Preparation method and application of resveratrol / piperine composite nanoparticles for improving ulcerative colitis

Abstract

The invention provides a preparation method and application of resveratrol / piperine composite nanoparticles for improving ulcerative colitis, and belongs to the technical fields of biological medicines, nano delivery systems and natural product utilization. Resveratrol-zein nanoparticles constructed by an anti-solvent precipitation method are used as a core, propylene glycol alginate is used as an intermediate layer and wraps piperine, and finally carboxymethyl chitosan is used as an outer layer coating material, so that synergistic encapsulation and slow release of difunctional components are realized. The composite nano-particles constructed by the invention have relatively high encapsulation efficiency and excellent stability and biocompatibility, the bioavailability of resveratrol and piperine is remarkably improved, and the composite nano-particles have a good ulcerative colitis resisting effect; the method has a great application prospect in the fields of medicines for adjuvant therapy of ulcerative colitis, prevention and health care products, functional foods and the like.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine, nanodelivery systems and natural product utilization technology, specifically to a method for preparing and applying resveratrol / piperine composite nanoparticles for improving ulcerative colitis. Background Technology

[0002] Piperine, an alkaloid found in black pepper, is often used as a bioavailability enhancer because it significantly inhibits hepatic drug-metabolizing enzyme systems (such as CYP3A4 and UGT) and transport proteins (such as P-gp), thereby prolonging the half-life of various drugs in vivo and increasing their systemic exposure concentrations. Studies have shown that the co-administration of piperine with resveratrol can significantly improve the latter's blood concentration and efficacy. However, piperine itself also has problems such as high irritation and poor water solubility, making it unsuitable for direct high-dose use in pharmaceutical design. Therefore, there is an urgent need to develop a delivery platform that can simultaneously load and protect resveratrol and piperine to achieve synergistic release, targeted delivery, and stable transport of both.

[0003] Nanoparticle delivery systems, due to their small size, large specific surface area, and ease of modification, have been widely used in the drug development of natural products. Zein, an amphiphilic protein containing approximately 75% hydrophobic amino acids, can form colloidal particles through molecular self-assembly, making it suitable for constructing functional factor delivery systems. However, standalone Zein particles exhibit low stability and encapsulation capacity. To improve these properties, many researchers have attempted to modify Zein particles with polysaccharides. Propylene glycol alginate (PGA) is a natural anionic polypeptide with good biocompatibility and readily combines with hydrophobic particles. Carboxymethyl chitosan (CH) overcomes the poor water solubility of chitosan, possessing good water solubility and exhibiting anti-inflammatory and anti-gastric acid digestive activities, making it an ideal material for constructing the outer shell of nanoparticles. Although Zein, PGA, and CH have their own applications in nanoparticle formulation, no research has yet synergistically integrated these three materials to construct multi-level synergistic encapsulation structures and achieve stable dual-drug delivery.

[0004] Anti-solvent precipitation (ASP) is commonly used to prepare protein-polysaccharide nanoparticles. This method is mainly based on the difference in solubility of substances in solvents and antisolvents. First, an ethanol-water solution containing zein is prepared, and then added to the polysaccharide solution. Since the solubility of zein in water is significantly lower than its solubility in ethanol, as the ethanol-water solution is added, the zein gradually reaches saturation in the aqueous environment and then self-assembles into small spherical nanoparticles. Due to its significant high hydrophobicity, zein usually acts as the core component in the formation of nanocomposites. Polysaccharides, on the other hand, due to their strong hydrophilicity, tend to form a protective shell on the outside. Therefore, the ASP method can prepare zein-polysaccharide nanocomposites with a core-shell structure. Resveratrol is easily metabolized in vivo and undergoes phase II reactions, being affected by glucuronidation and sulfation, which impacts its bioactivity. Nanodelivery systems can mitigate this problem to some extent.

[0005] Furthermore, previous studies have found that the synergistic use of piperine and other compounds with resveratrol can inhibit metabolic enzyme activity, reduce drug glucuronidation, and thus improve the bioavailability of resveratrol. This invention aims to further enhance the bioefficacy of resveratrol in nanoparticles by encapsulating piperine in a nanodelivery system. Previous studies have used Zein particles to encapsulate curcumin and piperine, two hydrophobic functional factors, within their internal hydrophobic regions, and then used carrageenan to form a protective layer on the particle surface. While this structural design successfully achieved the goal of carrying two functional factors (curcumin and piperine), it also has limitations: due to the limited carrying space in the hydrophobic regions within the Zein particles, and the competitive binding of the two functional factors to Zein, the loading rate is relatively low.

[0006] Therefore, this invention designs a nanodelivery system with a well-defined structure, complementary functions, and layered loading capacity to synergistically deliver resveratrol and piperine, and possesses good sustained-release, targeted, and biocompatible properties to solve the aforementioned technical problems. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides a method for preparing and applying resveratrol / piperine composite nanoparticles for improving ulcerative colitis, thereby overcoming the deficiencies of existing technologies in terms of dual-component synergistic delivery, improved drug bioavailability, and targeted controlled-release therapy for intestinal inflammation.

[0008] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing resveratrol / piperine composite nanoparticles for improving ulcerative colitis, comprising the following steps:

[0010] Hydrophobic resveratrol (Res) and zein (Zein) nanoparticle dispersions were prepared by antisolvent precipitation after uniform mixing of resveratrol (Res) and zein (Zein) in ethanol.

[0011] A resveratrol-zein nanoparticle dispersion was added dropwise to an aqueous solution of propylene glycol alginate (PGA). An intermediate shell of propylene glycol alginate was formed through electrostatic interactions and hydrogen bonding, resulting in a resveratrol-zein-propylene glycol alginate (RZP) nanoparticle dispersion.

[0012] Piperine ethanol solution was added dropwise to resveratrol-zein-propylene glycol alginate nanoparticle dispersion to allow piperine particles to be uniformly adsorbed onto RZP nanoparticles, thus obtaining resveratrol-zein-propylene glycol alginate-piperine (RZPP) nanoparticle dispersion.

[0013] Under acidic conditions, a dispersion of resveratrol-zein-propylene glycol alginate-piperine nanoparticles was added dropwise to an aqueous solution of carboxymethyl chitosan. After reaction and drying, resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan (RZPPC) composite nanoparticles were obtained.

[0014] The resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles prepared in this invention have a well-defined three-layer structure. Resveratrol is loaded within a hydrophobic core, piperine is distributed in the propylene glycol alginate middle layer, and the outer layer is stably coated with carboxymethyl chitosan (this invention chooses carboxymethyl chitosan as the outer shell material because, compared to chitosan, it has good water solubility, is easy to use, and, as the outer shell material, can resist the digestive erosion of gastric acid, which is beneficial for transporting more drug to the intestinal tract). This invention improves the dispersibility and stability of the composite nanoparticles by dropwise addition of a resveratrol-zein-propylene glycol alginate-piperine nanoparticle dispersion to a carboxymethyl chitosan aqueous solution under acidic conditions, constructing the carboxymethyl chitosan outer shell structure through electrostatic adsorption. The composite nanoparticles of this invention exhibit good controlled-release performance and stability in a simulated gastrointestinal environment.

[0015] Furthermore, the mass ratio of resveratrol, zein, propylene glycol alginate, piperine, and carboxymethyl chitosan is 30:100:(10-50):3:(10-25).

[0016] Furthermore, the mass ratio of propylene glycol alginate to piperine is (5-10):1.

[0017] In one example of the present invention, the mass ratio of resveratrol, zein, propylene glycol alginate, piperine, and carboxymethyl chitosan is 30:100:30:3:20.

[0018] Further, the specific operation of the antisolvent precipitation method is as follows: resveratrol and zein are mixed evenly in ethanol to obtain a resveratrol-zein solution; under the conditions of room temperature and stirring speed of 1000-2000 rpm, the resveratrol-zein solution is added dropwise to water to obtain a hydrophobic resveratrol-zein (RZ) nanoparticle dispersion.

[0019] Furthermore, the volume fraction of the ethanol is 60%-80%.

[0020] Furthermore, the acidic conditions are a pH value of 4.0-6.0.

[0021] Secondly, the present invention provides resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles prepared by the method described above.

[0022] Furthermore, the resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles have a particle size of 120-220 nm and an absolute value of zeta potential of not less than 25 mV.

[0023] Furthermore, the encapsulation rates of resveratrol and piperine in the resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles are both above 85%.

[0024] Thirdly, the present invention provides the application of the resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles in the preparation of products for the treatment of ulcerative colitis. The resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles improve intestinal barrier function and reduce the expression of inflammatory factors through a sustained-release mechanism, thereby achieving the effect of treating ulcerative colitis.

[0025] Furthermore, the products include, but are not limited to, pharmaceuticals, functional foods, or health supplements.

[0026] Fourthly, the present invention provides a medicament for treating ulcerative colitis, the active ingredient of which includes the resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles.

[0027] Furthermore, the dosage form of the drug includes, but is not limited to, granules, capsules, solutions, or gels.

[0028] Furthermore, the delivery method of the drug for treating ulcerative colitis includes, but is not limited to, oral administration. After oral administration, the drug passes stably through the stomach and releases the active ingredients resveratrol and piperine in the small intestine in response to pH triggering. The released resveratrol exerts anti-inflammatory and antioxidant functions, while piperine enhances the biological effects of the former by inhibiting metabolic enzymes, thereby achieving the therapeutic goal of synergistically relieving colonic inflammation and repairing the mucosal barrier.

[0029] Compared with the prior art, the advantages of the present invention are:

[0030] (1) This invention is the first to construct a "core-shell-shell" multifunctional resveratrol-zelamin-propylene alginate-piperine-carboxymethyl chitosan composite nanoparticle structure with zelamin as the core, propylene glycol alginate as the intermediate functional layer and carboxymethyl chitosan as the protective layer, which solves the technical problems of poor stability and mutual interference in the joint delivery of natural polyphenols and alkaloids.

[0031] (2) This invention constructs a composite nanoparticle structure of resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan through multi-level electrostatic interaction and intermolecular interaction, thereby increasing the encapsulation efficiency of resveratrol and piperine to over 90%, and the encapsulation efficiency is significantly better than that of single-layer or double-layer systems.

[0032] (3) The resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticle structure of the present invention has good gastrointestinal adaptability and pH-responsive controlled release performance. It can maintain structural stability in simulated gastric juice and gradually release drugs in the small intestine, effectively achieving targeted delivery.

[0033] (4) In a mouse model of ulcerative colitis, the resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles of the present invention showed significant anti-inflammatory effects, significantly improved colon tissue morphology and reduced the level of inflammatory factors, and its therapeutic effect was better than that of free resveratrol or piperine alone.

[0034] (5) The materials used in the resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles of the present invention are all natural biodegradable polymers, which are safe to obtain and easy to prepare for industrialization. They are applicable to multiple fields such as food, medicine, and functional beverages, and have good market prospects and promotion value. Attached Figure Description

[0035] Figure 1 A diagram showing the resveratrol-zein interaction; in which... Figure 1 a represents the global graph; Figure 1 b is a partial view; Figure 1c is a schematic diagram of receptor-ligand planar interaction;

[0036] Figure 2 The diagram shows the interaction between propylene glycol alginate and zein. Figure 2 a represents the global graph; Figure 2 b is a partial view; Figure 2 c is a schematic diagram of receptor-ligand planar interaction;

[0037] Figure 3 Microstructure diagrams of Zein, PGA, and CH observed by FE-SEM;

[0038] Figure 4 Microstructure diagrams of RZ, RZP, RZPP, and RZPPC composite nanoparticles observed by FE-SEM;

[0039] Figure 5 Fourier transform infrared spectra of Res, Pip, Zein, PGA, CH, RZ, RZP, RZPP, and RZPPC composite nanoparticles;

[0040] Figure 6 The photostability of Res / Pip, RZP / RZPP, and RZPPC composite nanoparticles; among them, Figure 6 a represents the photostability of the Res, RZP, and RZPPC composite nanoparticles; Figure 6 b represents the photostability of the Pip, RZPP, and RZPPC composite nanoparticles;

[0041] Figure 7 Thermal stability of Res / Pip, RZPP, and RZPPC composite nanoparticles;

[0042] Figure 8 The results of the cytotoxicity experiment of RZPPC composite nanoparticles;

[0043] Figure 9 The results of the blood compatibility test for RZPPC composite nanoparticles are shown; among them, Figure 9 Image a shows the results of centrifugation of red blood cell mixtures from different treatment groups; Figure 9 b represents the quantitative analysis of hemolysis in different treatment groups (n=3);

[0044] Figure 10 The graph shows the release behavior of RZ, RZP / RZPP, and RZPPC composite nanoparticles in simulated gastrointestinal fluid; among them, Figure 10 a represents the release rate of resveratrol in the RZ, RZP, and RZPPC composite nanoparticles; Figure 10 b represents the release rate of piperine from the RZPP and RZPPC composite nanoparticles;

[0045] Figure 11This is a graph showing the effects of different treatment groups on the basic physiological indicators of mice; among them... Figure 11 a represents the change in mouse body weight; Figure 11 b represents the Disease Activity Index (DAI) score; Figure 11 c and 11d represent the length of the mouse colon; Figure 11 e represents the mouse spleen index;

[0046] Figure 12 Images of colon tissue sections from mice in the normal control group (a), DSS group (b), MSZ group (c), ZPC group (d), R group (e), RP group (f), and RZPPC group (g) observed by HE staining, magnified 15×.

[0047] Figure 13 To observe the ultrastructure of intestinal epithelial cells of mice in the normal control group (a), DSS group (b), MSZ group (c), ZPC group (d), R group (e), RP group (f), and RZPPC group (g) under transmission electron microscopy, the magnification was 10000×.

[0048] Figure 14 Immunofluorescence staining results of Mucin 2 (a), claudin-1 (b), ZO-1 (c), and occludin (d) proteins in mouse colon tissue;

[0049] Figure 15 The statistical results of the expression area of ​​Mucin 2 (a), claudin-1 (b), ZO-1 (c), and occludin (d) proteins in mouse colon tissue are shown. All results are expressed as mean ± SEM. # indicates a significant difference compared with the control group (#, p < 0.05; ##, p < 0.01; ###, p < 0.001; *, p < 0.05; **, p < 0.01; ***, p < 0.001), and * indicates a significant difference compared with the DSS group. Detailed Implementation

[0050] 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.

[0051] This invention provides a method for preparing resveratrol / piperine composite nanoparticles for improving ulcerative colitis, comprising the following steps: resveratrol (Res) and zein are mixed uniformly in ethanol, and a hydrophobic resveratrol-zein (RZ) nanoparticle dispersion is prepared by antisolvent precipitation; the resveratrol-zein nanoparticle dispersion is dropwise added to an aqueous solution of propylene glycol alginate (PGA), and an intermediate shell of propylene glycol alginate is formed through electrostatic interactions and hydrogen bonding to obtain resveratrol-zein-propylene glycol alginate (RZP) nanoparticles. Rice particle dispersion; piperine ethanol solution was added dropwise to resveratrol-zein-propylene glycol alginate nanoparticle dispersion, allowing piperine particles to be uniformly adsorbed onto RZP nanoparticles, resulting in resveratrol-zein-propylene glycol alginate-piperine (RZPP) nanoparticle dispersion; under acidic conditions, resveratrol-zein-propylene glycol alginate-piperine nanoparticle dispersion was added dropwise to carboxymethyl chitosan aqueous solution, reacted, and dried to obtain resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan (RZPPC) composite nanoparticles.

[0052] In some examples, the mass ratio of resveratrol, zein, propylene glycol alginate, piperine, and carboxymethyl chitosan is 30:100:(10-50):3:(10-25).

[0053] Further, the specific operation of the antisolvent precipitation method is as follows: resveratrol and zein are mixed evenly in ethanol to obtain a resveratrol-zein solution; under the conditions of room temperature and stirring speed of 1000-2000 rpm, the resveratrol-zein solution is added dropwise to water to obtain a hydrophobic resveratrol-zein (RZ) nanoparticle dispersion.

[0054] In some examples, the volume fraction of ethanol is 60%-80%.

[0055] In some examples, the pH value of the acidic conditions is 4.0-6.0.

[0056] Example 1: Preparation and characterization of resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles

[0057] 1. Raw material preparation

[0058] Accurately weigh a certain amount of zein and resveratrol powder, and dissolve them together in a certain amount of 70% ethanol aqueous solution to achieve concentrations of 10 mg / mL for zein and 3 mg / mL for resveratrol. Store in a dark place and sealed with plastic wrap. Accurately weigh a certain amount of propylene glycol alginate (PGA) and dissolve it in distilled water. Store in a dark place and sealed with plastic wrap. Accurately weigh a certain amount of carboxymethyl chitosan (CH) powder and dissolve it in distilled water. Store in a dark place and sealed with plastic wrap. Accurately weigh a certain amount of piperine (Pip) powder and dissolve it in a 70% ethanol aqueous solution. Store in a dark place and sealed with plastic wrap. Stir continuously at 600 rpm for 10 h using a temperature-controlled magnetic stirrer to ensure complete dissolution and dispersion of the reagents.

[0059] 2. Preparation of composite nanoparticles

[0060] 2.1 Preparation of resveratrol-zein (RZ) nanoparticles

[0061] Using a syringe, a certain amount of resveratrol-zein solution was slowly injected into distilled water at a volume ratio of 1:3 under magnetic stirring at 1200 rpm. The solution was then continuously stirred at 1200 rpm for 30 minutes under light-protected conditions using a thermostatic magnetic stirrer to form an RZ nanoparticle dispersion.

[0062] 2.2 Preparation of resveratrol-zein-propylene glycol alginate (RZP) nanoparticles

[0063] Accurately measure the RZ nanoparticle dispersion and slowly drop it dropwise into a series of propylene glycol alginate solutions of varying concentrations at a volume ratio of 1:3. Stir continuously for 30 minutes at 1200 rpm using a constant-temperature magnetic stirrer under light-protected conditions to obtain RZP nanoparticle dispersions with mass ratios of zein, propylene glycol alginate, and resveratrol of 100:50:30, 100:40:30, 100:30:30, 100:20:30, and 100:10:30, respectively.

[0064] 2.3 Preparation of Resveratrol-Zezyme-Propylene Glycol Alginate-Piperine-Carboxymethyl Chitosan (RZPPC) Composite Nanoparticles

[0065] Accurately measure a certain amount of piperine ethanol aqueous solution, and use a syringe to slowly drip the piperine ethanol aqueous solution into the RZP nanoparticle dispersion under magnetic stirring at 1200 rpm, so that piperine is uniformly adsorbed on the RZP nanoparticles. The mass ratio of resveratrol to piperine is 10:1, and the RZPP nanoparticle dispersion is obtained.

[0066] Accurately measure the RZPP nanoparticle dispersion and, under acidic conditions (pH 4.5), dropwise add it dropwise into a series of carboxymethyl chitosan aqueous solutions with a concentration gradient using a syringe and magnetic stirring at 1200 rpm, so that the mass ratio of zein to carboxymethyl chitosan is 100:10, 100:15, 100:20, and 100:25, respectively. Stir continuously for 30 min at 1200 rpm using a temperature-controlled magnetic stirrer under light-protected conditions to obtain the RZPP composite nanoparticle dispersion. Freeze-dry to obtain the RZPP composite nanoparticles.

[0067] 3. Characterization of RZPPC composite nanoparticles

[0068] This embodiment performs a series of characterizations on RZPPC composite nanoparticles to study the preparation effect of the method of the present invention.

[0069] Figure 1 , 2 The figures show the interaction diagrams of resveratrol-zein and propylene glycol alginate-zein, respectively. As can be seen from the figures, resveratrol and zein form a stable bond through hydrophobic and hydrogen bonding mechanisms.

[0070] Table 1 shows the encapsulation efficiency and loading rate of RZPPC composite nanoparticles with different ratios of zein, propylene glycol alginate, and carboxymethyl chitosan for resveratrol and piperine. As shown in Table 1, when the mass ratio of zein, propylene glycol alginate, and carboxymethyl chitosan is 100:30:20, the RZPPC composite nanoparticles have a high encapsulation efficiency (87.03% and 85.31%, respectively) for resveratrol and piperine, and the photothermal stability is significantly enhanced.

[0071] Table 1: Encapsulation efficiency and loading rate of resveratrol and piperine by composite nanoparticles with different proportions

[0072]

[0073] Note: RZPPC(100:30:10) indicates that the mass ratio of zein, propylene glycol alginate, and carboxymethyl chitosan is 100:30:10; the mass ratio of zein and propylene glycol alginate in RZPP nanoparticles is 100:30; different letters in the upper right corner of the same column of data indicate statistical differences.

[0074] Figure 3 Microstructure diagrams of zein, propylene glycol alginate, and carboxymethyl chitosan observed by FE-SEM. Figure 4 Microstructure diagrams of RZ, RZP, RZPP, and RZPPC composite nanoparticles observed by FE-SEM. Figure 3The results showed that simple zein particles were well-defined small spherical particles, simple propylene glycol alginate particles exhibited a fine filamentous network structure, and simple carboxymethyl chitosan exhibited a leaf-like structure. Meanwhile... Figure 4 The results show that the RZ nanoparticles exhibit a spherical structure, indicating that resveratrol was successfully encapsulated within the zein shell. After the addition of propylene glycol alginate, the surface of the RZP nanoparticles (zein and propylene glycol alginate in a mass ratio of 100:30) became rougher, exhibiting a dendritic pattern. This is due to the interaction between propylene glycol alginate and zein, forming a coating on their surface. When piperine was further added to form RZPP nanoparticles, piperine was adsorbed onto propylene glycol alginate, and the nanoparticles maintained their original morphology. Finally, the RZPPC composite nanoparticles (zein, propylene glycol alginate, and carboxymethyl chitosan in a mass ratio of 100:30:10) formed by adding carboxymethyl chitosan exhibited a denser microstructure and increased particle size, indicating that carboxymethyl chitosan successfully coated the outermost layer, forming a stable "core-shell-shell" structure.

[0075] Figure 5 Fourier transform infrared (FTIR) spectra of Res, Pip, Zein, PGA, CH, RZ, RZP, RZPP, and RZPPC composite nanoparticles (with a mass ratio of 100:30:10 for zein, propylene glycol alginate, and carboxymethyl chitosan). The figures show that zein has a peak density at 3289.8 cm⁻¹. -1 1652.6 cm -1 and 1533.4 cm -1 Typical absorption bands are present at 1583.4 cm⁻¹, corresponding to the OH stretching vibration, amide I band, and amide II band, respectively; resveratrol shows an absorption band at 1583.4 cm⁻¹. -1 1385.6 cm -1 1137.8 cm -1 The appearance of sharp peaks is related to the C=C stretching vibration of aromatic rings, the C=C stretching vibration of olefins, and the C=O stretching vibration. Upon the formation of RZ and RZP nanoparticles, the position of the OH characteristic peak shifts, indicating the formation of hydrogen bonds between resveratrol and zein; simultaneously, the position of the amide I band characteristic peak of the RZP nanoparticles also shifts, indicating a hydrophobic effect between zein molecules and propylene glycol alginate.

[0076] Figure 6The photostability of Res / Pip, RZP / RZPP, and RZPPC composite nanoparticles (with a mass ratio of zein, propylene glycol alginate, and carboxymethyl chitosan of 100:30:10) was measured. As shown in the figure, free resveratrol and piperine degraded rapidly under light irradiation, with a retention rate of less than 20% after 100 min. However, the photodegradation rate of resveratrol in RZPP nanoparticles decreased, and the retention rate remained above 75% after 100 min. After further coating with carboxymethyl chitosan, the resveratrol retention rate exceeded 80%. The photodegradation rate of piperine in RZPP nanoparticles slowed down, with a retention rate greater than 50% after 100 min. Further coating with carboxymethyl chitosan resulted in a piperine retention rate as high as 70%, indicating that the composite nanoparticle delivery system of this invention effectively improved the photostability of resveratrol and piperine.

[0077] Figure 7 The thermal stability of Res / Pip, RZPP, and RZPPC composite nanoparticles (with a mass ratio of 100:30:10 for zein, propylene glycol alginate, and carboxymethyl chitosan) was measured. As shown in the figure, after heat treatment, the retention rates of resveratrol in the RZPP and RZPPC composite nanoparticles were 81.9% and 88.0%, respectively, significantly higher than the 71.8% in the free group. The retention rate of piperine in the RZPPC composite nanoparticles was 75.0%, significantly higher than the 62.6% in the free group. This indicates that the RZPPC composite nanoparticle delivery system of this invention effectively improves the thermal stability of resveratrol and piperine.

[0078] The following is an evaluation of the bioefficacy of RZPPC composite nanoparticles with a mass ratio of 100:30:10 of zein, propylene glycol alginate, and carboxymethyl chitosan.

[0079] Example 2: In vitro biological evaluation of RZPPC composite nanoparticles

[0080] 1. Cytotoxicity test

[0081] The toxicity of RZPPC composite nanoparticles to RAW 264.7 cells was determined using the CCK-8 assay. RAW 264.7 cells were cultured at 8.0 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells / well in 96-well plates and incubated overnight to promote cell adhesion. Subsequently, the original culture medium was removed, and the cells were washed three times with PBS. Serum-free medium containing different concentrations (1.8, 18, 90, 180, 360, 900 μg / mL) of RZPPC composite nanoparticles was added, and the cells were co-incubated for 6 h, 12 h, and 24 h, respectively. After incubation, the co-incubation medium containing RZPPC composite nanoparticles was removed, and 100 μL of CCK-8 reagent was added. The cells were incubated in the dark for 1 h. Finally, the absorbance of each well at 450 nm was measured using a microplate reader.

[0082] The results are as follows Figure 8 As shown in the figure, after incubating RAW 264.7 cells with low concentrations of RZPPC composite nanoparticles for 12 h, the cell viability in all experimental groups was above 75%, demonstrating good cell compatibility. No significant toxicity of the RZPPC composite nanoparticles to the cells was observed after 24 h. While cell viability decreased to some extent with increasing RZPPC composite nanoparticle concentration, overall, the RZPPC composite nanoparticles exhibited good cell compatibility.

[0083] 2. Blood compatibility test

[0084] One mL of whole blood from C57 / BL6 mice was collected and mixed in a heparin anticoagulant tube. The erythrocytes were collected by centrifugation and washed 3 to 5 times with sterile PBS to prepare a 4% erythrocyte suspension. The erythrocyte suspension was co-incubated with equal volumes of RZPPC composite nanoparticles at different concentrations (1, 5, 10, 20, 50, 100 μg / mL). In the positive control group, the erythrocyte suspension was co-incubated with an equal volume of deionized water; in the negative control group, the erythrocyte suspension was co-incubated with an equal volume of sterile PBS. After incubation at 37°C for 1 h, the supernatant was centrifuged, and the absorbance at 570 nm was measured to calculate the hemolysis rate for each experimental group.

[0085] The results are as follows Figure 9 As shown in the figure, after mixing and incubating different concentrations of RZPPC composite nanoparticles with red blood cells, the hemolysis phenomenon was not significant, proving that RZPPC composite nanoparticles have good blood compatibility.

[0086] 3. In vitro simulated gastrointestinal digestion experiment

[0087] 30 mL of RZ, RZP, RZPP, and RZPPC composite nanoparticle dispersions were separately mixed with 30 mL of simulated gastric fluid (mainly composed of 3.2 mg / mL pepsin and 2.0 mg / mL sodium chloride solution), and the pH value of the mixture was adjusted to 1.2 with 1.0 mol / L hydrochloric acid solution. Then, each sample was placed in a water bath at 37 °C and heated at a rotation speed of 100 rpm. Samples were collected at time intervals of 30 and 60 minutes for analysis. To terminate the gastric fluid digestion process, the pH value of the mixture was adjusted to 7.5 with 1.0 mol / L sodium hydroxide solution. Subsequently, 30 mL of simulated intestinal fluid (containing 2.0 mg / mL pancreatin, 12.0 mg / mL bile salts, 8.8 mg / mL sodium chloride solution, and 6.8 mg / mL potassium dihydrogen phosphate solution) was measured and mixed with an equal volume of gastric fluid mixture. It was then placed in a water bath at 37 °C again, heated at a rotation speed of 100 rpm, and samples were collected at time intervals of 90, 120, 150, and 180 minutes for determination.

[0088] The results are as Figure 10 shown. As can be seen from the figure, within the first 30 min of digestion in simulated gastric fluid, resveratrol was rapidly released from the nanoparticles, and then the release rate slowed down. The release rate of resveratrol in the RZPPC composite nanoparticles was the lowest ( Figure 10 a); relative to RZPP, the release rate of piperine in the RZPPC composite nanoparticles was also significantly reduced ( Figure 10 b). It indicates that the RZPPC composite nanoparticles prepared in this invention have obvious sustained-release performance.

[0089] Example 3 Pharmacokinetic Study of RZPPC Composite Nanoparticles

[0090] 1. Animal Feeding and Grouping

[0091] Twelve 8-week-old male SD rats were selected and purchased from Vital River Laboratory Animal Technology Co., Ltd., with the certificate number: SYXK (E) 2019-0013. All rats were housed in a SPF-class animal room and adaptively fed for 5 days. The environmental parameters were strictly controlled, the temperature was maintained at 22-24 °C, the humidity was kept at 50%-60%, and the lighting was cycled for 12 hours each in light and dark. During the adaptive feeding period, food and water were given normally, and water was not restricted during the fasting period of the animals. The twelve rats were randomly divided into two groups: the RZPPC composite nanoparticle group and the free resveratrol + piperine mixture group.

[0092] 2. Oral Absorption Experiment of Rats

[0093] Before the experiment, the animals were fasted for 12 hours but their water intake was not restricted. Then, rats were treated by gavage or oral administration with RZPPC composite nanoparticles and a mixture of free resveratrol and piperine, respectively. The resveratrol dose was 50 mg / kg, and the piperine dose was 5 mg / kg. Blood was collected from the tail vein of the rats at 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h after gavage in 1.5 mL heparin sodium anticoagulant centrifuge tubes. The tubes were centrifuged at 12000 rpm for 20 min at 4°C, and the supernatant plasma was collected. For each sample, 100 μL of plasma was mixed with 1 mL of ethyl acetate, vortexed for 5 min for complete extraction, centrifuged at 12000 rpm for 10 min, and 800 μL of the supernatant was collected into a centrifuge tube. After nitrogen blowing for 20 min and allowing the organic solvent to evaporate completely, 80 μL of methanol was added for redissolution, vortexed for 5 min, filtered through a 0.45 μm filter membrane, and the concentrations of resveratrol and piperine in the plasma sample were detected using a liquid chromatography system.

[0094] 3. Results and Analysis

[0095] The pharmacokinetic results are shown in Table 2. As can be seen from Table 2, the peak plasma concentration of resveratrol in the RZPPC composite nanoparticles occurred later than that of the free resveratrol + piperine mixture group. The mean retention time (MRT) was 4.115 ± 0.204 h, which was 2.32 times that of the free mixture group. The area under the concentration-time curve (AUC) was 8.387 ± 0.167 (μg / mL)·h, which was 4.8 times that of the free mixture group. This indicates that the relative bioavailability of the RZPPC composite nanoparticles is 4.8 times that of resveratrol. These results demonstrate that the RZPPC composite nanoparticle delivery system of this invention can significantly increase the bioavailability of resveratrol in rats, improve resveratrol plasma concentration, and thus enhance its anti-inflammatory efficacy.

[0096] Table 2: Pharmacokinetic Results of RZPPC Composite Nanoparticles Administered by Gavage to Rats

[0097]

[0098] Example 4: Interventional effect of RZPPC composite nanoparticles on acute ulcerative colitis.

[0099] 1. Animal model establishment and grouping

[0100] Seventy C57BL / 6 mice were randomly divided into seven groups (n=10 per group) according to body weight: Normal control group, Model control group (DSS group), Mesalazine (MSZ) positive treatment group, Carrier (ZPC) group, Free resveratrol group (R group), Free resveratrol + capsaicin mixture group (RP group), and RZPPC composite nanoparticle group. Mice in the Normal group received normal drinking water, while mice in the other groups received 3% (w / v) DSS (sodium dextran sulfate) solution instead of drinking water. The drug treatments for each group were as follows:

[0101] Normal group: 0.5% sodium carboxymethyl cellulose was administered by gavage;

[0102] DSS group: 0.5% sodium carboxymethyl cellulose administered by gavage;

[0103] MSZ group: Mesalazine 200 mg / kg administered by gavage;

[0104] ZPC group: Gavage administration of zein-propylene glycol alginate-carboxymethyl chitosan carrier nanoparticles (0.5% sodium carboxymethyl cellulose for dissolution);

[0105] Group R: 50 mg / kg of free resveratrol administered by gavage (with 0.5% sodium carboxymethyl cellulose as a dissolving agent);

[0106] RP group: 50 mg / kg of free resveratrol plus 5 mg / kg of piperine (0.5% sodium carboxymethyl cellulose for dissolution) was administered by gavage.

[0107] RZPPC group: RZPPC composite nanoparticles (0.5% sodium carboxymethyl cellulose for dissolution) were administered orally at a dose of 50 mg / kg of resveratrol and 5 mg / kg of piperine.

[0108] Each group received an equal volume of the drug once daily for 7 days.

[0109] 2. Evaluation of basic physiological condition of animals

[0110] Mouse weight, fecal characteristics, and bleeding status were observed and recorded daily. After the experiment, the spleen and colon tissue of the mice were harvested for colonic villus electron microscopy. The morphological damage of the mouse colon tissue was observed using hematoxylin-eosin (HE) staining and alicin blue-periodic acid-Schiff (AB-PAS) staining. The morphology of the mouse colonic epithelial cells was observed using transmission electron microscopy, and the damage to the mouse colonic barrier was assessed using fluorescence immunoassay.

[0111] 3 Results and Analysis

[0112] Physiological indicators of mice, such as Figure 11As shown in the figure, the body weight of mice in the DSS group decreased significantly from the third day of intervention, while the weight loss trend of mice treated with RZPPC composite nanoparticles was significantly alleviated; compared with the DSS group, the body weight of mice in the RZPPC group decreased by only 8%, which is close to that of the mesalazine drug intervention group. Figure 11 a). DAI score results showed that the DAI score of mice in the DSS group increased significantly from the fourth day of intervention, while the DAI scores of other intervention groups decreased significantly, with the RZPPC group having the lowest DAI score. Figure 11 b). Colon length measurements showed that the colon length of mice in the DSS group was significantly shortened, while the colon length of the RZPPC group recovered to some extent compared to the DSS group. Figure 11 c, 11d). Spleen index analysis showed that the spleen index of mice in the DSS group was significantly increased, while the spleen index of mice in the RZPPC group was significantly decreased. Figure 11 e). The above results indicate that the RZPPC composite nanoparticles of the present invention can significantly enhance the bioefficacy of resveratrol and piperine, and alleviate DSS-induced UC symptoms in mice.

[0113] HE staining results are as follows Figure 12 As shown in the figure, the DSS group mice exhibited severe ulceration of the colonic mucosa, disordered colonic structure, ruptured crypt glands, damaged surface epithelium, loss or even disappearance of intestinal villi, thickening of the submucosa and muscular layer, and edema and inflammatory cell infiltration. Figure 12 b). In contrast, the colonic epithelial cells of the RZPPC group mice exhibited striated borders, normal morphology and structure, and goblet cells were distributed among the epithelial cells, with no obvious lesions or inflammatory changes observed. Figure 12 g).

[0114] Transmission electron microscopy observation results as follows Figure 13 As shown in the figure, the microvilli on the surface of epithelial cells of mice in the DSS group are irregular in morphology, varying in length and sparse, mitochondria are swollen, the number of goblet cells is reduced, vacuoles appear in the cytoplasm, and the intercellular junctions are widened. Figure 13 b). In contrast, the RZPPC group mice had denser microvilli with normal morphology, a greater number of goblet cells, less obvious rough endoplasmic reticulum lesions, more intact tight junctions between cells, and regular mitochondrial morphology. Figure 13 g). This indicates that the RZPPC composite nanoparticles of the present invention can improve the inflammatory symptoms of intestinal epithelial cells in mice with DSS-induced colitis.

[0115] The results of immunofluorescence staining and the statistical results of expression area are as follows: Figure 14 , 15As shown in the figure, the levels of tight junction proteins Mucin 2, Claudin-1, ZO-1, and occludin in the colonic tissue of mice in the DSS group were significantly reduced. After treatment with RZPPC composite nanoparticles, the levels of tight junction proteins significantly increased compared to the DSS group, indicating that the RZPPC composite nanoparticle delivery system of this invention can maintain the integrity of the colonic mucosal barrier, protect colonic barrier function, and thus effectively alleviate the symptoms of ulcerative colitis in mice.

[0116] In summary, this invention utilizes resveratrol-zein nanoparticles constructed via antisolvent precipitation as the core, with propylene glycol alginate as the intermediate layer encapsulating piperine, and finally carboxymethyl chitosan as the outer coating material, thereby achieving synergistic encapsulation and sustained-release of the dual-functional components. The composite nanoparticles constructed in this invention possess high encapsulation efficiency, excellent stability and biocompatibility, and significantly improve the bioavailability of resveratrol and piperine, exhibiting good anti-ulcerative colitis effects. They show great application potential in the fields of adjuvant therapy for ulcerative colitis, preventive health products, and functional foods.

[0117] 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 / piperine composite nanoparticles for improving ulcerative colitis, characterized in that, Includes the following steps: Resveratrol and zein were mixed evenly in ethanol, and then a resveratrol-zein nanoparticle dispersion was prepared by antisolvent precipitation. The resveratrol-zein nanoparticle dispersion was added dropwise to an aqueous solution of propylene glycol alginate. Through electrostatic interaction and hydrogen bonding, an intermediate shell of propylene glycol alginate was formed, thus obtaining the resveratrol-zein-propylene glycol alginate nanoparticle dispersion. Piperine ethanol solution was added dropwise to resveratrol-zein-propylene glycol alginate nanoparticle dispersion to obtain resveratrol-zein-propylene glycol alginate-piperine nanoparticle dispersion. Under acidic conditions, a dispersion of resveratrol-zein-propylene glycol alginate-piperine nanoparticles was added dropwise to an aqueous solution of carboxymethyl chitosan. After reaction, resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles were obtained.

2. The method for preparing resveratrol / piperine composite nanoparticles for improving ulcerative colitis according to claim 1, characterized in that, The mass ratio of resveratrol, zein, propylene glycol alginate, piperine, and carboxymethyl chitosan is 30:100:(10-50):3:(10-25).

3. The method for preparing resveratrol / piperine composite nanoparticles for improving ulcerative colitis according to claim 2, characterized in that, The mass ratio of propylene glycol alginate to piperine is (5-10):

1.

4. The method for preparing resveratrol / piperine composite nanoparticles for improving ulcerative colitis according to claim 2, characterized in that, The mass ratio of resveratrol, zein, propylene glycol alginate, piperine, and carboxymethyl chitosan is 30:100:30:3:

20.

5. The method for preparing resveratrol / piperine composite nanoparticles for improving ulcerative colitis according to claim 1, characterized in that, The acidic conditions are defined as a pH value of 4.0-6.

0.

6. The method for preparing resveratrol / piperine composite nanoparticles for improving ulcerative colitis according to claim 1, characterized in that, The specific operation of the antisolvent precipitation method is as follows: resveratrol and zein are mixed evenly in ethanol to obtain a resveratrol-zein solution; under the conditions of room temperature and stirring speed of 1000-2000 rpm, the resveratrol-zein solution is added dropwise to water to obtain a hydrophobic resveratrol-zein nanoparticle dispersion.

7. Resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles prepared by the method according to any one of claims 1-6.

8. The use of the resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles according to claim 7 in the preparation of products for the treatment of ulcerative colitis.

9. The application according to claim 8, characterized in that, The products mentioned include, but are not limited to, pharmaceuticals, functional foods, or health supplements.

10. A drug for treating ulcerative colitis, characterized in that, The active ingredient includes the resveratrol-zein-propylene glycol alginate-piperine-carboxymethyl chitosan composite nanoparticles as described in claim 7.

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