Genipin crosslinked selenium nanoparticles, methods of making and using the same

Abstract

The application provides a genipin cross-linked selenium nanoparticle and a preparation method and application thereof, and belongs to the field of functional nanomaterials and intelligent nutrition delivery technology. The preparation method comprises the following steps: dissolving a protein substance in water to obtain a protein solution, adding a selenous acid compound, then adding vitamin C and genipin, performing cross-linking reaction, and performing dialysis on the obtained reaction solution to obtain the genipin cross-linked selenium nanoparticle. The application innovatively uses the concentration of genipin as a control switch, realizes active and accurate control of the release rate of selenium in the intestinal tract, realizes the treatment effect on colitis through instantaneous high-concentration release under a low-concentration selenium administration dose, and effectively solves the defect that the "nutrition-toxicity" dose range of selenium is narrow.

Description

Technical Field

[0001] This invention belongs to the field of functional nanomaterials and intelligent nutrient delivery technology, specifically relating to a genipin crosslinked selenium nanoparticle, its preparation method, and its application. Background Technology

[0002] Selenium is an essential trace element for the human body, participating in various physiological activities and playing a crucial role in maintaining intestinal immune homeostasis and suppressing inflammation. Studies have shown that selenium supplementation has positive intervention potential for inflammatory bowel diseases such as colitis. However, selenium has a narrow "nutrition-toxicity" dosage range; excessive intake is toxic, while insufficient intake fails to provide nutritional and health benefits. Therefore, constructing an oral delivery system is an effective way to achieve precise targeting, appropriate release, and improved intestinal health, but it also presents significant challenges in preparation technology. Furthermore, the acidic environment of the stomach and digestive enzymes easily damage the structure of protein selenium nanoparticles, causing premature release of the loaded selenium in the stomach. This not only prevents the small intestine, the primary absorption site, from obtaining sufficient selenium, reducing bioavailability, but also fails to establish an effective concentration at the site of inflammation, thus limiting its therapeutic effect on colitis.

[0003] To ensure the active ingredients reach the intestines smoothly, a pectin cross-linking system is used for encapsulation. This type of material exhibits good biocompatibility and remains stable in the acidic environment of the stomach, gradually swelling under intestinal pH conditions to achieve the goal of protecting the nano-selenium in the stomach and releasing it in the intestines. However, this method lacks the ability to actively and precisely regulate the release behavior of selenium at the target release site. Summary of the Invention

[0004] This invention provides genipin-crosslinked selenium nanoparticles, their preparation method, and applications. By crosslinking protein selenium nanoparticles with genipin, the release behavior of selenium is directionally regulated, meaning that the release rate of selenium in a simulated gastrointestinal environment can be significantly increased with increasing genipin concentration.

[0005] This invention proposes a method for preparing genipin crosslinked selenium nanoparticles, comprising the following steps:

[0006] Proteins were dissolved in water to obtain a protein solution, then selenite compounds were added, followed by vitamin C and genipin. A cross-linking reaction was initiated, and the resulting reaction solution was dialyzed to obtain genipin-crosslinked selenium nanoparticles.

[0007] Furthermore, at least one of the following conditions must be met:

[0008] (1) The protein-like substances include proteins or acid hydrolysis products of proteins;

[0009] (2) The concentration of the protein solution is 0.5-2 mg / mL;

[0010] (3) The selenite compounds include at least one of selenite salts or selenite acid;

[0011] (4) The amount of selenite compound added is such that the final concentration of the selenite compound in the system is 2 ~ 5 mmol / L.

[0012] Furthermore, the protein includes at least one of casein, lactoglobulin, whey protein, soy protein, or milk protein;

[0013] The selenite includes at least one of sodium selenite, potassium selenite, ammonium selenite, calcium selenite, or magnesium selenite.

[0014] Furthermore, at least one of the following conditions must be met:

[0015] (1) The amount of vitamin C added is such that the final concentration of vitamin C in the system is 4~10 mmol / L;

[0016] (2) The amount of genipin added is such that the final concentration of genipin in the system is 0.01 mmol / L-20 mmol / L;

[0017] (3) The amount of genipin added is divided into low concentration, medium concentration and high concentration; wherein, the low concentration of genipin is 0.01-0.99 mmol / L; the medium concentration of genipin is 1-9.99 mmol / L; and the high concentration of genipin is 10-20 mmol / L.

[0018] (4) The addition of vitamin C and genipin is as follows: first add genipin for pre-crosslinking, then add vitamin C for reduction reaction; or mix vitamin C and genipin in advance and add them to the reaction system at the same time.

[0019] (5) The temperature of the cross-linking reaction is 15-45℃; the time of the cross-linking reaction is 8-16 hours.

[0020] This invention also proposes genipin crosslinked selenium nanoparticles prepared by any of the preparation methods described above.

[0021] The present invention also proposes a method for preparing pectin gel nanoparticles, comprising the following steps: mixing any of the above-mentioned genipin crosslinked selenium nanoparticles with a pectin solution, then adding calcium chloride solution dropwise, and performing an ionic crosslinking reaction to obtain pectin gel nanoparticles.

[0022] Furthermore, the genipin cross-linked selenium nanoparticles were added in the form of a dispersion of genipin cross-linked selenium nanoparticles; wherein the volume ratio of the dispersion of genipin cross-linked selenium nanoparticles, pectin solution, and calcium chloride solution was (1-4):(3-10):(0.5-2); wherein the concentration of the pectin solution was 0.5~2 mg / mL; and the concentration of the calcium chloride solution was 0.5~2 mg / mL.

[0023] This invention also proposes pectin gel nanoparticles prepared by any of the preparation methods described above.

[0024] The present invention also proposes the use of any of the above-described pectin gel nanoparticles in the preparation of medicaments for the prevention and / or treatment of colitis.

[0025] Furthermore, the dosage forms of the drug include oral preparations and enemas.

[0026] This invention has the following advantages:

[0027] The present invention proposes a method for preparing genipin-crosslinked selenium nanoparticles. This method utilizes genipin to crosslink protein-selenium nanoparticles. Since the concentration of genipin directly determines the density of the crosslinked network, the selenium release behavior can be directionally controlled by adjusting the genipin concentration. The outer pectin-Ca... 2+ The microgel structure modification gives it gastric protection and intestinal release properties, effectively avoiding the loss of active ingredients in the stomach. This invention innovatively utilizes genipin concentration as a regulatory switch to achieve active and precise control of the selenium release rate in the intestine, improving the therapeutic effect on colitis and effectively overcoming the narrow "nutritional-toxic" dosage range limitation of selenium. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram of the reaction of pectin-protein-genipin-selenium microgel nanoparticles in an embodiment of the present invention;

[0030] Figure 2 The nanoparticle size, polydispersity index, and potential diagram are shown in Test Example 1 of this invention.

[0031] Figure 3 This is a microscopic morphology diagram of the nanoparticles in Test Example 1 of the present invention;

[0032] Figure 4The release rate curve of the nanoparticles in Test Example 2 of this invention;

[0033] Figure 5 The release rate curve of the nanoparticles in Test Example 2 of this invention;

[0034] Figure 6 The colon length in test example 3 of this invention;

[0035] Figure 7 The change in mouse body weight in Test Example 3 of this invention;

[0036] Figure 8 The disease activity index of the mice in Test Example 3 of this invention;

[0037] Figure 9 HE staining results of colitis mice in Test Example 3 of this invention;

[0038] Figure 10 The levels of inflammatory factors in the mice used in Test Example 3 of this invention are measured. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0040] Genipin is a natural, biocompatible cross-linking agent that can react with the amino groups of proteins to form stable covalent cross-links. The inventors of this application have discovered that genipin cross-linking of protein-selenium nanoparticles significantly increases the release rate of selenium in simulated gastrointestinal fluid; within a certain range, the selenium release rate is positively correlated with the genipin concentration.

[0041] In a first aspect, embodiments of the present invention provide a method for preparing genipin-crosslinked selenium nanoparticles, comprising the following steps:

[0042] Proteins were dissolved in water to obtain a protein solution, then selenite compounds were added, followed by vitamin C and genipin. A cross-linking reaction was initiated, and the resulting reaction solution was dialyzed to obtain genipin-crosslinked selenium nanoparticles.

[0043] The method for preparing genipin-crosslinked selenium nanoparticles proposed in this invention involves dissolving proteins in water for hydration. The polar groups (amino-NH2, carboxyl-COOH, etc.) on the surface of the protein molecules form hydrogen bonds with water molecules, thus forming a hydration layer. This allows the protein molecules to be dispersed in the solution, preventing aggregation. At the same time, it exposes the hidden active sites inside the protein, providing binding sites for subsequent crosslinking reactions and the loading of selenium nanoparticles.

[0044] When selenite compounds are added, selenite can be grafted onto proteins. Then, the selenite compounds act as a selenium source, and vitamin C acts as a reducing agent. The two undergo a redox reaction to generate selenium nanoparticles (SeNPs). Genipin, as a biocrosslinking agent, can undergo a specific nucleophilic addition reaction with the amino groups of protein molecules to form crosslinked network structures with varying degrees of density.

[0045] The cross-linking effect of genipin may occur within the protein-selenium nanoparticles, or it may encapsulate the selenium nanoparticles within the cross-linked network. This avoids selenium nanoparticle aggregation while achieving selenium dispersion and fixation, which is crucial for the subsequent adjustable release rate. Since the concentration of genipin directly determines the density of the cross-linked network, as the concentration increases, the density of cross-linking points may not linearly form a uniform and dense structure. Instead, it may create more and more extensive microporous channels or make the network structure more aggregated. This process significantly increases the specific surface area. Although the overall particle size increases, its porous and irregular surface provides more sites for enzymatic reactions and ion exchange, thereby accelerating selenium release.

[0046] The method for preparing genipin crosslinked selenium nanoparticles proposed in this invention is carried out at room temperature and in an aqueous phase. The conditions are mild, the operation is simple, and the energy consumption is extremely low. This avoids the use of toxic chemical reagents or harsh conditions of high temperature and high pressure in traditional methods.

[0047] In one embodiment of the present invention, the hydration is carried out by stirring; the stirring time is 5-24 hours.

[0048] In one embodiment of the present invention, the protein-like substance includes protein or acid hydrolysate of protein. Preferably, the protein includes at least one of casein, lactoglobulin, whey protein, soy protein, or milk protein. Preferably, the acid hydrolysate of protein includes acid hydrolysate of casein, acid hydrolysate of lactoglobulin, acid hydrolysate of whey protein, acid hydrolysate of soy protein, or acid hydrolysate of milk protein. For example, the acid hydrolysate of protein includes acid hydrolysate of casein, i.e., casein hydrolysate powder (CAH). The protein or its acid hydrolysate is readily soluble in water but insoluble in organic solvents.

[0049] In one embodiment of the present invention, the concentration of the protein solution is 0.5-2 mg / mL. Preferably, the concentration of the protein solution is 1 mg / mL.

[0050] In one embodiment of the present invention, the selenite compound includes at least one of selenite or selenic acid. Selenite includes at least one of sodium selenite, potassium selenite, ammonium selenite, calcium selenite, or magnesium selenite. Preferably, the selenite is sodium selenite, which has good water solubility and high biocompatibility, and can react efficiently with vitamin C to generate selenium nanoparticles.

[0051] In one embodiment of the present invention, the amount of selenite compound added is such that the final concentration of selenite or selenite in the system is 2 to 5 mmol / L, preferably 4 mmol / L.

[0052] In one embodiment of the present invention, the process further includes adding a selenite compound, stirring, and then adding genipin and vitamin C. Preferably, the stirring time is 0.2 to 2 hours. In this embodiment of the present invention, selenite or selenite acid can be grafted onto the protein, and through stirring, a stable protein-selenium complex is formed, which is beneficial for subsequent cross-linking reactions.

[0053] In one embodiment of the present invention, the amount of vitamin C added is such that the final concentration of vitamin C in the system is 4~10 mmol / L, preferably 8 mmol / L.

[0054] In one embodiment of the present invention, the amount of genipin added is such that the final concentration of genipin in the system is 0.01 mmol / L-20 mmol / L. Specifically, the final concentration of genipin in the system can be 0.01 mmol / L, 0.05 mmol / L, 0.1 mmol / L, 0.3 mmol / L, 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, etc. In this embodiment of the invention, by precisely controlling the concentration of genipin, the degree of cross-linking of selenium nanoparticles can be precisely controlled, thereby regulating the release behavior of the active ingredient selenium in the gastrointestinal environment.

[0055] Preferably, the dosage of genipin can be divided into low concentration, medium concentration, and high concentration. Specifically, the low concentration of genipin is 0.01-0.99 mmol / L; the medium concentration is 1-9.99 mmol / L; and the high concentration is 10-20 mmol / L.

[0056] In one embodiment of the present invention, vitamin C and genipin are added in the following ways: genipin is added first for pre-crosslinking, followed by vitamin C for reduction; or vitamin C and genipin are pre-mixed and then added to the reaction system simultaneously. Preferably, vitamin C and genipin are pre-mixed and then added to the reaction system simultaneously. In this embodiment of the present invention, both of the above-mentioned addition methods can effectively prepare genipin-crosslinked protein-selenium nanoparticles, providing flexibility for process optimization.

[0057] In one embodiment of the present invention, the crosslinking reaction temperature is 15-45°C; the crosslinking reaction time is 8-16 hours. The pH of the crosslinking reaction is maintained in the range of 4-7. These mild reaction conditions ensure effective crosslinking of genipin with the amino groups in protein molecules while avoiding excessive protein denaturation or aggregation of selenium nanoparticles, thus ensuring the formation of structurally stable composite nanoparticles.

[0058] In one embodiment of the present invention, after the obtained reaction solution is dialyzed, it is further freeze-dried to obtain particles.

[0059] Secondly, this invention also provides genipin-crosslinked selenium nanoparticles prepared by any of the above-mentioned preparation methods. This invention proposes a green and efficient preparation strategy for the release of selenium nanoparticles. The protein carrier and genipin used are derived from natural products, exhibiting good biocompatibility and degradability, and high safety.

[0060] In one embodiment of the present invention, the obtained genipin crosslinked selenium nanoparticles have a particle size distribution of 50 nm to 500 nm and a polydispersity index (PDI) of less than 0.4. This characteristic indicates that the obtained nanoparticle system has excellent monodispersity and colloidal stability, which is beneficial to its absorption and distribution in vivo.

[0061] Thirdly, the embodiments of the present invention also propose the application of genipin cross-linked selenium nanoparticles prepared by any of the above preparation methods, or the application of any of the above genipin cross-linked selenium nanoparticles in the preparation of general functional foods that help maintain normal gastrointestinal function, or in the preparation of health products that help with antioxidation, or help improve bone density, or help maintain healthy blood sugar levels, or have an auxiliary protective effect against chemical liver damage, or help protect the gastric mucosa.

[0062] This invention also proposes the application of genipin cross-linked selenium nanoparticles prepared by any of the above-described methods, or the application of any of the above-described genipin cross-linked selenium nanoparticles in pharmaceuticals and cosmetics. Genipin cross-linked selenium nanoparticles are used in the pharmaceutical field to achieve effects such as immunomodulation, antioxidation, and regulation of glucose and lipid metabolism, and in cosmetics to achieve anti-inflammatory effects.

[0063] This invention reveals the positive regulatory mechanism of genipin crosslinking on selenium release rate, achieving precise control of selenium release. The study found that genipin crosslinking significantly enhances the release rate of selenium nanoparticles, and this release rate is positively correlated with genipin concentration. This discovery makes it possible to achieve "gradient regulation" of selenium release rate by precisely controlling genipin concentration, providing a novel technical means for the precise delivery and safe application of selenium.

[0064] Fourthly, this invention also proposes a method for preparing pectin gel nanoparticles, comprising the following steps:

[0065] After mixing the above-mentioned genipin cross-linked selenium nanoparticles with pectin solution, calcium chloride solution was added dropwise to induce an ionic cross-linking reaction, resulting in pectin gel nanoparticles.

[0066] In this embodiment of the invention, the surface of the genipin-crosslinked protein selenium nanoparticles carries an electric charge (determined by the amino acid residues of the protein and the crosslinking sites of genipin), while pectin is a water-soluble anionic polysaccharide whose molecular chain contains a large number of carboxyl groups (-COO). - When the two are mixed, pectin molecules are uniformly adsorbed onto the surface of protein selenium nanoparticles through electrostatic attraction and hydrogen bonding, forming a "protein selenium nanoparticle-pectin" composite dispersion system. Ca 2+ Upon addition, it cross-links with the carboxyl groups on different pectin molecular chains, forming a dense three-dimensional gel network. Ultimately, the protein selenium nanoparticles are completely encapsulated within the pectin gel, yielding pectin gel nanoparticles.

[0067] In one embodiment of the present invention, genipin crosslinked selenium nanoparticles are added in the form of a dispersion of genipin crosslinked selenium nanoparticles. The dispersion of genipin crosslinked selenium nanoparticles is prepared by any of the above-described methods for preparing genipin crosslinked selenium nanoparticles. Specifically, the obtained reaction solution is dialyzed to obtain the dispersion of genipin crosslinked selenium nanoparticles.

[0068] In one embodiment of the present invention, the volume ratio of the genipin crosslinked selenium nanoparticle dispersion, pectin solution, and calcium chloride solution is (1-4):(3-10):(0.5-2). For example, the volume ratio of the genipin crosslinked protein selenium nanoparticles, pectin solution, and calcium chloride solution is 2 mL:5 mL:1 mL.

[0069] In one embodiment of the present invention, the concentration of the pectin solution is 0.5~2 mg / mL; the concentration of the calcium chloride solution is 0.5~2 mg / mL. Preferably, the concentration of the pectin solution is 1 mg / mL; the concentration of the calcium chloride solution is 1 mg / mL.

[0070] Fifthly, embodiments of the present invention also provide pectin gel nanoparticles prepared by any of the above preparation methods.

[0071] Sixthly, embodiments of the present invention also propose the use of any of the above-mentioned pectin gel nanoparticles in the preparation of medicaments for the prevention and / or treatment of colitis.

[0072] The "core regulation-shell protection" synergistic delivery system constructed in this invention achieves precise intestinal-targeted release of selenium. This invention creatively embeds genipin-crosslinked protein selenium nanoparticles within a pectin gel. This gel system is stable in the acidic environment of the stomach, effectively inhibiting premature selenium release; upon entering the intestine, the gel swells at a neutral pH and, in conjunction with the regulatory effect of genipin crosslinking, achieves controllable and enhanced selenium release at the target site, significantly improving bioavailability.

[0073] This invention innovatively utilizes genipin concentration as a control switch to achieve active and precise control of the selenium release rate in the intestine. That is, even at low selenium dosages, the release of high concentrations can enhance the therapeutic effect on colitis, effectively overcoming the narrow "nutritional-toxic" dosage range defect of selenium.

[0074] This invention achieves precise pharmacokinetic characteristics at the site of inflammation by regulating the release rate, significantly improving the therapeutic effect in the intervention of colitis. This will provide a technological supplement for the rapid, precise, and safe delivery of selenium into the intestine, and further offer a new technical approach to improving and efficiently alleviating colitis delivery systems.

[0075] In one embodiment of the present invention, the dosage form of the drug includes oral preparations, enemas, etc.

[0076] The present invention will now be described in detail with reference to the embodiments.

[0077] Example 1A: A method for preparing genipin-crosslinked selenium nanoparticles with adjustable release rate, comprising the following steps:

[0078] (1) Accurately weigh 100 mg of casein hydrolysate powder (CAH), dissolve it in 100 mL of ultrapure water to prepare a solution with a concentration of 1.0 mg / mL. Stir magnetically overnight (12 h) at room temperature to ensure full protein hydration. Add sodium selenite (Na2SeO3) powder to the obtained protein solution to make the final concentration in the reaction system 4 mmol / L. Stir the mixture continuously for 1 hour at room temperature in the dark to form a stable protein-selenium complex;

[0079] (2) Add vitamin C solution to the solution obtained in step (1) to make the final concentration of vitamin C in the system 8 mmol / L, and add genipin to make the final concentration in the system 0.1 mmol / L. Place the reaction system in a dark environment and stir continuously at room temperature (25℃) for 12 hours. The pH of the cross-linking reaction is maintained at 4-7 to form a reaction solution containing stable selenium nanoparticles.

[0080] (3) Transfer the reaction solution obtained in step (2) to a dialysis bag to remove unreacted ions, small molecule organic matter and byproducts, and finally obtain a pure dispersion of genipin crosslinked selenium nanoparticles, denoted as CAH-SeNPs-G0.1.

[0081] Example 1B: A method for preparing pectin gel nanoparticles, comprising the following steps:

[0082] The purified dispersion of genipin-crosslinked selenium nanoparticles obtained in Example 1A was mixed with 1 mg / mL pectin solution at a volume ratio of 2:5 and magnetically stirred at room temperature for 4 hours to ensure thorough mixing. Subsequently, under continuous stirring, 1 mg / mL calcium chloride solution was slowly added dropwise at a volume ratio of 5:1 (pectin solution to calcium chloride solution) until obvious gelation occurred in the mixture, ultimately forming an encapsulated selenium nanogel. A schematic diagram of the reaction can be found in [reference needed]. Figure 1 .

[0083] Example 2A

[0084] Same as Example 1A, except that in step (2), genipin is added to make its final concentration in the system 0.5 mmol / L, denoted as CAH-SeNPs-G0.5.

[0085] Example 2B

[0086] Same as Example 1B, except that the nanoparticle dispersion obtained in Example 1A is replaced with the nanoparticle dispersion obtained in Example 2A, denoted as P-CAH-SeNPs-GL.

[0087] Example 3A

[0088] Same as Example 1A, except that in step (2), genipin is added to make its final concentration in the system 1 mmol / L, denoted as CAH-SeNPs-G1.

[0089] Example 3B

[0090] Same as Example 1B, except that the nanoparticle dispersion obtained in Example 1A is replaced with the nanoparticle dispersion obtained in Example 3A.

[0091] Example 4A

[0092] Same as Example 1A, except that in step (2), genipin is added to make its final concentration in the system 5 mmol / L, denoted as CAH-SeNPs-G5.

[0093] Example 4 B

[0094] Same as Example 1B, except that the nanoparticle dispersion obtained in Example 1A is replaced with the nanoparticle dispersion obtained in Example 4A, denoted as P-CAH-SeNPs-GM.

[0095] Example 5A

[0096] Same as Example 1A, except that in step (2), genipin is added to make its final concentration in the system 10 mmol / L, denoted as CAH-SeNPs-G10.

[0097] Example 5 B

[0098] Same as Example 1B, except that the nanoparticle dispersion obtained in Example 1A is replaced with the nanoparticle dispersion obtained in Example 5A. A schematic diagram of the reaction can be found in [reference needed]. Figure 1 .

[0099] Example 6A

[0100] Similar to Example 1A, except that in step (2), genipin is added to make its final concentration in the system 15 mmol / L, denoted as CAH-SeNPs-G15.

[0101] Example 6 B

[0102] Same as Example 1B, except that the nanoparticle dispersion obtained in Example 1A is replaced with the nanoparticle dispersion obtained in Example 6A, denoted as P-CAH-SeNPs-GH.

[0103] Example 7A

[0104] Same as Example 1A, except that in step (2), genipin is added to make its final concentration in the system 20 mmol / L, denoted as CAH-SeNPs-G20.

[0105] Example 7 B

[0106] Same as Example 1B, except that the nanoparticle dispersion obtained in Example 1A is replaced with the nanoparticle dispersion obtained in Example 7A.

[0107] Comparative Example 1A

[0108] Same as Example 1A, except that genipin was not added in step (2), denoted as CAH-SeNPs.

[0109] Comparative Example 1B

[0110] Same as Example 1B, except that the nanoparticle dispersion obtained in Example 1A is replaced with the nanoparticle dispersion obtained in Comparative Example 1A, denoted as P-CAH-SeNPs. A schematic diagram of the reaction can be found in [reference needed]. Figure 1 .

[0111] Test Example 1: Characterization of Physicochemical Properties of Nanoparticles

[0112] (1) Characterization of dispersibility and stability

[0113] Methods: The hydrodynamic diameter, polydispersity index and zeta potential of selenium nanoparticles were determined using a nanolaser particle size analyzer. The dispersion medium was water. 1 mL of selenium nanoparticles (1 mg / mL) were transferred to particle size dish and potentiometer for measurement. Each sample was measured 3 times, and each measurement was scanned 10 times.

[0114] result: Figure 2 Figure A in the figure shows the particle size and polydispersity index (PDI) of Comparative Example 1A, Example 2A, Example 4A, Example 6A, Comparative Example 1B, and Example 6B. Figure 2 Figure B in the figure is a potential diagram of the above embodiment and comparative example.

[0115] Without the addition of genistin, the average particle size of CAH-SeNPs (protein-selenium nanoparticles) was 88.72 ± 0.78 nm (n = 3), and the polydispersity index (PDI) was 0.21 ± 0.02 (n = 3), indicating that the nanoparticles were uniformly dispersed. All PDI values ​​were below 0.5, reflecting that the particle size distribution was relatively concentrated.

[0116] As the genipin concentration increased from 0 mmol / L to 15 mmol / L, a certain degree of reduction in nanoparticle size was observed, attributed to the shrinkage of casein hydrolysate material caused by genipin crosslinking. Simultaneously, the PDI decreased significantly, indicating that under these crosslinking conditions, the uniformity of the genipin-protein-selenium nanoparticle dispersion system was further improved, suggesting a more compact nanoparticle structure.

[0117] When the concentration of genipin was further increased to 15 mmol / L, the average particle size of the nanoparticles increased accordingly, which can be used as evidence for cross-linking reactions between protein molecules. Higher concentrations of genipin can adsorb onto the CAH surface, increasing the probability of intermolecular cross-linking, thus leading to particle aggregation and increased particle size, while also increasing solution heterogeneity.

[0118] After being encapsulated in pectin, the size of the gel spheres increased significantly, and the PDI increased accordingly, indicating that the system was transformed from a uniform nano-dispersion system into micron-sized gel particles. This phenomenon is consistent with the typical characteristics of particle aggregation and network formation during the gelation process.

[0119] Notably, when the encapsulated material was genipin-crosslinked protein-selenium nanoparticles, the particle size of the resulting composite gel spheres further increased, and the PDI also increased accordingly. This indicates that the crosslinking effect of genipin not only occurred within the protein-selenium nanoparticles but may also have promoted the enhancement and aggregation of the pectin gel network structure, thereby forming denser and larger composite gel particles. Throughout the process, the Zeta potential of the nanoparticles and the subsequent gel system remained within the range of -30 to -10 mV, indicating that the system possessed good colloidal stability at each stage.

[0120] (2) Microstructure characterization

[0121] Methods: The morphology of selenium nanoparticles was characterized by transmission electron microscopy (TEM). 20 μL of sample (1 mg / mL) was dropped onto a 200-mesh carbon-supported copper grid, allowed to stand for 10 min, and then naturally dried in air for 2 h. The dried sample was stored in a desiccator for later analysis. The morphology of the sample was observed under an accelerating voltage of 30 kV.

[0122] Figure 3 Images A, C, B, and D in the table are TEM images of Comparative Example 1A (protein selenium nanoparticles, scale bar 1µm), Comparative Example 1B (pectin-embedded protein selenium nanoparticles, scale bar 0.5µm), Example 6A (genipin-crosslinked protein selenium nanoparticles, scale bar 1µm), and Example 6B (pectin-embedded genipin-crosslinked protein selenium nanoparticles, scale bar 0.5µm), respectively. TEM results show that all samples have a uniform spherical shape. Genipin promotes crosslinking between particles or between particles and proteins, forming a tighter covalent network.

[0123] Test Example 2: In vitro release performance test of genipin cross-linked selenium nanoparticles

[0124] Mix 5 mg of sample with 10 mL of simulated gastric fluid and place the mixture in a constant-temperature shaker to simulate gastric digestion. After gastric digestion, adjust the pH of the digestion system with 1 M NaOH solution, then add 10 mL of simulated intestinal fluid and continue digestion under the same conditions (37℃, 200 r / min) to simulate the intestinal environment. Samples were taken at 0, 0.5, 1, 2, 3, and 4 hours of gastric digestion and 0.5, 1, 2, 3, 6, and 12 hours of intestinal digestion, with 0.4 mL of digestion solution taken each time. The samples were centrifuged at 4℃ and 10000 r / min for 20 minutes (using a 3.0 kDa ultrafiltration centrifuge tube), and the supernatant was used for subsequent analysis.

[0125] Take 1 mL of the digestion supernatant, add 7 mL of nitric acid and 1 mL of hydrochloric acid, and then place the digestion vessel in a microwave digester for digestion. Transfer the digest to a temperature-controlled hot plate and heat at 100 °C to concentrate the solution until the volume is reduced to 1 mL and the solution is clear and transparent. Make up to 10 mL with ultrapure water in a volumetric flask, take 5 mL of the above solution, filter it through a 0.22 μm filter membrane, collect it in a centrifuge tube, and determine the selenium content using ICP-MS. Calculate the actual selenium concentration in the sample based on the selenium standard curve.

[0126] The following processing groups are set up in this case:

[0127] Comparative Example 1A (CAH-SeNPs): Protein selenium nanoparticles without genipin crosslinking;

[0128] Example 1A (CAH-SeNPs-G0.1): Protein selenium nanoparticles treated with 0.1 mmol / L genipin;

[0129] Example 2A (CAH-SeNPs-G0.5): Protein selenium nanoparticles treated with 0.5 mmol / L genipin;

[0130] Example 3A (CAH-SeNPs-G1): Protein selenium nanoparticles treated with 1 mmol / L genipin;

[0131] Example 4A (CAH-SeNPs-G5): Protein selenium nanoparticles treated with 5 mmol / L genipin;

[0132] Example 5A (CAH-SeNPs-G10): Protein selenium nanoparticles treated with 10 mmol / L genipin;

[0133] Example 6A (CAH-SeNPs-G15): Protein selenium nanoparticles treated with 15 mmol / L genipin;

[0134] Example 7A (CAH-SeNPs-G20): Protein selenium nanoparticles treated with 20 mmol / L genipin;

[0135] Comparative Example 1B (P-CAH-SeNPs): Pectin-encapsulated protein selenium nanogels;

[0136] Example 2B (P-CAH-SeNPs-GL): Pectin-encapsulated protein selenium nanogels, low-concentration genipin treatment group;

[0137] Example 4B (P-CAH-SeNPs-GM): Pectin-encapsulated protein selenium nanogels, treated with medium concentrations of genipin;

[0138] Example 6B (P-CAH-SeNPs-GH): Pectin-encapsulated protein selenium nanogel, high-concentration genipin treatment group.

[0139] See results Figure 4 . Figure 4 Figure A shows that the selenium accumulation and release rate of unencapsulated protein selenium nanoparticles (CAH-SeNPs) increases significantly with increasing concentration of the crosslinking agent genipin, which directly proves that genipin crosslinking has a clear positive regulatory effect on selenium release behavior.

[0140] Specifically, the selenium release rate of low-concentration genipin-crosslinked selenium nanoparticles is 0.059-0.073 mg / L. -1 h -1 The selenium release rate of medium-concentration genipin-crosslinked selenium nanoparticles ranged from 0.056 to 0.127 mg / L. -1 h -1 The selenium release rate of high-concentration genipin-crosslinked selenium nanoparticles ranged from 0.108 to 0.172 mg / L. -1 h -1 .

[0141] The release curves of CAH-SeNPs-GH show that it has a high release rate in the initial stage (0-3 h), with a cumulative release of over 50%; subsequently, the release rate decreases significantly and tends to stabilize. This may mean that most of the easily released selenium has already been rapidly released in the early stage. Figure 4 Figure B in the figure shows that the release behavior of the gel system clearly reproduces the regulatory pattern of genipin: although the release initiation is delayed due to encapsulation, the final intestinal release rate is still positively correlated with the concentration of genipin.

[0142] Figure 5 The cumulative selenium release rate curves of each experimental group during the simulated gastrointestinal digestion process in vitro are shown.

[0143] Figure 5Figure A in the figure shows the premature release of pectin-unencapsulated nanoparticles in the stomach. We further constructed a pectin-encapsulated composite gel system. Figure 5 Figure B shows that the release of all pectin-encapsulated protein selenium nanogels was effectively inhibited in the gastric stage, with a flat curve and extremely low cumulative release rate, fully demonstrating the excellent gastric protective function of the pectin gel. This indicates that the "physical barrier" effect of the gel and the "chemical regulation" effect of genipin crosslinking achieved a highly efficient synergy, jointly achieving the precise delivery target of "low release in the stomach, high release in the intestine, and controllable release".

[0144] Evaluation of the relief effect of test case 3 on colitis

[0145] To verify the intervention effect of the genipin crosslinked protein-selenium nanoparticle / pectin calcium composite gel system prepared in this invention on colitis, this study conducted a systematic evaluation through animal experiments. SPF-grade healthy C57BL / 6 mice were used in the experiment. After acclimatization under standard laboratory conditions, a colitis model was established by inducing the use of sodium dextran sulfate (DSS).

[0146] The experiment was divided into the following groups:

[0147] Normal group (Nor), Model group (Mod)

[0148] Experimental group (LP-CAH-SeNPs): Low concentration group of pectin-embedded protein selenium nanogels. The nano-dispersed particle liquid used in this experiment was prepared by Comparative Example 1B. The low concentration was 0.5 mg / kg, specifically 0.5 mg of selenium was added per kg of mouse body weight.

[0149] Experimental group (HP-CAH-SeNPs): High concentration group of pectin-embedded protein selenium nanogels. The nano-dispersed particle liquid used in this experiment was prepared by Comparative Example 1B. The high concentration was 1 mg / kg, specifically 1 mg of selenium was added per kg of mouse body weight.

[0150] Experimental group (LP-CAH-SeNPs-G): low concentration group treated with pectin-embedded protein selenium nanogels and genipin. The nano-dispersed particles used in this experiment were prepared in Example 5B. The low concentration was 0.5 mg / kg, specifically 0.5 mg of selenium per kg of mouse body weight.

[0151] Experimental group (HP-CAH-SeNPs-G): High concentration group treated with pectin-embedded protein selenium nanogels and genipin, wherein the nano-dispersed particles used in this experiment were prepared in Example 5B; the high concentration was 1 mg / kg, specifically 1 mg of selenium was added per kg of mouse body weight.

[0152] In terms of the evaluation method, this study established a systematic evaluation system. By regularly recording the body weight changes of mice daily, combined with the fecal character score (0 points: formed feces; 2 points: pasty soft feces; 4 points: watery diarrhea) and the blood in feces degree score (0 points: no blood in feces; 2 points: positive occult blood test; 4 points: visible blood in feces with the naked eye), the disease activity index was accurately calculated to comprehensively evaluate the improvement degree of animal clinical symptoms. At the same time, ELISA was used to detect tumor necrosis factor-α (TNF-α) to evaluate its core role in the inflammatory cascade reaction; the content of interleukin-6 (IL-6) was measured to reflect the degree of acute inflammatory reaction; and the level of interleukin-1β (IL-1β) was quantified.

[0153] This study successfully constructed a "genipin-crosslinked protein-selenium nanoparticles-pectin" composite gel system and studied the role of this delivery system in alleviating colitis. The results are shown in Figures 6-10 .

[0154] Figure 6 showed the colon length. It can be seen from Figure 6 that the colon length of mice in the Mod group was significantly shortened, showing a statistical difference compared with the Nor group (8.1 ± 0.4 cm). The order of the colon length of the experimental groups was L-P-CAH-SeNPs < H-P-CAH-SeNPs < L-P-CAH-SeNPs-G < H-P-CAH-SeNPs-G. H-P-CAH-SeNPs-G was closest to the normal control group level and was significantly better than other intervention groups.

[0155] Figure 7 showed the body weight changes. It can be seen from Figure 7 that the body weight of mice in the model group continued to decline. The weight loss rates of L-P-CAH-SeNPs were 14.9%, and those of H-P-CAH-SeNPs and L-P-CAH-SeNPs-G groups were 12.3% and 11.2% respectively, while the weight loss rate of the high-concentration genipin-crosslinked group H-P-CAH-SeNPs-G was only 7.9%, showing the best weight maintenance effect.

[0156] Figure 8 showed the disease activity index. It can be seen from Figure 8Therefore, in studies of intestinal diseases such as colitis, the disease activity index (DIA) is a comprehensive clinical scoring indicator. It is typically quantified by observing and measuring the degree of weight loss, fecal characteristics (such as diarrhea and bloody stools), and the general activity level of the animal. A higher DIA indicates more severe intestinal inflammation, more severe mucosal damage, and a worse overall disease state. Experimental results showed significant differences in DIA among the groups, with trends consistent with the measurement results of colon length. Specifically, the LP-CAH-SeNPs group had the highest DIA, significantly higher than other groups. However, the DIA of P-CAH-SeNPs-G crosslinked with different concentrations of genipin showed a clear dose-dependent decreasing trend, i.e., HP-CAH-SeNPs-G group > LP-CAH-SeNPs-G group. This indicates that pectin gel containing genipin-crosslinked selenium nanoparticles can effectively alleviate the disease severity in the experimental model.

[0157] Figure 9 The results of HE staining in colitis mice are shown. Figure 9 It was found that the colonic mucosal structure in the Mod group was severely damaged, with diffuse infiltration of a large number of inflammatory cells (such as lymphocytes and neutrophils), distortion and disappearance of crypt structures, and deep ulcers and epithelial cell shedding in some areas. The colonic tissue of the LP-CAH-SeNPs group still showed obvious inflammatory cell infiltration and mucosal edema, with more significant damage to the crypt structure. The degree of inflammatory cell infiltration in the HP-CAH-SeNPs and LP-CAH-SeNPs-G groups was moderate, with reduced submucosal edema, and the integrity and depth of the crypt structure were well maintained. The colonic mucosal structure of the HP-CAH-SeNPs-G group was intact, with only a small amount of focal inflammatory cell infiltration. The crypt structure was clear, neatly arranged, and close to the morphology of normal tissue, with almost no ulcers or epithelial shedding.

[0158] Figure 10 It shows the level of inflammatory factors, by Figure 10 It can be concluded that IL-6, IL-1β, and TNF-α are among the factors driving the inflammatory response in the pathogenesis of colitis. Experimental results showed that the model group (Mod) had the highest levels of pro-inflammatory cytokines, indicating the most severe local and systemic inflammatory response in the intestine. After treatment with pectin gel containing genipin-crosslinked selenium nanoparticles, the levels of all inflammatory factors were significantly reduced, exhibiting a clear dose-dependent inhibitory effect. Among them, the HP-CAH-SeNPs-G group showed the most significant inhibitory effect, with the lowest levels of IL-6, IL-1β, and TNF-α, even approaching normal levels, which was superior to the LP-CAH-SeNPs-G, LP-CAH-SeNPs, and HP-CAH-SeNPs groups.

[0159] The results of this study indicate that the high-concentration genipin crosslinking group (HP-CAH-SeNPs-G) significantly improved colitis-related indicators, effectively maintaining normal colonic physiological length, controlling weight loss, and significantly reducing the disease activity index, demonstrating its good intervention effect on colitis, and the effect showed a clear genipin concentration-dependent relationship. Previous studies have shown that as the concentration of genipin increases, its selenium release rate in the intestinal environment increases accordingly. This positive correlation directly determines the intervention effect of the system: HP-CAH-SeNPs-G, due to its fastest release rate, can rapidly establish an effective selenium concentration during the acute phase of colitis, timely neutralizing excess reactive oxygen species and blocking the inflammatory cascade response, thus exhibiting optimal effects on various indicators.

[0160] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing pectin gel nanoparticles, characterized in that, Includes the following steps: After mixing genipin cross-linked selenium nanoparticles with pectin solution, calcium chloride solution was added dropwise to induce an ionic cross-linking reaction, resulting in pectin gel nanoparticles. The genipin crosslinked selenium nanoparticles are prepared by the following steps: Proteins are dissolved in water to hydrate them into a protein solution. Selenite compounds are then added to form a protein-selenium complex solution. Vitamin C and genipin are then added to initiate a cross-linking reaction. The resulting reaction solution is dialyzed to obtain genipin-crosslinked selenium nanoparticles. The amount of genipin added is such that the final concentration of genipin in the protein-selenium complex solution is 0.1 mmol / L to 20 mmol / L. The methods for adding vitamin C and genipin are as follows: first add genipin for pre-crosslinking, then add vitamin C for reduction reaction; or mix vitamin C and genipin beforehand and add them to the protein-selenium complex solution at the same time. The protein-like substances include proteins or acid hydrolysates of proteins; the proteins include at least one of casein, lactoglobulin, whey protein, or milk protein.

2. The preparation method according to claim 1, characterized in that, At least one of the following conditions must be met: (1) The concentration of the protein solution is 0.5-2 mg / mL; (2) The selenite compounds include at least one of selenite salts or selenite acid; (3) The amount of selenite compound added is such that the final concentration of the selenite compound in the protein-selenium complex solution is 2 ~ 5 mmol / L.

3. The preparation method according to claim 2, characterized in that, The selenite includes at least one of sodium selenite, potassium selenite, ammonium selenite, calcium selenite, or magnesium selenite.

4. The preparation method according to claim 1, characterized in that, At least one of the following conditions must be met: (1) The amount of vitamin C added is such that the final concentration of vitamin C in the protein-selenium complex solution is 4~10 mmol / L; (2) The amount of genipin added is divided into low concentration, medium concentration and high concentration; wherein, the low concentration of genipin is 0.1-0.99 mmol / L; the medium concentration of genipin is 1-9.99 mmol / L; and the high concentration of genipin is 10-20 mmol / L. (3) The temperature of the cross-linking reaction is 15-45℃; the time of the cross-linking reaction is 8-16 hours.

5. The preparation method according to claim 1, characterized in that, Genipin cross-linked selenium nanoparticles were added in the form of a dispersion of genipin cross-linked selenium nanoparticles; wherein the volume ratio of the dispersion of genipin cross-linked selenium nanoparticles, pectin solution, and calcium chloride solution was (1-4):(3-10):(0.5-2); wherein the concentration of the pectin solution was 0.5~2 mg / mL; and the concentration of the calcium chloride solution was 0.5~2 mg / mL.

6. Pectin gel nanoparticles prepared by the preparation method according to any one of claims 1 to 5.

7. The use of the pectin gel nanoparticles of claim 6 in the preparation of a medicament for the prevention and / or treatment of colitis.

8. The application according to claim 7, characterized in that, The dosage forms of the drug include oral preparations and enemas.

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