injection flow rate of 1 mL / min, an injection volume of 10 μL, and a column temperature of 35 °C; EGCG and V E The mobile phases were acetonitrile: 0.2% acetic acid = 3:7, 100% methanol. E The detection wavelengths were 278 nm and 294 nm, respectively.

[0046] Scanning electron microscopy (SEM) was used to evaluate the morphological changes of the hydrogel microspheres before and after the reaction, and the mass loss of the hydrogel microspheres in various liquids was quantitatively evaluated.

[0047] The results are as follows Figure 6 As shown, Figure 6 a in the figure shows the EGCG / V in the supernatant of the microspheres after being immersed in simulated gastrointestinal fluid. E The content shows the digestion stability of the microspheres in the gastrointestinal tract. The residual amounts of EGCG in SSF / SGF / SIF are 99.32±0.08, 98.46±0.06, 96.91±0.09, V E The residual amounts in SSF / SGF / SIF were 99.39±0.05, 98.67±0.05, and 97.41±0.05, respectively; b is the difference in weight of the microspheres after they were soaked in simulated gastrointestinal fluid, indicating the digestive stability of the microspheres in the gastrointestinal tract, and the residual amounts were 97.80±0.47, 96.45±0.54, and 93.57±0.18, respectively; c is the difference in microsphere structure after they were soaked in simulated gastrointestinal fluid, indicating the digestive stability of the microspheres in the gastrointestinal tract. The results showed that the hydrogel microspheres had good stability in the gastrointestinal tract and maintained the integrity of the microsphere structure.

[0048] 2.5 Testing the absorption capacity of hydrogel microspheres for dietary fat The ability of hydrogel microspheres to absorb fat from emulsified fat was determined to test the absorbability of dietary fat by hydrogel microspheres. The specific method was as follows: First, various lipids (soybean oil, peanut oil, olive oil, lard, butter, and peanut butter) and free fatty acids (oleic acid, linoleic acid, and docosahexaenoic acid (DHA)) were mixed with artificial bile acid and simulated intestinal fluid in a ratio of 1:2:99, and the emulsified fat was prepared by stirring at 2000 rpm for 1 h. Then, the hydrogel microspheres were placed in the emulsified fat at 37 °C and 100 rpm for 2 h. The transmittance (T) of the emulsified fat before and after treatment was observed by an ultraviolet spectrophotometer (UV), and the fat content in the emulsified fat was observed by a microscope. The liposorption ability of the hydrogel microspheres was also evaluated by the weight difference of the hydrogel microspheres.

[0049] The results are as follows Figure 7 As shown in a, the digital photo and fluorescence microscope image of soybean oil show that the emulsified fat has obviously changed from an opaque state to a transparent state, and only sparse coumarin 6 fluorescently labeled fat droplets can be seen, indicating that the hydrogel microspheres have effectively absorbed fat. The weight difference of the hydrogel microspheres in b further shows that the emulsified fat has decreased. Compared with the hydrogel microspheres without polyphenol-based micelles (PFFT), the weight gain of the hydrogel microspheres (PFAT) after absorbing fat has increased by 19.8%, showing excellent fat absorption capacity. Figure c shows that the liposuction efficiency of the hydrogel for all dietary lipids is 63.10%-81.13%, and the liposuction efficiency from left to right is 72.65±3.31, 68.16±5.05, 63.10±9.27, 66.33±14.36, 65.07±10.49, 81.13±1.01, 73.58±4.70, 74.25±3.36, and 76.74±6.67, which proves the super liposuction ability of the hydrogel microspheres for various dietary fats.

[0050] Example 3 Application of hydrogel microspheres in the preparation of drugs for preventing intestinal absorption of dietary fat To investigate the ability of hydrogel microspheres to prevent the absorption of dietary fat in the gastrointestinal tract, 8-week-old male Sprague Dawley (SD) rats (200 ± 20 g) of specific pathogen-free (SPF) grade were used as research subjects, and soybean oil was fluorescently labeled with coumarin 6.

[0051] The specific method is that the rats were divided into three treatment groups: oral administration of 1 mL soybean oil (Fat), oral administration of 1 mL soybean oil and 50 mg orlistat (Fat+orlistat), and oral administration of 1 mL soybean oil and 100 mg hydrogel microspheres prepared in Example 2 (Fat+PFAT). After oral administration, blood samples were drawn from the rat tail vein every 2 h until 12 h. The collected blood samples were centrifuged at 3000 rpm for 15 min, and then the fat and free fatty acid content in the plasma were analyzed using the corresponding detection kit. In order to explore the biological deposition of oil in the rat intestine, the rats were euthanized and their gastrointestinal tract was removed at two different time points after oral administration: 6 h and 16 h, and fluorescence imaging was performed using IVIS, with excitation and emission wavelengths of 465 nm and 520 nm, respectively.

[0052] The results are as follows Figure 8 As shown, Figure 8 Figure a shows the fluorescence imaging of the gastrointestinal tract, and figure b shows the triglyceride and free fatty acid content in the blood. The results show that the hydrogel microspheres effectively prevent the absorption of dietary fat in the gastrointestinal tract.

[0053] Example 4 Application of hydrogel microspheres in the preparation of drugs for treating obesity 4.1 Testing the efficacy of hydrogel microspheres in obesity management Based on the in vitro verification that the hydrogel microspheres have excellent absorption capacity, their therapeutic potential in obesity treatment was further evaluated using rats fed for 30 days. Male Sprague Dawley (SD) rats were used as the research subjects. After being fed a high-fat diet and orally taking hydrogel microspheres for 30 days, body weight-related indicators were measured to verify the application effect of hydrogel microspheres in obesity management. The rats were randomly divided into four groups: a normal diet group (ND), a high-fat diet group (HFD), a high-fat diet group (HFD + PFAT) with the addition of hydrogel microspheres prepared in Example 2, and a high-fat diet group (HFD + PFFT) with the addition of traditional hydrogel (i.e., hydrogel prepared using only sodium alginate in Example 2); during the observation period, the body weight of each rat was recorded every 5 days, and the rats were fed once a day, with the amount of food being as follows: Fig. 9 As shown, after 30 days of feeding, the rats were fasted for 16 h, and then blood was drawn from the abdominal aorta for analysis of fat content, adipose tissue was collected and weighed, and major organs (heart, liver, spleen, lung, kidney, stomach, and intestine) were collected and sectioned for hematoxylin and eosin (H&E) staining, especially the liver was weighed and measured. Fig. 9The feeding amount of ND group on days 5, 10, 15, 20, 25, and 30 was 30.18±2.43, 60.80±1.99, 91.26±3.58, 120.93±1.77, 149.02±2.23, and 176.18±1.82, respectively; that of HFD group was 20.68±1.39, 41.06±2.84, 60.00±1.75, 77.21±2.39, 92.24±2.18, and 105.26±1.74, respectively; The weights of the PFAT group were 20.85±2.21, 41.24±2.17, 59.74±1.00, 76.51±2.00, 90.97±2.65, and 103.70±1.81, respectively; the weights of the HFD + PFFT group were 20.38±1.98, 40.77±1.99, 58.56±2.42, 74.75±1.81, 88.63±2.30, and 99.18±1.75, respectively (unit: g).

[0054] The results are as follows Fig.10As shown in the figure, a is a graph showing the weight changes of rats in different treatment groups after 30 days of feeding. The weight analysis showed that the weight of the HFD + PFFT group and the HFD + PFAT group increased by 90.90 ± 7.74 g (40.78%) and 77.34 ± 7.12 g (33.49%), respectively, which was lower than the 116.42 ± 11.06 g (50.51%) of the HFD group. The weight gain of the ND group was equivalent to that of the HFD + PFAT group, which was 69.14 ± 10.91 g (30.10%). These results show that the administration of hydrogel microspheres can reduce the weight gain of the HFD group by 17.02%, which is almost consistent with the weight change of the ND group. b is a morphological graph of rats in different treatment groups after 30 days of feeding. From the perspective of rat morphology, the rats in the HFD + PFAT group are very similar in size to the rats in the ND group, and are significantly slimmer than rats treated with HFD alone. c is a graph showing the weight of the liver and adipose tissue of rats in different treatment groups after 30 days of feeding, which is a typical indicator of obesity. HE staining of liver tissue showed that the lipid droplets (white circles) in the HFD + PFAT group were significantly lower than those in the HFD group, indicating that the degree of liver damage was reduced. The data graphs from left to right are the weight of liver, peri-epididymal adipose tissue, and peri-renal adipose tissue. Through the comparison of the morphology and quality of these weight-related liver and adipose tissues, it was found that compared with the HFD group (12.69 ± 1.80 g, 5.08 ± 0.27 g, 6.30 ± 0.85 g), the weight of liver, epididymal fat, and peri-renal fat in the HFD + PFAT group was lower (9.59 ± 1.66 g, 3.59 ± 0.41 g, 3.54 ± 0.56 g), and the content level was similar to that of the ND group (9.83 ± 1.44 g, 3.16 ± 0.17 g, 3.50 ± 0.92 g).

[0055] 4.2 In vitro test of the ability of hydrogel microspheres to eliminate dietary fat In order to solve the key problem of completely inhibiting intestinal absorption and subsequent disposal of fat in obesity treatment, the present invention closely monitored the dietary intervention in 4.1 for 30 days, collected the feces of rats, and examined the in vitro elimination ability of hydrogel microspheres for dietary fat by observing the morphology, lipid content, and water content of the feces.

[0056] The results are as follows Fig.11 As shown, Fig.11 The feces excreted by the rats in the ND group in a were brown and slightly dry, while the feces excreted by the rats in the HFD group were grass green and soft in texture. In particular, the feces showed obvious yellow particles in the case of oral administration of hydrogel microspheres, indicating that the fat-absorbing hydrogel was excreted from the body. To verify this hypothesis, the fecal lipid levels of the rats were analyzed, and the results are shown in Figure 2. Fig.11As shown in b, the four figures in b respectively represent the contents of triglyceride, cholesterol, free fatty acid and bile acid in feces. Compared with rats that only consumed HFD, the contents of triglyceride, cholesterol, free fatty acid and bile acid in feces of rats that consumed HFD + PFAT increased, quantitatively revealing that dietary fat was carried out of the body by hydrogel microspheres.

[0057] Comparative Example Several different phenols are provided to replace V in Example 1. E To prepare the hydrophobic cavity, Quercetin, Rutin, Curcumin, Caffeic acid, V E The micelle sizes formed with EGCG are as follows Fig.12 a, which are 825.4±24.3nm, 342.6±11.7nm, 164.6±7.5nm, 255.4±9.7nm, and 574±32.5nm, respectively. Fig.12 a and Figure 2 The size of the micelles in the medium proves that only EGCE and V E The reaction can prepare specific hydrophobic micelles, and the prepared hydrogel microspheres have hydrophobic cavities.

[0058] Several different phenols were provided to replace the EGCG in Example 1 to prepare microspheres. E The micelles were obtained by the reaction, and the micelle particle size was Fig.12 As shown in b, theabrownin, caffeic acid, kaempferol, quercetin, and EGCG were respectively E The particle sizes of the formed micelles were 712.4±39.3nm, 295.3±15.6nm, 458.7±26.5nm, 336.5±17.4nm, and 574±32.5nm.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing hydrogel microspheres, characterized in that: The method is: Epigallocatechin gallate and vitamin E were self-assembled in an ethanol solution to form hydrophobic polyphenol-based micelles; The hydrophobic polyphenol-based micelles are uniformly mixed with a sodium alginate solution to form a pre-gel; The pregel was cross-linked with calcium ions, washed, and freeze-dried.

2. The preparation method according to claim 1, characterized in that The mass ratio of epigallocatechin gallate, vitamin E and sodium alginate is 1:(0.7-0.9):(0.6-1.2).

3. The preparation method according to claim 1, characterized in that: The mass concentration of the sodium alginate is 1.5-3%.

4. The preparation method according to claim 1, characterized in that: The freeze drying step is freeze drying at −80 °C for 36-48 h.

5. The preparation method according to claim 1, characterized in that: The calcium ion concentration is 1.5-2%.

6. The hydrogel microspheres obtained according to any one of the preparation methods of claims 1-5.

7. Use of the hydrogel microspheres as claimed in claim 6 in the preparation of a medicament for preventing intestinal absorption of dietary fat.

8. Use of the hydrogel microspheres as claimed in claim 6 in preparing a drug for treating obesity.

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