Hydrogel microspheres, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of hydrogels. Disclosed are hydrogel microspheres, a preparation method therefor, and use thereof. According to the present invention, epigallocatechin gallate and vitamin E are self-assembled into a hydrophobic polyphenol-based micelle, wherein the hydrophobic polyphenol-based micelle, with EGCG as the core, forms a stable structure by means of non-covalent interactions (such as hydrophobic interactions, hydrogen bonding, and π-π stacking), thereby enhancing the stability and functionality of the hydrophobic core. Then, the hydrophobic structure is mixed with hydrophilic sodium alginate to form a gel, ultimately resulting in hydrogel microspheres with a unique amphiphilic heterogeneous structure. The hydrogel microspheres can exist stably in an aqueous environment, and actively capture dietary fats, thereby improving the ability to capture dietary fats, and improving the stability of the hydrogel microspheres in the gastrointestinal tract.

Description

Hydrogel microspheres, preparation method and application thereof TECHNICAL FIELD

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

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

[0003] The currently reported biomaterials for targeting the intestinal absorption of dietary fat mainly include ionic liquids, sucrose acid salts and dietary fibers. Ionic liquids and sucrose acid salts reduce the absorption of dietary fat by forming a barrier between intestinal cells and dietary fat, but undigested fat remaining in the intestine can cause physiological problems such as steatorrhea. Dietary fibers (resistant starch and nanocellulose) can reduce such intestinal side effects, and based on their unique macromolecular complex structure, they can effectively reduce the absorption of dietary fat in the intestine, however, due to their non-intelligent response structure, they lack the ability to dynamically target and capture dietary fat. SUMMARY

[0004] To solve the problems in the background art, the present application provides a hydrogel microsphere, a preparation method and application thereof. The hydrogel microsphere provided by the present application 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-mentioned purpose, the first technical solution adopted by the present application is:

[0006] The preparation method of the hydrogel microsphere is as follows:

[0007] Self-assembling epigallocatechin gallate and vitamin E in an ethanol solution to form a hydrophobic polyphenol-based micelle;

[0008] Uniformly mixing the hydrophobic polyphenol-based micelle with a sodium alginate solution to form a pre-gel;

[0009] Crosslinking the pre-gel with calcium ions, and washing and freeze-drying.

[0010] Preferably, the mass ratio of epigallocatechin gallate, vitamin E and sodium alginate is 1: (0.7-0.9): (0.6-1.2).

[0011] Preferably, the mass concentration of sodium alginate is 1.5-3%.

[0012] Preferably, the freeze-drying is freeze-drying at -80 DEG C for 36-48 h.

[0013] Preferably, the calcium ion concentration is 1.5-2%.

[0014] The second technical solution adopted by the present application is:

[0015] The hydrogel microspheres obtained by the preparation method of the first technical solution.

[0016] The third technical solution adopted by the present application is:

[0017] The application of the hydrogel microspheres in the second technical solution in the preparation of a drug for preventing intestinal absorption of dietary fat.

[0018] The fourth technical solution adopted by the present application is:

[0019] The application of the hydrogel microspheres in the second technical solution in the preparation of a drug for treating obesity.

[0020] Compared with the prior art, the present application has the following effects:

[0021] The present application forms a hydrophobic polyphenol-based micelle by self-assembling epigallocatechin gallate and vitamin E, and the hydrophobic polyphenol-based micelle takes EGCG as a core and forms a stable structure through non-covalent interaction (such as hydrophobic interaction, hydrogen bond and π-π stacking), thereby enhancing the stability and functionality of the hydrophobic core; then the hydrophobic structure is mixed with a hydrophilic sodium alginate gel, and finally a hydrogel microsphere with a unique amphiphilic heterogeneous structure is obtained, which can exist stably in a water environment, actively capture dietary fat, improve the capture capacity of dietary fat, and improve the stability of the hydrogel microsphere in the gastrointestinal tract. BRIEF DESCRIPTION OF DRAWINGS

[0022] Fig. 1 is the demulsification effect of the hydrogel microspheres with different proportions in Example 1, a is the transmittance of the emulsion, and b is the weight difference of the hydrogel microspheres before and after fat absorption;

[0023] Fig. 2 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;

[0024] Figure 3 is the amphiphilic property 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 taken by fluorescence microscope, c is the contact angle test result of the amphiphilic property of the hydrogel microspheres;

[0025] Figure 4 is the 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 chart, c, d are the thermogravimetric analysis charts;

[0026] Figure 5 is the biocompatibility of the hydrogel microspheres prepared in Example 2, a is the cytotoxicity, b is the cell viability;

[0027] Figure 6 is the gastrointestinal stability of the hydrogel microspheres prepared in Example 2, a is the detection of EGCG / V E content in the supernatant after the microspheres are soaked in simulated gastrointestinal fluid, b is the detection of weight difference of the microspheres after the microspheres are soaked in simulated gastrointestinal fluid, c is the detection of structural difference of the microspheres after the microspheres are soaked in simulated gastrointestinal fluid;

[0028] Figure 7 is the absorption capacity of the hydrogel microspheres prepared in Example 2 to dietary fat, a is the digital photo and fluorescence microscope image of soybean oil, b is the weight difference of the hydrogel microspheres before and after absorbing fat, c is the absorption rate of the hydrogel to all dietary lipids;

[0029] Figure 8 is the fluorescence imaging of the gastrointestinal tract of rats in Example 3, b is the content of triglyceride and free fatty acid in the blood of rats;

[0030] Figure 9 is a statistical chart of food intake of animals in each group in Example 4;

[0031] Figure 10 is the application effect of the hydrogel microspheres in obesity management in Example 4, a is the weight change chart of rats in different treatment groups after 30 days of feeding, b is the morphology chart of rats in different treatment groups after 30 days of feeding, c is the weight chart of liver and adipose tissue of rats in different treatment groups after 30 days of feeding;

[0032] Figure 11 is the in vitro exclusion capacity of the hydrogel microspheres to dietary fat in Example 4, a is the feces excreted by each group of rats; b from left to right is the content of triglyceride, cholesterol, free fatty acid and bile acid in the feces of each group;

[0033] Figure 12 is the particle size of different micelles in the comparative example. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present application. In addition, in the following description, the description of well-known structures and techniques is omitted to avoid unnecessary confusion of the concept of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased on the market.

[0035] The first embodiment of the present application provides a preparation method of hydrogel microspheres, which is as follows:

[0036] Self-assembling epigallocatechin gallate and vitamin E in an ethanol solution to form a hydrophobic polyphenol-based micelle;

[0037] Uniformly mixing the hydrophobic polyphenol-based micelle with a sodium alginate solution to form a pre-gel;

[0038] Crosslinking the pre-gel with calcium ions, and cleaning and freeze-drying.

[0039] In the embodiment of the present application, epigallocatechin gallate (EGCG) and vitamin E are self-assembled into a hydrophobic polyphenol-based micelle. The hydrophobic polyphenol-based micelle takes EGCG as the core and forms a stable structure through non-covalent interaction (such as hydrophobic interaction, hydrogen bonding, and π-π stacking), thereby enhancing the stability and functionality of the hydrophobic core. Then, the hydrophobic structure is mixed with a hydrophilic sodium alginate gel, and finally, a unique amphiphilic heterogeneous structure hydrogel microsphere is obtained. The hydrogel microsphere ingeniously combines the hydrophobic and hydrophilic characteristics in the same layer, forms an integrated heterogeneous network, and enables the hydrogel microsphere to exist stably in a water environment, actively capture dietary fat, improve the capture capacity of dietary fat, and improve the stability of the hydrogel microsphere in the gastrointestinal tract.

[0040] It should be noted that pure EGCG composition can only break emulsion, and the binding force between EGCG and dietary fat is insufficient, so that dietary fat is not easy to remain in the EGCG microsphere. It is found in the implementation process of the present application that only EGCG, a kind of polyphenol substance, can self-assemble with vitamin E to obtain a cavity hydrophobic polyphenol-based micelle, thereby enhancing the binding force with dietary fat, firmly locking the dietary fat inside the microsphere, and enhancing the functionality of the hydrophobic core. Then, the hydrophobic polyphenol-based micelle is filled in a dietary matrix to construct an amphiphilic heterogeneous structure hydrogel microsphere.

[0041] It should be further noted that the heterostructure hydrogel obtained by the present application specifically refers to the amphiphilic interface structure formed by different materials or different structures of the same material, and these heterostructure regions have different structures on the micro or macro level, resulting in different mechanical properties, water absorption performance or biological activity; these heterostructure hydrogels can have a layered structure, encapsulated particles or other types of composite structure. However, the heterostructure hydrogel in the prior art is a mixture containing different components, which is only a heterostructure concept proposed compared with homogeneous hydrogel: homogeneous hydrogel refers to a hydrogel composed of a single type of polymer network, and the chemical and physical properties are uniformly distributed in the whole system; in the homogeneous hydrogel, the polymer chains and crosslinking points are uniformly distributed on a macro scale; this means that the same structure and composition will be found in any part of the hydrogel, whether observed from a micro or macro perspective; the heterostructure hydrogel in the prior art is usually composed of different composite components for its function, and is mostly a heterostructure multi-level structure.

[0042] The ethanol solution is used to dissolve vitamin E and EGCG, and the amount thereof is preferably selected according to the actual situation so that the vitamin E and EGCG are fully dissolved, and the sodium alginate gel does not agglomerate after the addition of sodium alginate.

[0043] 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).

[0044] In some preferred embodiments, the mass concentration of the sodium alginate is 1.5-3%.

[0045] For the conditions of freeze-drying, there is no special limitation, and those skilled in the art can adaptively adjust according to the required product performance. For example, in some preferred embodiments, the freeze-drying is freeze-drying at -80 °C for 48 h.

[0046] In some preferred embodiments, in order to further increase the mechanical properties of the hydrogel, calcium salt can also be added when preparing the pre-gel, and after the subsequent crosslinking of the pre-gel with calcium ions, hydrochloric acid is added to release Ca 2+ , which is crosslinked with the hydrogel inside the microspheres, thereby improving the gelation inside the microspheres. The calcium salt can be calcium disodium ethylenediaminetetraacetate.

[0047] The second embodiment of the present application provides the hydrogel microspheres obtained according to the first embodiment.

[0048] The hydrogel microspheres of the embodiments of the present application are hydrogels with amphiphilic heterostructure, which can enhance the adsorption capacity of dietary fat and achieve efficient capture of dietary fat.

[0049] The third embodiment of the present application provides an application of the hydrogel microspheres of the second embodiment in the preparation of a drug for preventing the intestinal absorption of dietary fat.

[0050] The hydrogel microspheres of the embodiments of the present application have a dual effect in preventing the intestinal absorption of dietary fat: first, the hydrophilic regions on the surface thereof can be in contact with emulsified fat droplets to effectively capture them, laying a foundation for subsequent fat locking; second, the hydrophobic structure inside the hydrogel microspheres can interact with the oil released by demulsification, firmly locking the oil inside the microspheres and discharging it out of the body without causing adverse reactions such as steatorrhea. Under the dual driving of the interfacial activity of EGCG and the non-absorbability of the sodium alginate dietary matrix, the hydrogel microspheres can maintain the integrity of their structure in the gastrointestinal environment and safely wrap dietary fat inside the microspheres until it is naturally discharged out of the body. Therefore, the hydrogel microspheres of the embodiments of the present application can be applied to the preparation of a drug for preventing the intestinal absorption of dietary fat.

[0051] The fourth embodiment of the present application provides an application of the hydrogel microspheres of the second embodiment in the preparation of a drug for treating obesity.

[0052] The hydrogel microspheres of the embodiments of the present application achieve the treatment of obesity by preventing the intestinal absorption of dietary fat, effectively avoiding the common adverse reactions such as diarrhea, nausea and bloating in traditional anti-obesity biomaterial intervention measures, and showing great potential in obesity management. Therefore, the hydrogel microspheres of the embodiments of the present application can be applied to the preparation of a drug for treating obesity.

[0053] To make the technical solutions of the present application clearer, the preparation and performance of the hydrogel microspheres are described below through a plurality of specific examples. Example 1 1.1 Preparation of hydrogel microspheres

[0054] First, epigallocatechin gallate (EGCG) and vitamin E (Vitamin, V E ) are self-assembled in an ethanol solution to form a hydrophobic polyphenol-based micelle Fat lock. Then, the hydrophobic polyphenol-based micelle is added dropwise to a 2% sodium alginate solution, stirred to form a uniform pre-gel, and then cross-linked by Ca 2+ ions to form hydrogel microspheres. Finally, the residual ions are removed by washing with distilled water several times, and the amphiphilic hydrogel microspheres (Polyphenol-based fat trap, PFAT) are obtained by freeze-drying at -80 °C for 48 h.

[0055] 1.2 Study the effect of different raw material dosages on hydrogel microspheres

[0056] According to the method in 1.1, the mass ratio of sodium alginate: EGCG: V E of 0:0:0, 1:0:0, 1:1:0, 6:10:7, 6:10:9, 4:5:4, 12:10:9, and 12:10:7, respectively, to prepare hydrogel microspheres, and then detect the change in transmittance of the emulsion before and after treatment by ultraviolet spectrophotometer, and then explore the effect of demulsification and fat absorption. By determining the ability of hydrogel microspheres to absorb fat from emulsified fat, the fat absorption of hydrogel microspheres is tested.

[0057] The specific method is as follows: soybean oil is used as a fat model, which is mixed with artificial bile acid and simulated intestinal fluid at a ratio of 1:2:99, and stirred at a speed of 2000 rpm for 1 h to prepare emulsified fat. Then, the hydrogel microspheres are placed in the emulsified fat at 37 °C, and oscillated at 100 rpm for 2 h. The transmittance (T) of the emulsified fat before and after treatment is observed by ultraviolet spectrophotometer (UV), and the fat content in the emulsified fat is observed by microscope. The fat absorption capacity of the hydrogel microspheres is evaluated by the weight difference.

[0058] The results are shown in Figure 1, where a is the transmittance of the emulsion, and the transmittance from left to right is 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, and 63.93±1.26, respectively, in units of %; b is the weight difference of the hydrogel microspheres before and after fat absorption, and the weight difference from left to right is 0.149±0.027, 0.298±0.017, 0.210±0.017, 0.277±0.029, 0.347±0.021, 0.246±0.014, and 0.190±0.034, respectively, in units of g.

[0059] The mass ratio of sodium alginate: EGCG: V E of 4:5:4 is detected for its structure, and the results are shown in Figure 2, where a is the UV spectrum obtained by ultraviolet spectrophotometer, b is the dynamic light scattering particle size obtained by dynamic light scattering instrument, and c is the transmission electron microscope image of the hydrophobic cavity. In Figure b, the particle size of EGCG is 379.83±11.77 nm, the particle size of V E is 171.23±7.72 nm, and the particle size of Fat lock is 574±32.54 nm. As can be seen from Figure 2, the hydrogel microspheres prepared by the present application have a hydrophobic cavity.

[0060] Example 2 Preparation and performance detection of hydrogel microspheres

[0061] 2.1 Preparation of hydrogel microspheres

[0062] 1 g of EGCG and 0.8 g of vitamin E were dissolved in 3 mL of anhydrous ethanol solution, oscillated at a speed of 200 rpm for 30 min to form hydrophobic polyphenol-based micelles; then, a 2% w / v sodium alginate solution and a 2% w / v calcium disodium ethylenediaminetetraacetate 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 added dropwise to 100 mL of a 2% w / v calcium chloride (CaCl2) aqueous solution through a constant-flow pump and a thin hose (3 cm 3 ) at a speed of 300 rpm, and the resulting hydrogel microsphere intermediate was allowed to solidify in the CaCl2 aqueous solution for 4 h; then, the intermediate was immersed in a 0.1 mol / L hydrochloric acid (HC1) solution for 1 h to allow the EDTA-Ca to release Ca 2+ to crosslink the pre-gel inside the microspheres. Finally, the residual ions were removed by washing several times with distilled water, and the resulting hydrogel microspheres PFAT were freeze-dried at -80 °C for 48 h.

[0063] The contact angle of the hydrogel microspheres PFAT was detected, and the amphiphilic interface thereof was photographed using a fluorescence microscope, and the results are shown in FIG. 3. The results show that the prepared hydrogel microspheres have amphiphilicity, which demonstrates the potential for breaking emulsion and absorbing fat, and FIG. 3a is the macroscopic morphology of the hydrogel microspheres before freeze-drying (Fresh PFAT) and after freeze-drying (Dried PFAT). b is the amphiphilic interface of the hydrogel microspheres photographed by the fluorescence microscope: the rhodamine 6G interface is the sodium alginate matrix-hydrophilic; the coumarin 6 is the hydrophobic cavity-hydrophobic; the bright field picture; the hydrophilic and hydrophobic combined image. c is the contact angle test of the hydrogel microspheres, and the water contact angle is 17.5° and the oil contact angle is 51.1°.

[0064] 2.2 Preparation and characterization of hydrogel microspheres

[0065] The hydrogel microspheres were analyzed by Fourier transform infrared spectroscopy (FTIR), organic elemental analyzer (EA), and thermogravimetric analyzer (TGA). The specific method is as follows: the hydrogel microspheres were mixed with potassium bromide (KBr) at a ratio of 1:100 w / w, and then pressed into a transparent wafer, which was analyzed by FTIR at 25 °C, 400-4000 cm -1FTIR: The sample was scanned in the wavelength range; TGA curve was tested in the temperature range of 30-900 °C after 5 mg hydrogel microspheres were completely ground, the test conditions were nitrogen environment, the flow rate was 40 mL / min, and the heating speed was 10 °C / min; in order to quantify the content of carbon (C) and hydrogen (H), the sample was completely burned in an oxidation tube at 1150 °C in a pure oxygen environment; in order to analyze the oxygen (O) content, the hydrogel microspheres were pyrolyzed in a mixed gas stream of hydrogen (H2) and helium (He) at 1150 °C, and then the concentrations of C, H and O were determined by thermal conductivity detection.

[0066] The results show that the polyphenol cavity micelles are successfully filled in the dietary matrix to construct hydrogel microspheres, as shown in FIG. 4.

[0067] FIG. 4a is a Fourier infrared spectrum of the hydrogel microspheres, which is used to indicate the composition of the microspheres, each component has a specific peak, and the last microspheres contain the characteristic peaks of sodium alginate / EGCG / V E , indicating that the hydrogel microspheres are successfully prepared. b is an elemental analysis chart, PFFT is a microsphere prepared using pure sodium alginate in 2.1, PFAT is a hydrogel microsphere prepared in 2.1, and the change in the proportion of elements indicates that the hydrophobic polyphenol micelles are successfully filled in the sodium alginate matrix, i.e. the hydrogel microspheres are successfully prepared. c, d are thermogravimetric analysis charts, which analyze the temperature change of the decomposition of the microspheres PFFT prepared from pure sodium alginate and the microspheres PFAT prepared from sodium alginate + EGCG micelles, which show the difference in the composition of the material, and the results show that the hydrophobic polyphenol micelles are successfully filled in the sodium alginate matrix, i.e. the hydrogel microspheres are successfully prepared.

[0068] 2.3 Biological safety evaluation of hydrogel microspheres

[0069] NIH3T3 fibroblasts were used as the research object, and the biological safety of the 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) detection method. The effect of the hydrogel microspheres on cell proliferation was evaluated by Annexin V-FITC cell apoptosis detection kit and confocal laser scanning microscope (CLSM).

[0070] [Corrected according to Rule 91 on 27.08.2025] The results are shown in Figure 5, a is cell live and dead fluorescence staining, green represents live cells, red represents dead cells (circled in the figure), PBS is used as the control group, Fat locks are polyphenol-based micelles; the results show that there is no visible difference in red and green color compared with the control group, which qualitatively indicates that the cytotoxicity of the hydrogel prepared in the application is negligible; b is the CCK-8 detection of cell viability results, which quantitatively analyzes the safety of the material, and the cell viability values from left to right are 100.00±2.26, 103.71±3.17, 97.18±1.60, 98.18±2.05, respectively; the results show that there is no significant difference between the treatment group and the control group, indicating that the hydrogel microspheres prepared in the application have no significant effect on the cell viability.

[0071] 2.4 Detection of gastrointestinal stability of hydrogel microspheres

[0072] 50 mg of the hydrogel microspheres prepared in 2.1 were sequentially soaked in simulated saliva (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) for 10 min, 2 h and 2 h at 37 °C with 200 rpm oscillation reaction; after the reaction was completed, the hydrogel microspheres were separated, and 1 mL of SSF, SGF and SIF were filtered with a 0.22 μm filter, respectively, and then analyzed.

[0073] EGCG and V E in the simulated liquid were quantitatively determined by high performance liquid chromatography (HPLC): CAPCELL PAK high performance liquid chromatography column (C18, 250 mm × 4.6 mm, 5 μm) was used for high performance liquid chromatography analysis, and was equipped with an ultraviolet detector; the chromatographic conditions were set as follows: injection flow rate 1 mL / min, injection volume 10 μL, column temperature 35 °C; the mobile phases of EGCG and V E were acetonitrile:0.2% acetic acid =3:7, 100% methanol, respectively. The detection wavelengths of EGCG and V E were 278 nm and 294 nm, respectively.

[0074] 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.

[0075] The results are shown in Figure 6, a in Figure 6 is the detection of EGCG / V E content in the supernatant after the microspheres were soaked in simulated gastrointestinal fluid, which indicates the digestion stability of the microspheres in the gastrointestinal tract, and the residual amounts of EGCG in SSF / SGF / SIF are 99.32±0.08, 98.46±0.06, 96.91±0.09, respectively, V EThe residual amounts in SSF / SGF / SIF were 99.39±0.05, 98.67±0.05, and 97.41±0.05, respectively; b is the weight difference of the microspheres after immersion in simulated gastrointestinal fluid, which indicates the digestion 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 structural difference of the microspheres after immersion in simulated gastrointestinal fluid, which indicates the digestion stability of the microspheres in the gastrointestinal tract. The results show that the hydrogel microspheres have good stability in the gastrointestinal tract and maintain the integrity of the microsphere structure.

[0076] 2.5 Test of the absorption capacity of hydrogel microspheres for dietary fat

[0077] The absorption capacity of hydrogel microspheres for dietary fat was tested by measuring the ability of hydrogel microspheres to absorb fat from emulsified fat. The specific method is 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 DHA) were mixed with artificial bile acid and simulated intestinal fluid at a ratio of 1:2:99, and stirred at a speed of 2000 rpm for 1 h to prepare emulsified fat. Then, the hydrogel microspheres were placed in the emulsified fat at 37 °C, and oscillated at 100 rpm for 2 h. The transmittance (T) of the emulsified fat before and after treatment was observed using a UV spectrophotometer (UV), the fat content in the emulsified fat was observed using a microscope, and the fat absorption capacity of the hydrogel microspheres was evaluated by the weight difference of the hydrogel microspheres.

[0078] The results are shown in Figure 7, a is a digital photograph and fluorescence microscope image of soybean oil, which shows that the emulsified fat is obviously changed from an opaque state to a transparent state, and only sparse coumarin 6 fluorescently labeled fat droplets can be seen, which indicates that the hydrogel microspheres effectively absorbed the fat. The weight difference of the hydrogel microspheres in b further shows that the emulsified fat is reduced, and the weight gain of the hydrogel microspheres (PFAT) after absorbing fat is increased by 19.8% compared to the hydrogel microspheres without polyphenol-based micelles (PFFT), which shows excellent fat absorption capacity. c shows that the fat absorption efficiency of the hydrogel for all dietary lipids is 63.10%-81.13%, and the fat absorption 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, respectively, which proves the super strong fat absorption capacity of the hydrogel microspheres for various dietary fats.

[0079] Example 3 Application of hydrogel microspheres in the preparation of a drug for preventing the absorption of dietary fat in the intestinal tract

[0080] In order to study the absorption prevention ability of hydrogel microspheres to dietary fat in the gastrointestinal tract, 8-week-old male Sprague Dawley (SD) rats (200 ± 20 g) of specific pathogen free (SPF) level were used as research objects, and coumarin 6 fluorescently labeled soybean oil was used.

[0081] Specifically, the rats were divided into three treatment groups: oral administration of 1 mL of soybean oil (Fat), oral administration of 1 mL of soybean oil and 50 mg of orlistat (Fat + orlistat), and oral administration of 1 mL of soybean oil and 100 mg of hydrogel microspheres prepared in Example 2 (Fat + PFAT). After oral administration, blood samples were taken from the tail vein of the rats every 2 h until 12 h. The collected blood samples were centrifuged at a speed of 3000 rpm for 15 min, and then the content of fat and free fatty acid in the plasma was analyzed using the corresponding detection kit. In order to explore the biological deposition of oil in the intestinal tract of rats, at two different time points after oral administration: 6 h and 16 h, the rats were euthanized and their gastrointestinal tracts were removed, and fluorescence imaging was performed using IVIS with excitation and emission wavelengths of 465 nm and 520 nm, respectively.

[0082] The results are shown in Figure 8, where a is the fluorescence imaging of the gastrointestinal tract, and b is the content of triglyceride and free fatty acid in the blood. The results show that the hydrogel microspheres effectively prevent the absorption of dietary fat in the gastrointestinal tract.

[0083] Example 4 Application of hydrogel microspheres in the preparation of a drug for treating obesity

[0084] 4.1 Test of the application effect of hydrogel microspheres in obesity management

[0085] On the basis of in vitro verification of the excellent absorption capacity of the hydrogel microspheres, a 30-day-fed rat was further used to evaluate its therapeutic potential in the treatment of obesity. Male Sprague Dawley (SD) rats were used as the research object, and the body weight-related indicators were measured after high-fat diet feeding and oral administration of hydrogel microspheres for 30 days to test the application effect of hydrogel microspheres in obesity management. The rats were randomly divided into four groups: normal diet group (ND), high-fat diet group (HFD), high-fat diet group added with hydrogel microspheres prepared in Example 2 (HFD + PFAT), and high-fat diet group added with traditional hydrogel (i.e. hydrogel prepared only using sodium alginate in Example 2) (HFD + PFFT); during the observation period, the body weight of each rat was recorded every 5 days, and the rats were fed once a day, and the feeding amount is shown in FIG. 9. After feeding for 30 days, the rats were fasted for 16 h, and then blood was drawn from the abdominal aorta for analysis of fat content, fat tissue was collected and weighed, major organs (heart, liver, spleen, lung, kidney, stomach and intestine) were collected and sectioned for hematoxylin and eosin (H&E) staining, and the liver was particularly weighed and measured. In FIG. 9, the feeding amount of ND at 5, 10, 15, 20, 25, and 30 days 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 the 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; that of the HFD + PFAT group was 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; and that of the HFD + PFFT group was 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, with units of g.

[0086] Results are shown in Figure 10, a is the weight change graph of rats in different treatment groups after 30 days of feeding. Body weight analysis showed that the body 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 that of the HFD group, which was 116.42 ± 11.06 g (50.51%). The weight gain of the ND group was comparable to that of the HFD + PFAT group, which was 69.14 ± 10.91 g (30.10%). These results showed that the use of hydrogel microspheres could reduce the weight gain of the HFD group by 17.02%, which was almost identical to 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 appearance of the rats, the rats in the HFD + PFAT group were very similar to the rats in the ND group, and were significantly slimmer than the rats treated with HFD only. c is a graph of the weight of 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 a decrease in liver damage. The data graph from left to right is a graph of the weight of liver, epididymal adipose tissue, and perirenal adipose tissue. By comparing the morphology and mass of these weight-related liver and adipose tissues, it was found that the weight of the liver, epididymal fat, and perirenal fat in the HFD + PFAT group (9.59 ± 1.66 g, 3.59 ± 0.41 g, 3.54 ± 0.56 g) was lower than that in the HFD group (12.69 ± 1.80 g, 5.08 ± 0.27 g, 6.30 ± 0.85 g), and the content level was similar to that in the ND group (9.83 ± 1.44 g, 3.16 ± 0.17 g, 3.50 ± 0.92 g).

[0087] In vitro exclusion ability test of hydrogel microspheres on dietary fat

[0088] To solve the key problem of completely inhibiting intestinal absorption and subsequent disposal of fat in obesity treatment, the present application closely monitors the feces of rats after 30 days of dietary intervention in 4.1, and tests the in vitro exclusion ability of hydrogel microspheres on dietary fat by observing the morphology, lipid content, and water content of the feces.

[0089] The results are shown in Figure 11, Figure 11a shows that the feces of the rats in the ND group are brown and slightly dry, while the feces of the rats in the HFD group are grass green and soft in texture. In particular, the feces present obvious yellow particles in the case of oral administration of hydrogel microspheres, which indicates that the hydrogel that absorbs fat is excreted outside the body. In order to verify this hypothesis, the rats were subjected to fecal lipid level analysis, and the results are shown in Figure 11b, which shows the contents of triglycerides, cholesterol, free fatty acids and bile acids in the feces. Compared with the rats that only ingested HFD, the contents of triglycerides, cholesterol, free fatty acids and bile acids in the feces of the rats that ingested HFD + PFAT increased, and the quantification revealed that dietary fat was taken out of the body by the hydrogel microspheres.

[0090] Comparative Example

[0091] Several different phenols were provided to replace EGCG in Example 1 to prepare microspheres, and the phenols were reacted with V E to prepare hydrophobic cavities, and Quercetin, Rutin, Curcumin, Caffeic acid, V E The micellar particle sizes formed by EGCG and the phenols, respectively, are shown in Figure 12a, which are 825.4±24.3nm, 342.6±11.7nm, 164.6±7.5nm, 255.4±9.7nm and 574±32.5nm, respectively. Comparing Figure 12a with the micellar particle sizes in Figure 2, it is proved that only EGCE can form micelles with V E to prepare specific hydrophobic micelles, and the prepared hydrogel microspheres have hydrophobic cavities.

[0092] Several different phenols were provided to replace EGCG in Example 1 to prepare microspheres, and the phenols were reacted with V E to prepare micelles, and the micellar particle sizes are shown in Figure 12b, which are 712.4±39.3nm, 295.3±15.6nm, 458.7±26.5nm, 336.5±17.4nm and 574±32.5nm, respectively. E

[0093] The above only describes preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. A method for preparing hydrogel microspheres, characterized by, The method comprises: Self-assembling epigallocatechin gallate and vitamin E in an ethanol solution to form hydrophobic polyphenol-based micelles; Mixing the hydrophobic polyphenol-based micelles with a sodium alginate solution to form a pre-gel; Cross-linking the pre-gel with calcium ions, and cleaning and freeze-drying.

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

3. The production method according to claim 1, wherein The mass concentration of sodium alginate is 1.5-3%.

4. The production method according to claim 1, wherein The freeze-drying is carried out at -80 °C for 36-48 h.

5. The production method according to claim 1, wherein The concentration of calcium ions is 1.5-2%.

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

7. The use of the hydrogel microspheres according to claim 6 in the preparation of a drug for preventing the absorption of dietary fat in the intestinal tract.

8. The use of the hydrogel microspheres according to claim 6 in the preparation of a drug for treating obesity.

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