A chitosan-coated curcumin / selenium composite particle, its preparation method and application

Abstract

This invention relates to the field of biomaterials technology, and discloses a chitosan-coated curcumin / selenium composite particle, its preparation method, and its application. The method includes the following steps: Step 1: Dissolve chitosan in acetic acid solution until fully dissolved to obtain mixed solution A; Step 2: Add sodium selenite to gum arabic solution and mix thoroughly to obtain mixed solution B; Step 3: Add curcumin, Tween, and lecithin to anhydrous ethanol and mix thoroughly to obtain mixed solution C; Step 4: Add mixed solution C to mixed solution A, then add mixed solution B dropwise; stir thoroughly, remove the solvent, and centrifuge to obtain the desired composite particle; The composite particle obtained by this invention improves the antioxidant performance and overall stability of the system while achieving synergistic delivery of curcumin and selenium.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a chitosan-coated curcumin / selenium composite particle, its preparation method, and its application. Background Technology

[0002] Curcumin is a natural polyphenol compound with broad biological functions, exhibiting significant activity in antioxidation, anti-inflammation, and antitumor activity. However, its physicochemical instability severely restricts its practical application. Studies have shown that curcumin is easily degraded under light, oxidative environments, and heating conditions, and its extremely low solubility in water makes it difficult to maintain an effective concentration during in vivo digestion and absorption, resulting in low oral bioavailability and thus limiting its bioavailability. Selenium, as an essential trace element for the human body, plays an important role in maintaining the body's antioxidant defense system and immune function. Currently, many regions worldwide still suffer from varying degrees of selenium deficiency, making efficient and safe selenium supplementation methods a focus of widespread attention. Compared to traditional inorganic or organic selenium forms, nano-selenium, as a zero-valent, amorphous form of selenium, is considered a promising new selenium supplement carrier due to its low toxicity and good biocompatibility. However, SeNPs are prone to aggregation in water-soluble systems, exhibiting insufficient stability and affecting their bioavailability.

[0003] Furthermore, curcumin is toxic and cannot be used directly; existing delivery systems cannot solve these problems. Summary of the Invention

[0004] This invention addresses the problems existing in the prior art by providing a chitosan-coated curcumin / selenium composite particle, its preparation method, and its application.

[0005] A method for preparing chitosan-coated curcumin / selenium composite particles includes the following steps: Step 1: Dissolve chitosan in acetic acid solution until fully dissolved to obtain mixed solution A; Step 2: Add sodium selenite to the gum arabic solution and mix thoroughly to obtain mixed solution B; Step 3: Add curcumin, Tween, and lecithin to anhydrous ethanol and mix thoroughly to obtain mixed solution C; Step 4: Add mixed solution C to mixed solution A, and then add mixed solution B dropwise; the mass ratio of chitosan to gum arabic in the mixed system is 3:1 to 1:2, and the mass ratio of curcumin to sodium selenite is 1 to 2:173; stir thoroughly, remove the solvent, and centrifuge to obtain the desired composite particles.

[0006] Furthermore, the mixed solution A in step 1 also includes ascorbic acid, and the molar ratio of ascorbic acid to sodium selenite is 3:1 to 5:1.

[0007] Furthermore, in step 3, the concentration of curcumin in the mixed solution C is 3–7 mg / mL.

[0008] Furthermore, in step 4, the pH value is maintained at 4.0.

[0009] Furthermore, stirring conditions are maintained in both steps 1 and 4; The stirring speed in step 1 is 600 rpm, and the stirring speed in step 4 is 800 rpm.

[0010] Furthermore, the concentration of the acetic acid solution in step 1 is 1% (w / v).

[0011] Furthermore, in step 3, curcumin, Tween, and lecithin are dissolved in anhydrous ethanol by ethanol injection.

[0012] Furthermore, in step 3, the mass ratio of Tween to lecithin is 1:1 to 4:1.

[0013] A chitosan-coated curcumin / selenium composite particle, wherein the composite particle has a core-shell structure, with chitosan as the shell and a mixture of curcumin and nano-selenium distributed in a chitosan and gum arabic composite matrix as the core.

[0014] Application of a chitosan-coated curcumin / selenium composite particle, said composite particle being used in drug preparation.

[0015] The beneficial effects of this invention are: In the preparation of composite particles, sodium selenite and ascorbic acid react in a polysaccharide matrix to generate nano-selenium, which is then co-distributed with curcumin in a chitosan-gum arabic composite matrix. The polysaccharide-based polyelectrolyte composite system not only stabilizes and encapsulates the nanoparticles through electrostatic interactions, hydrogen bonding, and steric hindrance, but also achieves synergistic enhancement at the functional level. The resulting composite particles, while stabilizing the nano-selenium structure, further improve the system's antioxidant properties and overall stability, and achieve synergistic delivery of curcumin and selenium. Attached Figure Description

[0016] Figure 1 This is a schematic diagram showing the amount of selenium bound in the composite particles obtained in Examples 1-3 and Comparative Examples 1, 5, and 6 of the present invention.

[0017] Figure 2 This is a schematic diagram showing the selenium content and polysaccharide content in the composite particles obtained in Examples 1-3 and Comparative Examples 5 and 6 of the present invention.

[0018] Figure 3This is a schematic diagram showing the DPPH scavenging rate of the composite particles and free curcumin obtained in Examples 1-3 and Comparative Examples 5 and 6 of the present invention.

[0019] Figure 4 This is a schematic diagram showing the ABTS scavenging rate of the composite particles and free curcumin obtained in Examples 1-3 and Comparative Examples 5 and 6 of the present invention.

[0020] Figure 5 The composite particles obtained in Examples 1-3, Comparative Examples 5 and 6 of this invention and the composite particles obtained in Comparative Example 1 simulate the curcumin release rate during the gastrointestinal digestion process, where a is the release rate in the stomach and b is the release rate in the intestine.

[0021] Figure 6 The values ​​represent the release rates of selenium in the gastrointestinal tract during the simulated effect of composite particles obtained in Examples 1-3 and Comparative Examples 5 and 6 of this invention. a represents the release rate in the stomach, and b represents the release rate in the intestine.

[0022] Figure 7 The images shown are SEM images of the composite particles obtained in Examples 1-3 and Comparative Examples 5 and 6 of this invention.

[0023] Figure 8 Fourier transform infrared spectra of composite particles (d), comparative example 2 (c), comparative example 3 (b), and free curcumin (a) obtained in Example 1 of the present invention.

[0024] Figure 9 The bioavailability of Examples 1-3, Comparative Examples 5 and 6, and free curcumin of the present invention.

[0025] Figure 10 The results of microscopic observation of the morphology of HepG2 cells by nano-selenium and free curcumin are shown in Example 1 and Comparative Example 4 of this invention.

[0026] Figure 11 The results show the effects of Example 1 of the present invention and different treatment groups on the survival rate of L-02 normal hepatocytes. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0028] A method for preparing chitosan-coated curcumin / selenium composite particles includes the following steps: Step 1: Dissolve chitosan in acetic acid solution until fully dissolved to obtain mixed solution A; also includes ascorbic acid, with a molar ratio of ascorbic acid to sodium selenite of 3:1 to 5:1. The concentration of the acetic acid solution is 1% (w / v).

[0029] Step 2: Add sodium selenite to the gum arabic solution and mix thoroughly to obtain mixed solution B; Step 3: Add curcumin, Tween, and lecithin to anhydrous ethanol and mix thoroughly; the concentration of curcumin in the mixed solution C is 3–7 mg / mL; the preferred concentration of curcumin is 5 mg / mL, which can be adjusted according to the amount to be encapsulated as needed. Dissolve curcumin, Tween, and lecithin in anhydrous ethanol using the ethanol injection method.

[0030] Step 4: Add mixed solution C to mixed solution A, then add mixed solution B dropwise; the mass ratio of chitosan to gum arabic in the mixed system is 3:1 to 1:2, and the mass ratio of curcumin to sodium selenite is 1 to 2:173; stir thoroughly, remove the solvent, and centrifuge to obtain the desired composite particles. Maintain the pH value at 4.0.

[0031] Example 1 A method for preparing chitosan-coated curcumin / selenium composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved. Add 5 mL of 0.2 M ascorbic acid solution to obtain mixed solution A.

[0032] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water. Add 2 mL of 0.1 M sodium selenite to the gum arabic solution to obtain mixed solution B.

[0033] Step 3: Using the ethanol injection method, dissolve curcumin, Tween 80 and lecithin in anhydrous ethanol, keeping the concentration of curcumin at 5 mg / mL, with 0.05 g of curcumin, 0.05 g of Tween 80, 0.05 g of lecithin and 10 mL of anhydrous ethanol, to obtain mixed solution C.

[0034] Step 4: At 800 rpm, add solution C to solution A, and then add solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion (Cur-Se-Cs-Ga) is obtained.

[0035] Example 2 A method for preparing chitosan-coated curcumin / selenium composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved. Add 3 mL of 0.2 M ascorbic acid solution to obtain mixed solution A.

[0036] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water. Add 2 mL of 0.1 M sodium selenite to the gum arabic solution to obtain mixed solution B.

[0037] Step 3: Using the ethanol injection method, dissolve curcumin, Tween 80 and lecithin in anhydrous ethanol, keeping the concentration of curcumin at 5 mg / mL, with 0.05 g of curcumin, 0.05 g of Tween 80, 0.05 g of lecithin and 10 mL of anhydrous ethanol, to obtain mixed solution C.

[0038] Step 4: At 800 rpm, add solution C to solution A, and then add solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion is obtained.

[0039] Example 3 A method for preparing chitosan-coated curcumin / selenium composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved. Add 4 mL of 0.2 M ascorbic acid solution to obtain mixed solution A.

[0040] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water. Add 2 mL of 0.1 M sodium selenite to the gum arabic solution to obtain mixed solution B.

[0041] Step 3: Using the ethanol injection method, dissolve curcumin, Tween 80 and lecithin in anhydrous ethanol, keeping the concentration of curcumin at 5 mg / mL, with 0.05 g of curcumin, 0.05 g of Tween 80, 0.05 g of lecithin and 10 mL of anhydrous ethanol, to obtain mixed solution C.

[0042] Step 4: At 800 rpm, add solution C to solution A, and then add solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion is obtained.

[0043] Comparative Example 1 A method for preparing chitosan-coated curcumin / selenium composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved to obtain mixed solution A.

[0044] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water. Add 2 mL of 0.1 M sodium selenite to the gum arabic solution to obtain mixed solution B.

[0045] Step 3: Using the ethanol injection method, dissolve curcumin, Tween 80 and lecithin in anhydrous ethanol, keeping the concentration of curcumin at 5 mg / mL, with 0.05 g of curcumin, 0.05 g of Tween 80, 0.05 g of lecithin and 10 mL of anhydrous ethanol, to obtain mixed solution C.

[0046] Step 4: At 800 rpm, add solution C to solution A, and then add solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion is obtained.

[0047] Comparative Example 2 A method for preparing composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved. Add 5 mL of 0.2 M ascorbic acid solution to obtain mixed solution A.

[0048] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water. Add 2 mL of 0.1 M sodium selenite to the gum arabic solution to obtain mixed solution B.

[0049] Step 3: Using the ethanol injection method, Tween 80 and lecithin were dissolved in anhydrous ethanol, wherein Tween 80 was 0.05 g, lecithin was 0.05 g, and anhydrous ethanol was 10 mL, to obtain mixed solution C.

[0050] Step 4: At 800 rpm, add mixed solution C to mixed solution A, and then add mixed solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the mixed solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion (Se-Cs-Ga) is obtained.

[0051] Comparative Example 3 A method for preparing composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved to obtain mixed solution A.

[0052] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water to obtain mixed solution B.

[0053] Step 3: Using the ethanol injection method, Tween 80 and lecithin were dissolved in anhydrous ethanol, with 0.05 g of Tween 80, 0.05 g of lecithin, and 10 mL of anhydrous ethanol, to obtain mixed solution C.

[0054] Step 4: At 800 rpm, add solution C to solution A, and then add solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion (Cs-Ga) is obtained.

[0055] Comparative Example 4 A method for preparing composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved to obtain mixed solution A.

[0056] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water to obtain mixed solution B.

[0057] Step 3: Using the ethanol injection method, dissolve curcumin, Tween 80 and lecithin in anhydrous ethanol, keeping the concentration of curcumin at 5 mg / mL, with 0.05 g of curcumin, 0.05 g of Tween 80, 0.05 g of lecithin and 10 mL of anhydrous ethanol, to obtain mixed solution C.

[0058] Step 4: At 800 rpm, add solution C to solution A, and then add solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion (Cur-Cs-Ga) is obtained.

[0059] Comparative Example 5 A method for preparing chitosan-coated curcumin / selenium composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved. Add 1 mL of 0.2 M ascorbic acid solution to obtain mixed solution A.

[0060] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water. Add 2 mL of 0.1 M sodium selenite to the gum arabic solution to obtain mixed solution B.

[0061] Step 3: Using the ethanol injection method, dissolve curcumin, Tween 80 and lecithin in anhydrous ethanol, keeping the concentration of curcumin at 5 mg / mL, with 0.05 g of curcumin, 0.05 g of Tween 80, 0.05 g of lecithin and 10 mL of anhydrous ethanol, to obtain mixed solution C.

[0062] Step 4: At 800 rpm, add solution C to solution A, and then add solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion is obtained.

[0063] Comparative Example 6 A method for preparing chitosan-coated curcumin / selenium composite particles includes the following steps: Step 1: Weigh 0.1 g of chitosan (Cs) and dissolve it in 30 mL of 1% (w / v) acetic acid solution. Stir at 600 rpm for 6 h at room temperature until completely dissolved. Add 2 mL of 0.2 M ascorbic acid solution to obtain mixed solution A.

[0064] Step 2: Weigh 0.1 g of gum arabic powder (Ga) and dissolve it in 30 mL of deionized water. Add 2 mL of 0.1 M sodium selenite to the gum arabic solution to obtain mixed solution B.

[0065] Step 3: Using the ethanol injection method, dissolve curcumin, Tween 80 and lecithin in anhydrous ethanol, keeping the concentration of curcumin at 5 mg / mL, with 0.05 g of curcumin, 0.05 g of Tween 80, 0.05 g of lecithin and 10 mL of anhydrous ethanol, to obtain mixed solution C.

[0066] Step 4: At 800 rpm, add solution C to solution A, and then add solution B dropwise using a syringe. After the addition is complete, continue stirring for 30 min, then rotary evaporate at 40 ℃ for 30 min to remove ethanol. Centrifuge the solution at 10000 rpm for 20 min at room temperature. Collect the supernatant and dialyze it to obtain the sample. Finally, a pure composite particle dispersion is obtained.

[0067] The selenium binding content in the composite particles obtained from Comparative Examples 1, 5, and 6, and Examples 1-3 was tested, and the results are as follows: Figure 1 As shown in the figure, the selenium binding amount of Comparative Example 1 was 17.96 μg / mg, or 1.8%, significantly lower than that of the Example group. The selenium binding amount was tested according to the method for determining selenium binding amount using 3,3′-diaminobenzidine. The addition of ascorbic acid significantly increased the selenium binding amount.

[0068] Vitamin C is a typical water-soluble antioxidant, easily absorbed and metabolized in the body, with no cumulative toxicity and extremely high safety, making it suitable for all types of people. Compared to fat-soluble vitamin E, vitamin C is more easily dispersed in aqueous systems (such as granules, solutions, and tablets), and has a wider range of applications. Furthermore, vitamin C is a colorless and odorless crystalline solid, which does not affect the sensory properties of the finished product. In contrast, some antioxidants, such as tea polyphenols and anthocyanins, may cause bitterness or color changes, which is detrimental to product quality control. Glutathione is also a commonly used antioxidant. Glutathione (GSH) itself is a tripeptide with strong reducing properties. GSH and bovine serum albumin are used together, which greatly increases the complexity of the system. BSA is a protein with a large molecular weight, easily binding to, coating, or adsorbing onto the surface of nanoparticles, affecting particle formation, stability, dispersibility, and even subsequent physicochemical properties. Moreover, glutathione is much more expensive than vitamin C, and the cost increases significantly with large dosages.

[0069] The encapsulation efficiency of curcumin in the composite particles obtained in Examples 1-3 and Comparative Example 4 was tested. Encapsulation efficiency EE (%) = ) × 100% The composite particles obtained in Comparative Example 4 showed a curcumin encapsulation rate of 86.77 ± 0.36%. In contrast, the composite particles obtained in Examples 1-5 all had a curcumin content greater than 90%. This demonstrates that the addition of nano-selenium and ascorbic acid can significantly improve the curcumin encapsulation rate of the particles.

[0070] Figure 2 The selenium and polysaccharide contents of the composite particles obtained in Examples 1-3 and Comparative Examples 5 and 6 of this invention are shown. It can be seen that the composite particles obtained in Comparative Example 6 have the lowest selenium binding capacity, while the composite particles obtained in Example 1 have the highest selenium binding capacity. There is a hydrogen bond interaction between the -OH group of the polysaccharide polyelectrolyte and Se, which induces the particles to self-assemble into a porous three-dimensional structure through weak electrostatic interactions. Excess ascorbic acid molecules (5 mL) form a dense hydrogen bond network with the surface of selenium nanoparticles (SeNPs) through hydroxyl and carboxylic acid groups. This exposes more encapsulated selenium active sites on the nanoparticles and prevents the SeNPs from being oxidized, thereby improving the binding efficiency. The polysaccharide content in the Cur-Se-Cs-Ga composite shows a trend of first decreasing and then increasing with the increase of Vc addition. With the increase of Vc addition, the particle size is smaller, and the hydroxyl groups (-OH) of the polyelectrolyte compete with the carboxylic acid groups (-COOH) of Vc for binding sites on the selenium surface, resulting in a decrease in polysaccharide-Se complexation and a decrease in polysaccharide content. As the concentration of vitamin C continues to increase, the amount of selenium bound increases significantly, which promotes the recombination of the polysaccharide network and causes the polysaccharide content to rebound.

[0071] Figure 3 and Figure 4The figures show the antioxidant activity test results of the embodiments of the present invention and free curcumin. As can be seen from the figures, the antioxidant activity of the embodiments is significantly higher than that of the comparative example. The DPPH results of the Cur-Se-Cs-Ga complex are given as the 50% inhibition concentration (IC50), and then the IC50 is obtained from the linear regression plot of the inhibition percentage relative to the sample concentration. The IC50 of the antioxidant activity of the free curcumin solution is 10.24 ± 0.29 μg / mL. When the amount of ascorbic acid added is 5 mL, the scavenging rate of the Cur-Se-Cs-Ga complex is better, with an antioxidant activity IC50 of 1.3 ± 0.22 μg / mL, indicating that the antioxidant activity is significantly improved compared to the free curcumin complex particles and the comparative example.

[0072] The ABTS assay results of the Cur-Se-Cs-Ga complex are given as 50% inhibition concentrations (IC50), which were then obtained from a linear regression plot of the inhibition percentage versus sample concentration. The IC50 for the antioxidant activity of the free curcumin solution was 9.80 ± 0.32 μg / mL. When the ascorbic acid addition was 5 mL, the Cur-Se-Cs-Ga complex showed better scavenging efficiency, with an IC50 of 3.83 ± 0.18 μg / mL, indicating a significantly improved antioxidant activity compared to the free curcumin complex particles and the comparative complex particles.

[0073] SeNPs generated from precursors are key components in the antioxidant process. Selenium nanoparticles achieved a maximum scavenging rate of 63% at a concentration of 5.69 × 10⁻⁵ mol / L; sodium selenite achieved its maximum scavenging rate (65%) at 2.64 × 10⁻⁴ mol / L, but this concentration was 4.64 times that of selenium nanoparticles; ascorbic acid achieved the highest scavenging rate (60%) at 2.15 × 10⁻⁴ mol / L. This is because SeNPs exhibit high antioxidant properties, and their antioxidant efficacy is significantly negatively correlated with size. Small-sized SeNPs showed significantly higher scavenging rates of hydroxyl radicals (·OH) and DPPH radicals than large-sized particles. The amount of ascorbic acid significantly affected the particle size of SeNPs. A Na₂SeO₃ to Vc molar ratio of 1:5 resulted in smaller SeNPs with a size of 112.734 nm, exhibiting higher carrier stability and thus a higher free radical scavenging rate.

[0074] When the molar ratio of Na₂SeO₃ to Vc is 1:5, selenium exhibits strong binding capacity, and the adsorption of more SeNPs with strong free radical scavenging capabilities further enhances the free radical scavenging ability of the Cur-Se-Cs-Ga complex. The nanocarrier form improves the solubility of curcumin, exposing more of its phenolic hydroxyl groups to the environment, thus enabling them to better function as electron donors or capture free radicals. Furthermore, the increased selenium content also synergistically contributes to the enhanced antioxidant properties of the Cur-Se-Cs-Ga complex. Nano-selenium forms a hydrogen bond network with the phenolic hydroxyl groups of curcumin through surface selenool groups (-SeH), synergistically scavenging intermediate products of free radical chain reactions.

[0075] Figure 5 The composite particles obtained in Examples 1-3, Comparative Examples 5 and 6 of this invention and the composite particles obtained in Comparative Example 1 simulate the curcumin release rate during the gastrointestinal digestion process, where a is the release rate in the stomach and b is the release rate in the intestine. Figure 6 The values ​​represent the release rates of selenium in the gastrointestinal tract during the simulated effect of composite particles obtained in Examples 1-3 and Comparative Examples 5 and 6 of this invention. a represents the release rate in the stomach, and b represents the release rate in the intestine.

[0076] The gastrointestinal stage was performed using simulated stomach and small intestine digestion solutions. For the stomach stage: simulating oral digestion, in vitro gastric digestion was conducted. 10.0 mL of the oral digested mixture was mixed with 7.5 mL of SGF, pepsin (25,000.0 U / mL), and 10.0 μL of CaCl2 (0.3 mol / L). The final pH of each mixture was then adjusted to 3.0 using HCl (1.0 mol / L) to simulate the gastric digestive environment. Finally, the mixture was continuously stirred at 37 °C for 2 h. (Oral stage: 5.0 mL of sample solution was mixed with 3.5 mL of SSF in a 15.0 mL centrifuge tube. Subsequently, 25.0 μL of CaCl2 (0.3 mol / L) and salivary amylase (1500.0 U / mL) were added to each tube. After thorough mixing, the mixture was stirred at 37 °C for 10 min.) Small intestine stage After simulating gastric digestion, 20.0 mL of gastric digestion products from different emulsion samples were mixed with 11.0 mL of SIF, 2.5 mL of bile solution, trypsin (10 mg / mL), pancreatic lipase (10 mg / mL), and 40.0 μL of CaCl2 (0.3 mol / L). The pH of the mixture was adjusted to 7.0. The final mixture was then continuously stirred at 37 °C for 2 h.

[0077] from Figure 5 and Figure 6As can be seen, the cumulative release rate of curcumin in the intestinal stage is significantly higher than that in the gastric stage. Between pH 3.5 and 5, over 90% of the primary amino groups are protonated, and over 90% of the carboxyl groups are deprotonated, resulting in the strongest interaction between chitosan and gum arabic. The interaction between gum arabic, chitosan, selenium nanoparticles, and the emulsifier makes the complex nanoparticles more compact, reducing damage to the nanoparticle structure by pepsin and inhibiting the release of curcumin from the complex nanoparticles. Under pH 7.0 conditions, due to the loose polymer network structure and the increased water solubility of curcumin under weakly alkaline conditions, curcumin is continuously released in the intestinal stage. Burst release occurs in both the intestinal and gastric stages within the first 30 minutes. When the molar ratio of sodium selenite to ascorbic acid is 1:5, the release rate of curcumin reaches its highest value at the end of the intestinal stage. At this point, the system exhibits high curcumin solubility, and the high binding amount of selenium and well-dispersed system promote the release of curcumin from the nanoparticles. The curcumin release rate of the Cur-Se-Cs-Ga complex during the gastrointestinal stage was significantly higher than that of curcumin powder. This indicates that the dense structure of nanoparticles can prevent the burst release of curcumin and promote its sustained release.

[0078] The bioavailability of curcumin was determined after intestinal digestion. The fully digested emulsion was centrifuged at 10,000 rpm for 10 min at room temperature to obtain micelles containing curcumin. The micelle solution was collected using a syringe and ethanol was added and vortexed. The supernatant was collected, and the absorbance was measured at 425 nm using a spectrophotometer. The curcumin content was calculated according to the standard curve, and the bioavailability of curcumin was calculated. Biological accessibility (%) = (Cmicelle / Cinitial) × 100 Wherein, Cmicelle represents the amount of curcumin in the micelles, and Cinitial represents the initial amount of curcumin in the digestive system. Samples were collected at 30, 60, 90, 120, 150, and 180 minutes for analysis. Samples collected during the SGF and SIF digestion stages were mixed with ethanol, centrifuged (2000 rpm, 10 minutes), and the curcumin release rate was calculated by spectrophotometry at 425 nm. Results are shown below. Figure 9 As shown.

[0079] Experiments showed that the bioavailability of free curcumin was 2.64%, while the bioavailability of the Cur-Se-Cs-Ga complex was significantly increased compared to free curcumin. The Cur-Se-Cs-Ga complex, through micellar encapsulation or carrier stabilization, significantly reduced precipitation, increasing bioavailability by 5-15 times compared to the free form. In the Cur-Se-Cs-Ga complex, curcumin was more concentrated in the micelle layer, improving its solubility and intestinal absorption efficiency.

[0080] Figure 7 The images show SEM images of the composite particles obtained in Examples 1-3 and Comparative Examples 5 and 6 of this invention. As can be seen from the images, the obtained composite particles are uniform.

[0081] Figure 8 Fourier transform infrared (FT-IR) spectra of the composite particles (d), comparative examples 2 (c), 3 (b), and free curcumin (a) obtained in Example 1 of this invention are shown. As can be seen from the FT-IR spectra, the characteristic peaks of Cur, Cs-Ga, Se-Cs-Ga, and Cur-Se-Cs-Ga change in several key regions, which can be used to infer the interaction modes of the composite system. First, in the 3600–3200 cm⁻¹ region… - ¹ The –OH ( / –NH) stretching vibration region, free Cur at 3512 cm⁻¹ - ¹, Cs-Ga complex at 3488 cm⁻¹ - ¹ Both showed obvious broad peaks; however, the broad peak of Se-Cs-Ga showed a significant red shift to 3336 cm⁻¹. - ¹ indicates that the microenvironment of the hydroxyl groups in the system is significantly altered after the introduction of the Cs-Ga polyelectrolyte complex, with enhanced hydrogen bonding and the formation of a denser hydrogen bond network. This redshift typically implies that the stretching vibration of –OH is subject to stronger intermolecular constraints, consistent with the characteristic of “numerous hydroxyl groups of polysaccharide segments undergoing hydrogen bonding with the surface of nano-selenium (adsorption layer / hydration layer) to form a stable coating layer.” Further composite with Cur resulted in the –OH broad peak position of Cur-Se-Cs-Ga being adjusted to 3396 cm⁻¹. - ¹, compared to Se-Cs-Ga (3336 cm⁻¹) - ¹) Some shift was observed, but it was still significantly lower than that of free Cur (3512 cm). - ¹), indicating a redistribution of the hydrogen bond network in the composite system: on the one hand, the hydrogen bond stabilizing layer on the polysaccharide-Se surface still exists and maintains the interfacial stability of SeNPs; on the other hand, the introduction of Cur allows some hydroxyl groups to participate in the polysaccharide-Cur interaction, thereby changing the original hydrogen bond strength and configuration. Secondly, at ~1700 cm⁻¹... - ¹ Nearby, Se-Cs-Ga 1704 cm - ¹Moves to 1688 cm in Cur-Se-Cs-Ga - ¹, suggesting that the group environment corresponding to the vibration in this region is subject to stronger interactions, namely the carbonyl (C=O) related stretching vibration in the composite system, further supporting the idea that Cur's entry into the support leads to changes in the local environment. Finally, in the fingerprint region (approximately 1500–900 cm⁻¹), -¹), a series of grouped peak position changes were observed between Se-Cs-Ga and Cur-Se-Cs-Ga: redshift from 1376 cm⁻¹ to 1364 cm⁻¹. - ¹(C–H bending) 1264, 1136 and 1044 cm - ¹Redshifted to 1260, 1132 and 1016 cm - ¹(Polysaccharide C–O / C–O–C stretching) indicates a change in the chemical environment surrounding the polysaccharide backbone and its hydroxyl / glycosidic bonds. This suggests that the polysaccharide hydroxyl groups participate in the formation of a stronger hydrogen-bonded network (including interactions with the SeNPs surface and with Cur), leading to a rearrangement of the composite structure and stable encapsulation. The polysaccharide backbone-related vibrations change significantly after composite formation. This reflects that the introduction of Cur not only causes band overlap but also adjusts the conformation / local interactions of the polysaccharide segments, transforming Cur from a "free state" to a state bound / encapsulated by the composite network. Based on the above evidence, it can be concluded that the construction of the Cur-Se-Cs-Ga complex mainly depends on the formation of a hydrogen-bonded stable coating layer on the surface of nano-selenium by a large number of polysaccharide hydroxyl groups, followed by hydrogen bond rearrangement and synergistic binding with Cur, ultimately forming a more stable composite delivery system with well-defined interactions.

[0082] Figure 10These figures show the microscopic observation results of the effects of nano-selenium and free curcumin on the morphology of HepG2 liver cancer cells in Examples 1 and 4 of this invention. As can be seen from the figures, in the control group (i.e., untreated) (a–d), the cells were intact, evenly distributed, and maintained normal adherent growth. The cells were tightly packed, exhibiting typical tumor cell growth characteristics, indicating that the cells were in a good growth state. However, in the treated groups (e–h), the differences in cell morphology caused by different treatments were very significant. In the free curcumin treatment group, the overall cell morphology did not change much compared to the control group; only a few cells showed slight shrinkage or changes in transparency, and most cells maintained adherent growth with a limited decrease in cell density. The nano-selenium treatment group also showed a similar trend; the overall cell morphology was relatively intact, with only some cells showing slight shrinkage, suggesting that under the same administration conditions, the effect of a single active ingredient on the morphology of HepG2 cells was relatively weak. In contrast, the HepG2 cell morphology in the Cur-Cs-Ga complex treatment group (g) showed more significant changes. Compared with the control group, cell density was significantly reduced, and some cells showed signs of shrinkage, irregular morphology, and weakened adhesion. A certain proportion of cells were observed floating in the culture medium, indicating that the introduction of the composite carrier enhanced the effect of curcumin on tumor cells. The most significant changes in cell morphology were observed in the Cur-Se-Cs-Ga complex treatment group (h). Microscopic observation showed a significant reduction in the number of adherent cells, increased intercellular spaces, and numerous cells exhibiting significant shrinkage, rounding, and even detachment, resulting in a substantial decrease in overall cell density. Compared with other treatment groups, the Cur–Se–Cs–Ga complex showed the most significant disruption to the growth state of HepG2 cells, suggesting a stronger inhibitory effect on tumor cells.

[0083] Figure 11 The figure shows the effect of different treatment groups on the survival rate of normal L-02 hepatocytes. As can be seen from the figure, the Cur-Se-Cs-Ga complex had the highest cell survival rate (lowest toxicity), followed by the Cur-Cs-Ga complex, while the nano-selenium alone group had the lowest cell survival rate (highest toxicity). The physical mixture of curcumin and nano-selenium / polymer polysaccharide had significantly higher toxicity than the complex formed by curcumin and polysaccharide polyelectrolytes, indicating that multi-component synergistic compounding (especially the introduction of nano-selenium and biocompatible carrier) can effectively reduce the cytotoxicity of bioactive substances.

[0084] The composite particles obtained by this invention improve the solubility, stability, and antioxidant capacity of the composite particles. Under different vitamin C addition levels, the solubility of curcumin in Cur-Se-Cs-Ga is significantly higher than that of free curcumin (0.8 μg / mL) (even though the antioxidant capacity of comparative examples 5 and 6 is weaker, other properties are significantly enhanced compared to those without added vitamin C).

[0085] In terms of antioxidant activity, the DPPH scavenging IC50 of free curcumin was 10.24±0.29 μg / mL, while the IC50 of Cur-Se-Cs-Ga decreased to 1.3±0.22 μg / mL when Vc was 5 mL, indicating that its antioxidant activity was significantly improved. This is consistent with the mechanism by which nano-selenium participates in the formation of hydrogen bond networks and synergistically scavenges intermediate products of free radical chain reactions.

[0086] In in vitro release and digestion experiments, the cumulative release rates of Cur-Se-Cs-Ga in pH 3.0 and 7.0 media over 24 h were 10.65% and 18.95%, respectively, showing faster release under neutral conditions. This was consistent with the Korsmeyer–Peppas model, which characterized the synergistic control of diffusion and polymer chain relaxation / erosion. Digestion experiments further demonstrated that Cur-Se-Cs-Ga had a significantly higher curcumin release rate in the gastrointestinal tract than curcumin powder. The dense structure inhibited gastric burst release and achieved sustained release in the intestinal tract. Bioavailability results showed that free curcumin was only 2.64%, while Cur-Se-Cs-Ga significantly increased it by 5–15 times, reaching its peak at a Vc concentration of 5 mL, indicating that this system can effectively reduce post-digestion precipitation and improve micellar layer distribution and absorption efficiency. Selenium release was slow in a simulated gastric environment, while some selenium was gradually and slowly released in a simulated intestinal environment. The burst release of selenium was achieved at 1.5 h, and the release rate at 2.0 h was 48.72%, which was significantly higher than the release rate of 21.71% in the simulated stomach stage.

[0087] Cellular experiments showed that the Cur-Se-Cs-Ga complex delivery system significantly inhibited HepG2 tumor cells in vitro, and the introduction of the complex structure significantly enhanced the anticancer effect of curcumin. Morphological observations revealed that compared with free curcumin and nano-selenium alone, the complex delivery system treatment resulted in decreased cell adhesion and significantly reduced cell density in HepG2 cells, with the Cur-Se-Cs-Ga treatment group exhibiting the most significant morphological damage. In L-02 normal hepatocytes, the Cur-Se-Cs-Ga complex showed the lowest cytotoxicity, significantly better than nano-selenium alone and the physical mixing system, indicating that the synergistic combination of the polysaccharide polyelectrolyte carrier and nano-selenium enhances anticancer activity while helping to reduce the risk of toxicity to normal cells.

[0088] The Cur-Se-Cs-Ga complex obtained in this invention achieves stable dispersion of selenium nanoparticles by forming hydrogen bonds or Se-O bonds on the surface of selenium nanoparticles through numerous hydroxyl groups of polysaccharides. Simultaneously, the strong biopolymer network matrix effectively delays the release of curcumin and selenium nanoparticles in the gastrointestinal environment. The composite particles obtained in this invention can serve as carriers for controlled release and delivery of curcumin and selenium supplementation, providing better antioxidant properties and stability, exhibiting good antitumor activity and stable biocompatibility. It shows great application potential in the food, pharmaceutical, and other health fields.

Claims

1. A method for preparing chitosan-coated curcumin / selenium composite particles, characterized in that, Includes the following steps: Step 1: Dissolve chitosan in acetic acid solution until fully dissolved to obtain mixed solution A; Step 2: Add sodium selenite to the gum arabic solution and mix thoroughly to obtain mixed solution B; Step 3: Add curcumin, Tween, and lecithin to anhydrous ethanol and mix thoroughly to obtain mixed solution C; Step 4: Add mixed solution C to mixed solution A, and then add mixed solution B dropwise; the mass ratio of chitosan to gum arabic in the mixed system is 3:1 to 1:2, and the mass ratio of curcumin to sodium selenite is 1 to 2:173; stir thoroughly, remove the solvent, and centrifuge to obtain the desired composite particles.

2. The method for preparing chitosan-coated curcumin / selenium composite particles according to claim 1, characterized in that, The mixed solution A in step 1 also includes ascorbic acid, and the molar ratio of ascorbic acid to sodium selenite is 3:1 to 5:

1.

3. The method for preparing chitosan-coated curcumin / selenium composite particles according to claim 1, characterized in that, In step 3, the concentration of curcumin in mixed solution C is 3–7 mg / mL.

4. The method for preparing chitosan-coated curcumin / selenium composite particles according to claim 1, characterized in that, In step 4, the pH value is maintained at 4.

0.

5. The method for preparing chitosan-coated curcumin / selenium composite particles according to claim 1, characterized in that, Both steps 1 and 4 are performed under stirring conditions. The stirring speed in step 1 is 600 rpm, and the stirring speed in step 4 is 800 rpm.

6. The method for preparing chitosan-coated curcumin / selenium composite particles according to claim 1, characterized in that, The concentration of the acetic acid solution in step 1 is 1% (w / v).

7. The method for preparing chitosan-coated curcumin / selenium composite particles according to claim 1, characterized in that, In step 3, curcumin, Tween, and lecithin are dissolved in anhydrous ethanol using the ethanol injection method.

8. The method for preparing chitosan-coated curcumin / selenium composite particles according to claim 1, characterized in that, In step 3, the mass ratio of Tween to lecithin is 1:1 to 4:

1.

9. The chitosan-coated curcumin / selenium composite particles obtained by any one of the preparation methods described in claims 1 to 8, characterized in that, The composite particles have a core-shell structure, with chitosan as the shell and a mixture of curcumin and nano-selenium distributed in a chitosan and gum arabic composite matrix as the core.

10. The application of the chitosan-coated curcumin / selenium composite particles as described in claim 9, characterized in that, The composite particles are used in drug preparation.

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