Resveratrol-tea polyphenol composite particles for preventing and treating non-alcoholic fatty liver disease and preparation method of resveratrol-tea polyphenol composite particles

Abstract

The invention provides resveratrol-tea polyphenol composite particles for preventing and treating non-alcoholic fatty liver diseases and a preparation method of the resveratrol-tea polyphenol composite particles, and belongs to the technical field of nutritional active ingredient delivery systems. According to the preparation method, a zein-carrageenan-carboxymethyl chitosan composite carrier is constructed through an anti-solvent coprecipitation method and a layer-by-layer self-assembly technology, and resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles are formed. Compared with free resveratrol and tea polyphenol, the composite particles prepared by the invention can effectively improve the physical and chemical stability and efficacy performance of resveratrol and tea polyphenol, significantly improve the bioavailability of resveratrol and tea polyphenol and the content of resveratrol and tea polyphenol in rat plasma, have a stronger function of improving fatty degeneration of the liver, and can be used for treating liver fatty degeneration. Good application prospects are realized in the aspect of development of functional products for preventing and treating the non-alcoholic fatty liver diseases.

Description

Technical Field

[0001] This invention relates to the field of nutritional active ingredient delivery system technology, specifically to a resveratrol-tea polyphenol composite particle for the prevention and treatment of non-alcoholic fatty liver disease and its preparation method. Background Technology

[0002] Resveratrol is a natural plant polyphenol widely found in plants such as grapes and Japanese knotweed. It possesses various physiological activities, including antioxidant, anti-inflammatory, hepatoprotective, and cardiovascular regulatory effects. It is particularly effective in reducing liver fat accumulation, slowing hepatic steatosis, and inhibiting hepatocyte apoptosis, thus attracting significant attention in the development of functional foods and hepatoprotective drugs. Tea polyphenols, such as epigallocatechin gallate (EGCG), have antioxidant, anti-inflammatory, and antibacterial properties, as well as good hepatoprotective effects, and can lower serum cholesterol, free fatty acids, and triglyceride levels. Multiple studies have shown that the combined use of resveratrol and tea polyphenols can enhance antioxidant, anti-inflammatory, and lipid metabolism-regulating effects. However, resveratrol has low water solubility and poor stability, resulting in low absorption and utilization rates in the body. Similarly, tea polyphenols are easily degraded by factors such as digestive juices, pH, and ionic strength, and most orally ingested EGCG is excreted before being absorbed by the gastrointestinal tract.

[0003] Zein is an amphiphilic plant protein containing 75% hydrophobic amino acids. It can self-assemble into nanoparticles, which can be used to encapsulate and protect active substances. Zein also exhibits good resistance to gastric acid, enhancing the stability of resveratrol during gastrointestinal digestion. However, using zein alone to encapsulate nutrients suffers from poor stability and leakage. Furthermore, this system is only suitable for two hydrophobic functional factors with similar molecular polarities and cannot be applied to the encapsulation of the hydrophilic functional factor EGCG. Carrageenan is a natural hydrophilic anionic linear polysaccharide with strong stability. It does not deteriorate over long periods and is stable in neutral and alkaline solutions, even without hydrolysis upon heating. Theoretically, the hydrophobic core of zein-polysaccharide core-shell nanoparticles can encapsulate hydrophobic functional factors, while the hydrophilic long chains of carrageenan form a dense shell on the surface of zein through electrostatic deposition. This not only provides binding sites for hydrophilic EGCG but also enhances the colloidal stability of the particles through steric hindrance. Carboxymethyl chitosan is an important water-soluble chitosan derivative with good biocompatibility and biodegradability. Furthermore, carboxymethyl chitosan is easily processed into nanoparticles, and functionalizing the surface of these particles with carboxymethyl chitosan makes them more suitable for drug delivery.

[0004] Although technologies such as nanoemulsification and liposomes have been used to improve the solubility of polyphenolic active ingredients, there are no reports on the synergistic effect of zein's gastric acid resistance and the carrageenan-carboxymethyl chitosan polysaccharide hierarchical network on liver protection. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a resveratrol-tea polyphenol composite granule for the prevention and treatment of non-alcoholic fatty liver disease (NAFLD) and its preparation method, thereby solving the problem of low bioavailability of active substances such as resveratrol and tea polyphenols due to poor stability and easy degradation in the existing technologies.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing resveratrol-tea polyphenol composite particles for the prevention and treatment of non-alcoholic fatty liver disease, comprising the following steps:

[0008] (1) Dissolve zein and resveratrol in ethanol to obtain a zein-resveratrol mixed solution;

[0009] (2) Prepare resveratrol-zein composite particles by reacting a zein-resveratrol mixed solution with water via antisolvent precipitation method;

[0010] (3) The resveratrol-zein protein composite particles were injected into a mixed solution of carrageenan and tea polyphenols to carry out a mixing reaction, thereby obtaining resveratrol-zein protein-tea polyphenol-carrageenan composite particles.

[0011] (4) Inject the resveratrol-zein-tea polyphenol-carrageenan composite particles into a carboxymethyl chitosan aqueous solution and remove the ethanol to obtain the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles.

[0012] To address the low bioavailability of resveratrol and tea polyphenols in existing technologies, this invention utilizes antisolvent precipitation and layer-by-layer self-assembly techniques to encapsulate hydrophobic resveratrol with zein as the core. A tea polyphenol / carrageenan composite layer and a carboxymethyl chitosan coating layer are then sequentially loaded via electrostatic adsorption, forming a pH-responsive core-shell-crown three-dimensional structure. This invention validates the photothermal stability, intestinal safety, bioavailability, and hepatoprotective function of the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles through photo / thermal stability testing, Caco-2 cytotoxicity evaluation, in vivo digestion models, and non-alcoholic fatty liver disease animal models. This provides theoretical support and a new technological pathway for the development of resveratrol-based products for the prevention and treatment of non-alcoholic fatty liver disease (such as health supplements, functional foods, or pharmaceuticals). Experiments have shown that the mass ratio of zein to resveratrol / carrageenan / carboxymethyl chitosan significantly affects the particle size, aggregation degree, and stability of the composite particles, thus impacting the efficacy of the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles. Therefore, this invention controls the mass ratio of zein, resveratrol, carrageenan, tea polyphenol, and carboxymethyl chitosan to 100:(20-40):(10-30):(5-20):(10-30) to maximize the therapeutic efficacy of resveratrol and tea polyphenols on non-alcoholic fatty liver disease. Compared to free resveratrol, the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles of this invention exhibit significantly improved physicochemical stability and efficacy in preventing and treating non-alcoholic fatty liver disease, demonstrating promising application prospects.

[0013] Furthermore, the mass ratio of zein, resveratrol, carrageenan, tea polyphenols, and carboxymethyl chitosan is 100:(20-40):(10-30):(5-20):(10-30).

[0014] Furthermore, the tea polyphenols include, but are not limited to, at least one of epigallocatechin gallate (EGCG), epicatechin (EC), epigallocatechin (EGC), and epicatechin gallate (ECG).

[0015] Further, in step (2), the antisolvent precipitation method is operated as follows: the zein-resveratrol mixed solution is added dropwise to water at a volume ratio of 1:3, and a constant temperature magnetic stirrer is used to continuously stir the solution during the process.

[0016] Furthermore, in step (3), the mixing reaction process is carried out by stirring with a constant temperature magnetic stirrer under light-proof conditions for 20-40 min.

[0017] Furthermore, in step (4), ethanol is removed by rotary evaporation at a temperature of 35-45°C and a pressure of -0.1 MPa.

[0018] Secondly, the present invention provides resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles prepared by the method described above.

[0019] Thirdly, the present invention provides the application of the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles in the preparation of products for the prevention or treatment of non-alcoholic fatty liver disease.

[0020] Fourthly, the present invention provides a product (e.g., a medicine) for the prevention or treatment of non-alcoholic fatty liver disease, comprising the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] This invention constructs a zein-carrageenan-carboxymethyl chitosan composite carrier using an antisolvent co-precipitation method and layer-by-layer self-assembly technology, forming resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles. The hydrophobic properties of zein efficiently encapsulate resveratrol, and its resistance to gastric acid significantly reduces the degradation of the active ingredient in the stomach. Simultaneously, the hydrophilic shell of carrageenan loads tea polyphenols, and the mucosal adhesion of carboxymethyl chitosan prolongs intestinal retention time, achieving synergistic delivery of hydrophobic and hydrophilic components. The resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles provided by this invention significantly reduce liver triglyceride (TG) levels, alleviate liver inflammation, and inhibit oxidative stress through synergistic effects, thereby significantly enhancing the prevention and treatment of non-alcoholic fatty liver disease. Attached Figure Description

[0023] Figure 1 The results of measuring the average particle size (A), polydispersity index (PDI) (B), and zeta potential (C) of the composite particles prepared in Examples 1-5 are shown.

[0024] Figure 2 The results show the encapsulation efficiency and loading rate of resveratrol and EGCG in the composite particles prepared in Examples 1-5.

[0025] Figure 3 The Fourier transform infrared spectroscopy analysis results are shown for the composite particles prepared in Example 1.

[0026] Figure 4 The X-ray diffraction analysis results are shown for the composite particles prepared in Example 1.

[0027] Figure 5 Field emission scanning electron microscope (FESEM) images of the microstructure of the composite particles prepared in Examples 1-5;

[0028] Figure 6 This is the result of a cytotoxicity assay;

[0029] Figure 7 The blood concentration curve of resveratrol after oral administration of RZECC compound granules;

[0030] Figure 8 This is a comparison chart of the body weight (A), adipose tissue content (B), and food energy intake (C) of mice in each group in Example 6;

[0031] Figure 9 This is a comparison chart of liver coefficient (A), serum ALT activity (B), and AST activity (C) of mice in each group in Example 6;

[0032] Figure 10 These are H&E stained sections of mouse liver tissue from each group in Example 6; Figure A is 10, scale bar is 200 μm; Figure B is 40, scale bar is 20 μm.

[0033] Figure 11 This is a schematic diagram of the oil red stained sections (20, scale bar is 100 μm) of mouse liver tissue in each group in Example 6 and the TG content.

[0034] Figure 12 This is a schematic diagram showing the levels of TG (A), TC (B), HDL-C (C), and LDL-C (D) in the serum of mice in each group in Example 6.

[0035] In the figure, * indicates p < 0.05. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] This invention provides a method for preparing resveratrol-tea polyphenol composite granules for the prevention and treatment of non-alcoholic fatty liver disease, comprising the following steps:

[0038] (1) Dissolve zein and resveratrol in ethanol to obtain a zein-resveratrol mixed solution;

[0039] (2) Prepare resveratrol-zein composite particles by mixing zein-resveratrol mixed solution with water at a volume ratio of 1:3 using an antisolvent precipitation method;

[0040] (3) The resveratrol-zein protein composite particles were injected into a mixed solution of carrageenan and tea polyphenols to carry out a mixing reaction, thereby obtaining resveratrol-zein protein-tea polyphenol-carrageenan composite particles.

[0041] (4) Inject the resveratrol-zein-tea polyphenol-carrageenan composite particles into a carboxymethyl chitosan aqueous solution and remove the ethanol to obtain the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles.

[0042] In some examples, the mass ratio of zein, resveratrol, carrageenan, tea polyphenols, and carboxymethyl chitosan is 100:(20-40):(10-30):(5-20):(10-30).

[0043] Example 1

[0044] A resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan (RZECC) composite particle is prepared by the following steps:

[0045] a. Accurately weigh 1.0 g of zein powder and place it in an Erlenmeyer flask, and add 100 mL of 70% ethanol aqueous solution. Seal the flask and stir continuously for 8-12 h using a constant temperature magnetic stirrer. Then, accurately weigh 300 mg of resveratrol and add it to the zein solution that has been stirred above. Seal and stir in the dark for at least 30 min.

[0046] b. Accurately measure 10 mL of the above resveratrol-zein mixture and use the antisolvent precipitation method to slowly add it dropwise into 30 mL of pure water at a volume ratio of 1:3. During this process, use a magnetic stirrer to continuously stir to obtain resveratrol-zein composite particles (Res-Zein, RZ). The mass ratio of zein to resveratrol is 100:30, and it is named RZ(100:30).

[0047] c. Accurately weigh 0.1 g of carrageenan powder and place it in 100 mL of pure water. Stir continuously on a magnetic stirrer for 8-12 h. Then, accurately weigh 10 mg of EGCG and add it to 20 mL of carrageenan aqueous solution. Stir for 30 min in the dark to obtain an EGCG-carrageenan mixture. Inject 40 mL of RZ (100:30) from step b into 20 mL of the above EGCG-carrageenan mixture at a uniform rate. After the injection is complete, continue stirring to obtain resveratrol-zein-EGCG-carrageenan composite particles (Res-Zein-EGCG-Car, RZEC), wherein the mass ratio of zein to carrageenan is 100:20, named RZEC (100:20).

[0048] d. Accurately weigh 0.1 g of carboxymethyl chitosan powder and place it in 100 mL of pure water. Stir continuously on a magnetic stirrer for 8-12 h. Inject 60 mL of RZEC (100:20) from step c into 20 mL of the above carboxymethyl chitosan solution at a uniform rate. After the injection is complete, continue stirring. Then, the solution after the antisolvent coprecipitation process is subjected to rotary evaporation in a rotary evaporator to remove ethanol (40℃, -0.1 MPa) to obtain RZECC composite particles, wherein the mass ratio of zein to carboxymethyl chitosan is 100:20, and named RZECC(CHI:20).

[0049] Example 2

[0050] The composite particles in this embodiment are basically the same as those in Example 1, except that step d is as follows: 0.05 g of carboxymethyl chitosan powder is accurately weighed and placed in 100 mL of pure water, and stirred continuously on a magnetic stirrer for 8-12 h. 60 mL of RZEC (100:20) from step c is uniformly injected into 20 mL of the above carboxymethyl chitosan solution. After the injection is complete, the mixture is stirred continuously. Then, the solution after the antisolvent coprecipitation process is subjected to rotary evaporation in a rotary evaporator to remove ethanol (40℃, -0.1 MPa) to obtain RZECC composite particles, wherein the mass ratio of zein to carboxymethyl chitosan is 100:10, and is named RZECC(CHI:10).

[0051] Example 3

[0052] The composite particles in this embodiment are basically the same as those in Example 1, except that step d is as follows: 0.075 g of carboxymethyl chitosan powder is accurately weighed and placed in 100 mL of pure water, and stirred continuously on a magnetic stirrer for 8-12 h. 60 mL of RZEC (100:20) from step c is uniformly injected into 20 mL of the above carboxymethyl chitosan solution. After the injection is complete, the mixture is stirred continuously. Then, the solution after the antisolvent coprecipitation process is subjected to rotary evaporation in a rotary evaporator to remove ethanol (40℃, -0.1 MPa) to obtain RZECC composite particles, wherein the mass ratio of zein to carboxymethyl chitosan is 100:15, and is named RZECC(CHI:15).

[0053] Example 4

[0054] The composite particles in this embodiment are basically the same as those in Example 1, except that step d is as follows: 0.125 g of carboxymethyl chitosan powder is accurately weighed and placed in 100 mL of pure water, and stirred continuously on a magnetic stirrer for 8-12 h. 60 mL of RZEC (100:20) from step c is uniformly injected into 20 mL of the above carboxymethyl chitosan solution. After the injection is complete, the mixture is stirred continuously. Then, the solution after the antisolvent coprecipitation process is subjected to rotary evaporation in a rotary evaporator to remove ethanol (40℃, -0.1 MPa) to obtain RZECC composite particles, wherein the mass ratio of zein to carboxymethyl chitosan is 100:25, and is named RZECC(CHI:25).

[0055] Example 5

[0056] The composite particles in this embodiment are basically the same as those in Example 1, except that step d is as follows: 0.15 g of carboxymethyl chitosan powder is accurately weighed and placed in 100 mL of pure water, and stirred continuously on a magnetic stirrer for 8-12 h. 60 mL of RZEC (100:20) from step c is uniformly injected into 20 mL of the above carboxymethyl chitosan solution. After the injection is complete, the mixture is stirred continuously. Then, the solution after the antisolvent coprecipitation process is subjected to rotary evaporation in a rotary evaporator to remove ethanol (40℃, -0.1 MPa) to obtain RZECC composite particles, wherein the mass ratio of zein to carboxymethyl chitosan is 100:30, and is named RZECC(CHI:30).

[0057] The composite particles prepared in Examples 1-5 were tested, and the specific test results are as follows:

[0058] 1. Determination of average particle size, polydispersity index (PDI), and zeta potential.

[0059] A suitable amount of composite particle sample was placed in a DTS0012 sample cell, and the particle size distribution (PDI) of the sample was measured at 25℃. Another suitable amount of composite particle sample was placed in a DTS1070 potential cell, and the zeta potential of the sample was measured after an equilibration time of 120 s and an interval of 30 s. All samples were measured three times consecutively, and the average value was taken.

[0060] Figure 1The results of measuring the average particle size (A), polydispersity index (PDI) (B), and zeta potential (C) of the composite particles are presented. The results show that the average particle size of the RZECC composite particles increases with increasing carboxymethyl chitosan content (407.97 nm → 598.93 nm), and the PDI trend is similar to that of the average particle size, increasing with increasing carboxymethyl chitosan content (0.4892 → 0.5746). This may be because excessive carboxymethyl chitosan content exceeds the adsorption site capacity on the composite particle surface, causing free carboxymethyl chitosan molecules to form micelles or aggregates in the solution, coexisting with the main particles and exacerbating the polydispersity of the system. Figure 1 The C-value shows that the Zeta potential of the RZECC composite particles ranges from -41.04 mV to -45.73 mV, and the negative charge density gradually increases with the increase of carboxymethyl chitosan content. This indicates that carboxymethyl chitosan and carrageenan produce charge synergy, further enhancing the negative charge on the particle surface, and depositing on the surface of zein molecules through electrostatic interactions.

[0061] 2. Determination of encapsulation efficiency and loading rate

[0062] (1) Determination of resveratrol encapsulation efficiency and loading rate: Accurately weigh 10 mg of resveratrol powder, dissolve it in 10 mL of 70% ethanol aqueous solution to prepare a resveratrol solution with a concentration of 1 mg / mL, and then dilute it with 70% ethanol aqueous solution to obtain standard solutions with concentrations of 2.0 ~ 6.0 µg / mL. Measure the absorbance value at the characteristic absorption peak of resveratrol at 306 nm, and establish a standard curve of resveratrol content (x) versus absorbance value (y): y = 0.1271x - 0.0078, R 2 =0.9992. The composite particle dispersions prepared in Examples 1-5 were centrifuged (1000 rpm, 10 min). The supernatant after centrifugation was diluted with 70% ethanol aqueous solution by an appropriate factor, and its absorbance value was measured at 306 nm. The concentration of free resveratrol was calculated according to the above standard curve. The amount of free resveratrol was obtained by subtracting the amount of free resveratrol from the total amount of resveratrol added. The encapsulation efficiency and loading rate of resveratrol in the composite particles were calculated by formula (1) and formula (2), respectively:

[0063]

[0064]

[0065] (2) Determination of EGCG encapsulation efficiency and loading rate: Accurately weigh 10 mg of EGCG powder, dissolve it in 10 mL of ultrapure water to obtain an EGCG stock solution with a concentration of 1 mg / mL, and then dilute it with ultrapure water to obtain standard EGCG aqueous solutions with concentrations of 10 ~ 30 µg / mL. Measure the absorbance value at the characteristic absorption peak of EGCG at 274 nm, and establish a standard curve of EGCG content (x) versus absorbance value (y): y = 0.0239x - 0.0006, R 2 = 0.9996. The composite particle dispersion prepared in Examples 1-5 was centrifuged (10000 rpm, 10 min). The supernatant after centrifugation was centrifuged again in a 3 kDa ultrafiltration centrifuge tube (3000 rpm, 20 min) to separate the free EGCG and measure its absorbance value. Finally, the concentration of free EGCG was determined according to the standard curve. The amount of EGCG encapsulated was obtained by subtracting the amount of free EGCG from the total amount of EGCG added. The encapsulation rate and loading rate of EGCG in the composite particles were calculated by formula (3) and formula (4), respectively:

[0066]

[0067]

[0068] The results of the encapsulation efficiency and loading rate of resveratrol and EGCG are shown in the figure. Figure 2 . Figure 2 As shown in A and 2B, when the amount of carboxymethyl chitosan added was 15 mg, the encapsulation efficiency and loading rate of resveratrol reached the maximum, which were 87.17% and 14.94%, respectively. When the amount added was 20 mg, the encapsulation efficiency (86.75%) and loading rate (14.46%) decreased slightly, but there was no significant difference (p > 0.05). Figure 2 As shown in C and 2D, the encapsulation efficiency of EGCG increased continuously with the increase of carboxymethyl chitosan addition (77.85% ~ 85.75%), while the loading initially increased and then decreased, reaching a maximum of 4.68% at an addition of 20 mg. Based on the combined results of the encapsulation efficiency and loading of resveratrol and EGCG, this invention selected a composite particle with a zein:carboxymethyl chitosan mass ratio of 100:20 (i.e., the composite particle prepared in Example 1) for subsequent testing.

[0069] 3. Fourier transform infrared spectroscopy analysis

[0070] The composite particles prepared in Example 1 were freeze-dried. The freeze-dried composite particle samples and their components (resveratrol, EGCG, zein, carrageenan, and carboxymethyl chitosan) were then subjected to Fourier transform infrared spectroscopy (FTIR). A certain mass of the sample was pressed into a potassium bromide pellet, and the scanning wavelength was 600-4000 cm⁻¹. -1 The resolution is 4 cm. -1 Each sample was scanned 32 times.

[0071] Fourier transform infrared spectroscopy analysis results are shown below Figure 3 In the figure, Res represents resveratrol, Zein represents zein, Car represents carrageenan, and CHI represents carboxymethyl chitosan. As shown in the figure, the intermolecular interactions significantly change after zein and carrageenan are combined: the hydroxyl (-OH) absorption peak in the composite particles shifts to lower wavenumbers compared to single zein, indicating that carrageenan binds to the hydroxyl groups of zein through hydrogen bonding; simultaneously, the characteristic peaks of amide I and amide II bands both show slight shifts, indicating the existence of hydrophobic interactions between the two. The characteristic peaks of resveratrol and EGCG completely disappear in the composite particles, proving that they are effectively embedded inside the composite particles and not exposed on the surface. Upon further introduction of carboxymethyl chitosan, the hydroxyl absorption peak of the composite particles continues to shift to lower wavenumbers, while the characteristic peak of amide II band shifts back to near its original position, suggesting that carboxymethyl chitosan and carrageenan reconstruct the molecular network through hydrogen bonding and may form new hydrophobic microregions. Furthermore, the continuous disappearance of the characteristic peaks of resveratrol and EGCG indicates that the multilayer encapsulation structure did not disrupt the encapsulation state of the active ingredients. These phenomena collectively demonstrate that the zein-carrageenan-carboxymethyl chitosan system of this invention achieves stable loading of functional factors and controllable assembly of hierarchical structures through the synergistic effect of hydrogen bonding and hydrophobicity.

[0072] 4. X-ray diffraction pattern analysis

[0073] The crystal structure of the composite particle sample prepared in Example 1 was analyzed by X-ray diffraction (XRD). The specific parameters were set as follows: the 2θ scan range was 4-60°. o The scan step size is 0.02. o The scanning mode is continuous scanning, the tube voltage is 40 kV, and the tube current is 50 mA.

[0074] The X-ray diffraction pattern analysis results are shown below. Figure 4The figure shows that zein, carrageenan, and carboxymethyl chitosan all exhibit broad peaks characteristic of amorphous structures, while resveratrol and EGCG alone show typical sharp diffraction peaks typical of crystalline states. However, in the RZECC composite particles, the crystalline peaks of resveratrol and EGCG completely disappear, indicating that they are successfully encapsulated in an amorphous state within the composite particles. Previous studies have confirmed that amorphous resveratrol can significantly improve water solubility, and this change in crystallinity further verifies the effect of composite particles on improving the solubility of active ingredients. This result, together with infrared spectroscopy analysis, demonstrates that multiple components achieve amorphous encapsulation and enhanced stability of functional factors through intermolecular forces.

[0075] 5. Microstructure analysis

[0076] The microstructure of the composite particles prepared in Examples 1-5 was observed using a field emission scanning electron microscope (FE-SEM). The freeze-dried composite particle samples were placed on a sample stage coated with conductive adhesive, loose powder was blown away, and the samples were sputter-coated with gold (30 mA, 60 s) to overcome the limitation of poor sample conductivity. The micromorphology of the composite particles was then observed at 50 K magnification.

[0077] Figure 5 The images show field emission scanning electron microscope (SEM) images of the microstructure of RZECC composite particles with different mass ratios. As can be seen from the images, all RZECC composite particles are spherical. When the carboxymethyl chitosan content is 100:10, 100:15, and 100:20, the boundaries of the composite particles are clear, consisting of individual spherical particles. However, when the content increases to 100:25 and 100:30, the boundaries of the composite particles are no longer clear and are instead linked together by an excess of carboxymethyl chitosan.

[0078] In summary, this invention prepares resveratrol-zein-EGCG-carrageenan-carboxymethyl chitosan composite particles via antisolvent coprecipitation and layer-by-layer self-assembly technology. When a small amount of carboxymethyl chitosan is added, the RZECC composite particles exhibit smaller average particle size, lower turbidity, and higher stability; however, as the proportion of carboxymethyl chitosan increases, both the average particle size and turbidity of the RZECC composite particles increase. These results collectively indicate that a mass ratio of zein to carboxymethyl chitosan of 100:20 is optimal for preparing RZECC composite particles.

[0079] Comparative Example 1 consisted of free resveratrol and EGCG.

[0080] Comparative Example 2 was a blank composite carrier consisting of zein-carrageenan-carboxymethyl chitosan (without resveratrol and EGCG).

[0081] The bioactivity of the composite particles prepared in Example 1 and the components in Comparative Examples 1-2 was studied. The specific research contents are as follows:

[0082] 1. Cytotoxicity test

[0083] The experiment was conducted following the operating instructions of the Elabscience Enhanced Cell Viability Assay Kit with slight adjustments.

[0084] (1) Take Caco-2 cells in good growth condition, adjust the cell density, and add 150 μL of cell suspension (containing 5 × 10⁻⁶ cells per well) to each well. 4 (100 cells) were seeded into 96-well plates, with blank wells containing the same volume of culture medium but without cells. 200 mL of PBS was added to the outermost ring of the 96-well plates to slow down liquid evaporation. The plates were then incubated in an incubator for 24 h (the cells adhere to the plate relatively slowly).

[0085] (2) Remove the old culture medium and add complete culture medium containing free resveratrol and EGCG (Res-EGCG Free), blank composite particle carrier (without resveratrol and EGCG, Blank NPs), and intact RZECC composite particles. The concentration gradient of resveratrol is 0, 1, 5, 10, 25, 50, 100, 150 μg / mL, and the concentration gradient of EGCG is 0, 0.33, 1.67, 3.33, 8.33, 16.67, 33.33, 50 μg / mL. Free resveratrol and EGCG are dissolved in DMSO and then diluted with complete culture medium to the above series of concentrations. The final concentration of DMSO after dilution is less than 0.1%. Weigh an appropriate amount of RZECC composite particles and blank composite particle carrier, and dilute them with complete culture medium to the above concentrations (the blank composite particle carrier should maintain the same carrier concentration as the RZECC composite particles). Then, add 150 μL of the prepared complete culture medium to each well and incubate for 24 h.

[0086] (3) Remove the old culture medium and wash three times with PBS buffer. Add 100 μL of the prepared CCK-8 reagent (10 μL Enhanced CCK-8 Buffer + 90 μL complete culture medium) to each well and incubate at 37°C in the dark.

[0087] (4) Measure the absorbance at 450 nm using an ELISA reader, and calculate the cell viability according to the following formula (5):

[0088]

[0089] Cytotoxicity test results as follows Figure 6As shown in the figure, the blank composite particle carrier showed no inhibitory effect on cell viability at any of these concentrations, indicating the safety of the composite particle carrier. At low concentrations of resveratrol and EGCG (resveratrol concentration below 10 μg / mL), neither free resveratrol and EGCG nor the RZECC composite particles exhibited cytotoxicity. When the resveratrol concentration reached 10 μg / mL (EGCG concentration was 3.33 μg / mL), the RZECC composite particles began to show some cytotoxicity, which further increased with increasing concentration. This indicates that through layer-by-layer encapsulation, the protein and polysaccharide outer layers of the RZECC composite particles provide protection and sustained release of resveratrol and EGCG, allowing for a continuous and slow release from the interior of the composite particles, thereby enhancing its inhibitory effect on Caco-2 cell viability.

[0090] 2. In vivo absorption experiment

[0091] Sixteen 6-8 week old SPF-grade male SD rats (200-230 g) were randomly divided into two groups after one week of acclimatization feeding: the RZECC compound granule group and the free resveratrol + EGCG (Res-EGCG Free) group, with 8 rats in each group. The rats were deprived of their food 12 hours before the formal experiment, but were allowed free water. RZECC composite particles were dissolved in sterile physiological saline. Free resveratrol and EGCG were dissolved together in 0.5% sodium carboxymethyl cellulose to prepare a suspension. The gavage dosage was 150 mg / kg bw for resveratrol and 50 mg / kg bw for EGCG. Blood was collected by tail clipping at 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h after gavage into heparin sodium anticoagulant tubes. The samples were centrifuged at 10,000 rpm for 10 min at 4 °C. 100 μL of the supernatant plasma was added to 1 mL of ethyl acetate and vortexed for 5 min. The sample was then centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was collected, filtered through a 0.45 μm nylon membrane, and dried under nitrogen. The sample was then reconstituted with 80 μL of methanol, and the concentration of resveratrol in the sample was detected by high performance liquid chromatography.

[0092] The changes in resveratrol concentration at different times are shown in the figure. Figure 7 ,from Figure 7The data clearly shows that the area under the curve (AUC) of resveratrol plasma concentration in the RZECC composite granule group was significantly larger than that in the Res-EGCG Free group. In the Res-EGCG Free group, the plasma concentration of resveratrol in rats initially increased and then decreased, reaching its maximum at 0.5 h, after which it began to decline. In the RZECC group, the plasma concentration of resveratrol generally increased from the first time point after gavage to 8 h, reaching its peak at 8 h, indicating that resveratrol was slowly released from the composite granules. Furthermore, the peak plasma concentration of resveratrol in the RZECC group was 2.3 times that of the Res-EGCG Free group, the MRT (mean retention time) was prolonged to 6.191 h, and the AUC increased to 16.761 (μg / mL)·h, indicating that the relative bioavailability of resveratrol in RZECC was nearly 6 times that of free resveratrol. Therefore, it can be seen that the RZECC composite granules can effectively deliver resveratrol.

[0093] Example 6

[0094] Research on the prevention and treatment of non-alcoholic fatty liver disease (NAFLD) using RZECC compound granules

[0095] A high-fat diet (HFD) model of NAFLD was induced in C57BL / 6N mice. The RZECC complex particles prepared in Example 1 were then used to intervene in the mice to explore the mechanism of action of RZECC on NAFLD. After one week of acclimatization, the mice were divided into 5 groups (n=8 per group) according to body weight: a normal control (Contorl, Con) group, a blank complex carrier (Veh) group, a high-fat model control (HFD) group, a free resveratrol + EGCG (Free) group, and an RZECC complex particle (RZECC) group. Details of mouse feeding and administration for each group are as follows:

[0096]

[0097] In the Free group, the dose of free resveratrol was 75 mg / kg bw, and the dose of EGCG was 25 mg / kg bw. The oral dose was 75 mg / kg bw of resveratrol and 25 mg / kg bw of EGCG. In the RZECC group, the doses, based on the active ingredients, were 75 mg / kg bw of free resveratrol and 25 mg / kg bw of EGCG. In the Veh group, the amounts of zein, carrageenan, and carboxymethyl chitosan were the same as in the RZECC group. All groups were administered the same volume of medication for 20 weeks.

[0098] Figure 8 A comparison of the changes in body weight (A), adipose tissue content (B), and dietary energy intake (C) of mice in each group over a period of 20 weeks. Figure 9Comparison of liver coefficient (A), serum ALT activity (B), and AST activity (C) among mice in each group; Figure 10 H&E stained sections of liver tissue from mice in each group; Figure 11 Schematic diagram of Oil Red stained sections and TG content of liver tissue from each group of mice; Figure 12 This diagram illustrates the serum TG (A), TC (B), HDL-C (C), and LDL-C (D) levels in each group of mice. The results showed that, compared to the HFD and Free groups, RZECC compound particle intervention had no significant effect on the daily food intake and energy metabolism of C57BL / 6N mice, but it effectively inhibited HFD-induced NAFLD weight gain and significantly reduced liver weight and serum transaminase (ALT / AST) activity. Histopathological analysis further revealed reduced lipid droplet deposition and significantly improved inflammatory cell infiltration in the hepatocytes of the RZECC group mice. This protective effect may stem from the synergistic mechanism of the multiple components in the compound particles: resveratrol and EGCG reduce hepatic steatosis by regulating lipid metabolism and oxidative stress pathways, while the composite structure of carboxymethyl chitosan and carrageenan enhances the targeted delivery of active ingredients and inhibits hepatic inflammatory responses.

[0099] In summary, this invention prepares resveratrol-zein composite particles via anti-solvent precipitation. The resveratrol-zein composite particles are injected into a mixed solution of carrageenan and tea polyphenols for a mixing reaction, yielding resveratrol-zein-tea polyphenol-carrageenan composite particles. These particles are then injected into a carboxymethyl chitosan aqueous solution to remove ethanol, resulting in resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles. Compared to free resveratrol and tea polyphenols, the composite particles prepared by this invention effectively improve the physicochemical stability and efficacy of resveratrol and tea polyphenols, significantly increase their bioavailability and content in rat plasma, and exhibit stronger function in improving hepatic steatosis. This invention shows promising application prospects in the development of functional products for the prevention and treatment of NAFLD.

[0100] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing resveratrol-tea polyphenol composite granules for the prevention and treatment of non-alcoholic fatty liver disease, characterized in that, Includes the following steps: (1) Dissolve zein and resveratrol in ethanol to obtain a zein-resveratrol mixed solution; (2) Prepare resveratrol-zein composite particles by reacting a zein-resveratrol mixed solution with water via antisolvent precipitation method; (3) The resveratrol-zein protein composite particles were injected into a mixed solution of carrageenan and tea polyphenols to carry out a mixing reaction, thereby obtaining resveratrol-zein protein-tea polyphenol-carrageenan composite particles. (4) Inject the resveratrol-zein-tea polyphenol-carrageenan composite particles into a carboxymethyl chitosan aqueous solution and remove the ethanol to obtain the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles.

2. The method for preparing resveratrol-tea polyphenol composite granules for the prevention and treatment of non-alcoholic fatty liver disease according to claim 1, characterized in that, The mass ratio of zein, resveratrol, carrageenan, tea polyphenols, and carboxymethyl chitosan is 100:(20-40):(10-30):(5-20):(10-30).

3. The method for preparing resveratrol-tea polyphenol composite granules for the prevention and treatment of non-alcoholic fatty liver disease according to claim 2, characterized in that, The tea polyphenols include, but are not limited to, at least one of epigallocatechin gallate, epicatechin, epigallocatechin, and epicatechin gallate.

4. The method for preparing resveratrol-tea polyphenol composite granules for the prevention and treatment of non-alcoholic fatty liver disease according to claim 1, characterized in that, The antisolvent precipitation method is operated as follows: a mixed solution of zein and resveratrol is added dropwise to water at a volume ratio of 1:3, and a constant temperature magnetic stirrer is used to continuously stir the solution during the process.

5. The method for preparing resveratrol-tea polyphenol composite granules for the prevention and treatment of non-alcoholic fatty liver disease according to claim 1, characterized in that, The mixing reaction process is carried out by stirring with a constant temperature magnetic stirrer under light-proof conditions for 20-40 minutes.

6. The method for preparing resveratrol-tea polyphenol composite granules for the prevention and treatment of non-alcoholic fatty liver disease according to claim 1, characterized in that, Ethanol is removed by rotary evaporation at a temperature of 35-45°C and a pressure of -0.1 MPa.

7. Resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles prepared by the method according to any one of claims 1-6.

8. The use of the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles according to claim 7 in the preparation of products for the prevention or treatment of non-alcoholic fatty liver disease.

9. A product for the prevention or treatment of non-alcoholic fatty liver disease, characterized in that, Includes the resveratrol-zein-tea polyphenol-carrageenan-carboxymethyl chitosan composite particles as described in claim 7.

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