A composite polysaccharide nano selenium complex and a preparation method and application thereof

Abstract

This invention provides a composite polysaccharide-selenium nanoparticle complex, its preparation method, and its applications. The complex comprises polysaccharide-modified selenium nanoparticles, obtained by a redox reaction of a polysaccharide composition with a selenium solution. The polysaccharide composition includes *Boletus edulis* polysaccharide, *Portulaca oleracea* polysaccharide, and *Astragalus membranaceus* polysaccharide, with a mass ratio of 0.5–3:0.5–3:0.5–3. The composite polysaccharide-selenium nanoparticle complex of this invention, through the synergistic modification of selenium nanoparticles by multiple polysaccharides, effectively prevents the aggregation and oxidation of selenium nanoparticles, significantly improves the dispersibility and stability of selenium nanoparticles, enhances their bioavailability and targeting, strengthens their antioxidant activity, reduces their cytotoxicity, and achieves multifunctionality, expanding the application potential of selenium nanoparticles in medicine, food, cosmetics, and other fields.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and in particular relates to a composite polysaccharide nano-selenium complex, its preparation method and application. Background Technology

[0002] Selenium is an essential trace element for the human body, possessing various physiological functions such as antioxidation, antitumor activity, and immune regulation. However, its low bioavailability and narrow toxicity window limit its application. Selenium nanoparticles (SeNPs) have become a research hotspot due to their high bioavailability and low toxicity, but their poor stability and tendency to aggregate remain problems that need to be addressed. Polysaccharides, as natural polymers, possess abundant functional groups (such as hydroxyl and carboxyl groups) and can serve as stabilizers and carriers for selenium nanoparticles, improving their dispersibility and bioactivity. Currently, single-polysaccharide-stabilized selenium nanoparticle complexes have been studied, but synergistically stabilized selenium nanoparticle complexes with multiple polysaccharides and their preparation methods have not yet been reported.

[0003] Existing polysaccharide nano-selenium complexes have at least the following problems:

[0004] (1) Narrow toxicity window: The safety window between the effective dose and the toxic dose of selenium is narrow, which limits its application in nutritional supplements and drugs; while monopolysaccharide-modified nano-selenium may increase its toxicity risk while improving its biological activity.

[0005] (2) Complex preparation process: The conditions are harsh, making it difficult to achieve large-scale production; single polysaccharide-modified selenium nanoparticles are prone to problems such as uneven particle size and poor dispersibility during the preparation process. Typical preparation techniques reported in the literature mainly include: ① Physical mixing method, such as the Chinese invention patent with publication number CN119978159A, which discloses a method for preparing high-purity jujube polysaccharide selenium nanoparticles, which simply mixes polysaccharide with pre-synthesized selenium nanoparticles, but the interfacial binding force is weak and it is easy to dissociate in the physiological environment; ② Reduction method, for example, Shi Menghua et al. disclosed in "Research Progress on Preparation, Characterization and Bioactivity of Polysaccharide Selenium Nanoparticles" that the method uses polysaccharide to reduce selenium salt (such as Na2SeO3) to generate selenium nanoparticles, but the particle size distribution is wide (50-200nm) and the loading rate is low (<5%); ③ Fermentation method (such as commercial selenium yeast), although it achieves intracellular deposition of selenium, the bioavailability is less than 30%;

[0006] (3) Limited application scope: The application of existing nano-selenium complexes in the fields of medicine, food, cosmetics, etc. is limited; nano-selenium modified with single polysaccharides has poor adaptability and stability in complex environments. Summary of the Invention

[0007] In view of this, the present invention aims to propose a composite polysaccharide nano-selenium complex, its preparation method and application, in order to develop a low-toxicity, high-safety, highly adaptable and highly stable nano-selenium complex, and to provide a simple, efficient and controllable preparation method suitable for industrial production.

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

[0009] In a first aspect, the present invention provides a composite polysaccharide-selenium nanocomposite, the composite comprising polysaccharide-modified selenium nanoparticles, wherein the polysaccharide-modified selenium nanoparticles are obtained by redox reaction of a polysaccharide composition and a selenium solution, and the polysaccharide composition comprises Boletus edulis polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide.

[0010] Furthermore, the particle size of the polysaccharide-modified selenium nanoparticles is 20–200 nm.

[0011] Further, the mass ratio of Boletus polysaccharide, Portulaca polysaccharide, and Astragalus polysaccharide is 0.5–3:0.5–3:0.5–3; preferably, the mass ratio is 0.8–1.2:0.8–1.2:0.8–1.2; even more preferably, the mass ratio is 1:1:1.

[0012] Secondly, the present invention provides a method for preparing a composite polysaccharide nano-selenium complex, the method comprising the following steps:

[0013] S1. Mix the polysaccharide composition with water to obtain a polysaccharide mixed solution, wherein the polysaccharide composition includes Boletus polysaccharide, Portulaca polysaccharide and Astragalus polysaccharide, and the mass ratio of Boletus polysaccharide, Portulaca polysaccharide and Astragalus polysaccharide is 0.5-3:0.5-3:0.5-3;

[0014] S2. Stir the selenium solution and the polysaccharide mixed solution evenly to obtain a polysaccharide nano-selenium suspension;

[0015] S3. Mix the stabilizer with the polysaccharide nano-selenium suspension and stir until the reaction is complete to obtain the reaction solution;

[0016] S4. Centrifuge, wash, and dry the reaction solution to obtain the composite polysaccharide nano-selenium complex.

[0017] Further, the polysaccharide composition includes Boletus edulis polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide, and the concentration of the polysaccharide composition in the polysaccharide mixed solution is 1-5 mg / mL, for example, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, or 5 mg / mL; preferably 1.5-3 mg / mL; more preferably 2 mg / mL;

[0018] Preferably, the mass ratio of Boletus polysaccharide, Portulaca polysaccharide, and Astragalus polysaccharide is 0.8–1.2:0.8–1.2:0.8–1.2; more preferably, the mass ratio of Boletus polysaccharide, Portulaca polysaccharide, and Astragalus polysaccharide is 1:1:1.

[0019] Furthermore, the extraction and purification of the *Boletus edulis* polysaccharide, *Portulaca oleracea* polysaccharide, and *Astragalus membranaceus* polysaccharide includes steps such as hot water extraction, alcohol precipitation, protein removal, ion exchange chromatography, and gel filtration chromatography, applicable to polysaccharides from various sources such as fungi and plants. Preferably, the extraction of the *Boletus edulis* polysaccharide, *Portulaca oleracea* polysaccharide, or *Astragalus membranaceus* polysaccharide is performed using a hot water extraction method, the specific process of which is as follows:

[0020] (1) Extraction of crude polysaccharides

[0021] The raw material powders (Boletus edulis, Portulaca oleracea, and Astragalus membranaceus) were added to deionized water at a material-to-liquid ratio of 1:20 (w / v), and extracted in a 90°C water bath for 3 hours. The supernatant was collected by centrifugation (6000 rpm, 10 min), and the extraction was repeated three times. The extracts were combined, concentrated to 1 / 4 of their original volume, and 4 times their volume of 95% ethanol was added. The mixture was precipitated overnight at 4°C. The precipitate was collected by centrifugation, redissolved in water, and the crude polysaccharide solution was obtained.

[0022] (2) Deproteinization treatment

[0023] The Sevage method (chloroform:n-butanol = 4:1, 5:1 v / v added to the sample) was used. After shaking for 20 min, the sample was centrifuged (4000 rpm, 10 min) and repeated until no protein precipitation was observed at the interface. After removing the organic solvent, the sample was dialyzed through a 3500 Da dialysis bag for 48 h and then lyophilized to obtain deproteinized crude polysaccharide.

[0024] (3) Ion exchange chromatography purification

[0025] The deproteinized crude polysaccharide was separated by a DEAE-52 cellulose column (6.0×100.0cm), eluted with a gradient of 0-0.5mol / L NaCl, and the polysaccharide peak was detected by the phenol-sulfuric acid method. The main peak was collected, dialyzed and lyophilized to obtain purified polysaccharide.

[0026] (4) Validation by gel filtration chromatography

[0027] The purified polysaccharide (20 mg) was further separated by a Sephadex G-100 column, eluted with deionized water, and the main peaks were detected and lyophilized to obtain homogeneous polysaccharide.

[0028] Further, the concentration of the selenium solution in step S2 is 30-80 mmol / L, for example, it can be 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, or 80 mmol / L; preferably 50-70 mmol / L; more preferably 60 mmol / L.

[0029] Further, the volume ratio of the selenium solution to the polysaccharide mixed solution is 1:3 to 8, for example, it can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, or 1:8; preferably 1:4-7, and more preferably 1:5.

[0030] Furthermore, the stabilizer is selected from sodium citrate solution and ascorbic acid solution.

[0031] Furthermore, the reaction time in step S3 is more than 24 hours.

[0032] Further, the concentration of the stabilizer is 30-80 mmol / L, for example, it can be 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, or 80 mmol / L; preferably 50-70 mmol / L; more preferably 60 mmol / L.

[0033] Preferably, the volume ratio of the stabilizer to the polysaccharide nano-selenium suspension is 2-5:5-10; more preferably 3-5:5-8; and even more preferably 4:6.

[0034] Thirdly, the present invention provides the application of the composite polysaccharide nano-selenium complex as described in the first aspect or the preparation method as described in the second aspect in the preparation of pharmaceuticals, food, and cosmetics.

[0035] Fourthly, the present invention provides the application of a polysaccharide composition in the preparation of a composite polysaccharide nano-selenium complex, wherein the polysaccharide composition comprises Boletus edulis polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide, wherein the mass ratio of Boletus edulis polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide is 0.5–3:0.5–3:0.5–3, and the application includes at least one of the following:

[0036] (1) Application in improving the stability of nano-selenium;

[0037] (2) Application in improving the antioxidant capacity of composite polysaccharide nano-selenium complex;

[0038] (3) Application in reducing the cytotoxicity of complex polysaccharide nano-selenium complex;

[0039] (4) Application in improving the bioavailability of complex polysaccharide nano-selenium complex.

[0040] Furthermore, the antioxidant capacity includes the ability to scavenge DPPH free radicals, the ability to scavenge ABTS free radicals, or the ability to scavenge hydroxyl free radicals.

[0041] Compared with existing technologies, the composite polysaccharide nano-selenium complex, its preparation method, and its application described in this invention have the following advantages:

[0042] (1) The composite polysaccharide nano-selenium complex of the present invention obtains a highly efficient and stable multi-polysaccharide nano-selenium complex by synergistic modification of nano-selenium with multiple polysaccharides, effectively preventing nano-selenium aggregation and oxidation, significantly improving the dispersibility and stability of nano-selenium, solving the technical problem of poor nano-selenium stability, and significantly improving the physicochemical properties (such as particle size, surface charge, and solubility) of the polysaccharide nano-selenium complex. In complex environments (such as the gastrointestinal tract and blood), it exhibits stronger adaptability and stability.

[0043] (2) The composite polysaccharide nano-selenium complex described in this invention improves the bioavailability and targeting of nano-selenium. The complementary and synergistic effects of different polysaccharides can enhance the antioxidant activity of nano-selenium and enhance its physiological functions.

[0044] (3) The composite polysaccharide nano-selenium complex described in this invention reduces the cytotoxicity of nano-selenium, broadens its safe application range, and realizes the multifunctionality of nano-selenium. The combination of multiple polysaccharides can endow nano-selenium with more functional properties, such as antibacterial, anti-inflammatory, and anti-aging properties, to meet the application needs of different fields and expand the application potential of nano-selenium in medicine, food, cosmetics and other fields.

[0045] (4) The preparation method of the composite polysaccharide nano-selenium complex of the present invention has the advantages of simple preparation process, mild conditions, high efficiency and controllability, and is suitable for large-scale production. Compared with single polysaccharide nano-selenium complex, it can reduce the amount of single polysaccharide, effectively reduce production costs, and also reduce the use of stabilizers, thereby improving the purity and safety of the product. Attached Figure Description

[0046] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0047] Figure 1 Fourier transform infrared spectra of the polysaccharide nano-selenium complexes prepared in Example 1 and Comparative Example 1.

[0048] Figure 2 This is a schematic diagram showing the stability test results of the polysaccharide nano-selenium complex prepared in Example 1;

[0049] Figure 3 This is a schematic diagram showing the antioxidant activity test results of the polysaccharide nano-selenium complexes prepared in Example 1 and Comparative Examples 1-3.

[0050] Figure 4 This is a schematic diagram showing the cytotoxicity test results of the polysaccharide nano-selenium complexes prepared in Example 1 and Comparative Examples 1-3. Detailed Implementation

[0051] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0052] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0053] In this embodiment and comparative example, the polysaccharides of Boletus edulis, Portulaca oleracea, and Astragalus membranaceus used were extracted using a hot water extraction method. The specific process is as follows:

[0054] (1) Extraction of crude polysaccharides

[0055] The raw material powders (Boletus edulis, Portulaca oleracea, and Astragalus membranaceus) were added to deionized water at a material-to-liquid ratio of 1:20 (w / v), and extracted in a 90°C water bath for 3 hours. The supernatant was collected by centrifugation (6000 rpm, 10 min), and the extraction was repeated three times. The extracts were combined, concentrated to 1 / 4 of their original volume, and 4 times their volume of 95% ethanol was added. The mixture was precipitated overnight at 4°C. The precipitate was collected by centrifugation, redissolved in water, and the crude polysaccharide solution was obtained.

[0056] (2) Deproteinization treatment

[0057] The Sevage method (chloroform:n-butanol = 4:1, 5:1 v / v added to the sample) was used. After shaking for 20 min, the sample was centrifuged (4000 rpm, 10 min) and repeated until no protein precipitation was observed at the interface. After removing the organic solvent, the sample was dialyzed through a 3500 Da dialysis bag for 48 h and then lyophilized to obtain deproteinized crude polysaccharide.

[0058] (3) Ion exchange chromatography purification

[0059] The deproteinized crude polysaccharide was separated by a DEAE-52 cellulose column (6.0×100.0cm), eluted with a gradient of 0-0.5mol / L NaCl, and the polysaccharide peak was detected by the phenol-sulfuric acid method. The main peak was collected, dialyzed and lyophilized to obtain purified polysaccharide.

[0060] (4) Validation by gel filtration chromatography

[0061] The purified polysaccharide (20 mg) was further separated by a Sephadex G-100 column, eluted with deionized water, and the main peaks were detected and lyophilized to obtain homogeneous polysaccharide.

[0062] Example 1

[0063] The preparation method of the composite polysaccharide nano-selenium complex in this embodiment includes the following steps:

[0064] (1) Dissolve Boletus polysaccharide, Portulaca polysaccharide and Astragalus polysaccharide in deionized water in a ratio of 1:1:1 to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0065] (2) Dissolve sodium selenite (Na2SeO3) in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0066] (3) Under stirring conditions, 1 mL of 60 mmol / L selenium source solution was added to 5 mL of polysaccharide mixed solution and stirred for 1 h to obtain polysaccharide nano selenium suspension;

[0067] (4) Add 4 mL of freshly prepared 60 mmol / L ascorbic acid solution dropwise to the polysaccharide nano-selenium suspension, and continue stirring in the dark for 24 hours to allow the polysaccharide and nano-selenium to fully combine and obtain the reaction solution.

[0068] (5) Centrifuge the reaction solution to collect the complex, wash it three times with deionized water, and freeze-dry it to obtain the polysaccharide nano-selenium complex.

[0069] Example 2

[0070] The preparation method of the composite polysaccharide nano-selenium complex in this embodiment includes the following steps:

[0071] (1) Dissolve Boletus polysaccharide, Portulaca polysaccharide and Astragalus polysaccharide in deionized water at a ratio of 0.8:1:1.2 to prepare a polysaccharide mixed solution with a concentration of 1.5 mg / mL;

[0072] (2) Dissolve sodium selenite (Na2SeO3) in deionized water to prepare a selenium source solution with a concentration of 50 mmol / L;

[0073] (3) Under stirring conditions, 1 mL of 50 mmol / L selenium source solution was added to 4 mL of polysaccharide mixed solution and stirred for 1 h to obtain polysaccharide nano selenium suspension.

[0074] (4) Add 3 mL of freshly prepared 50 mmol / L ascorbic acid solution dropwise to the polysaccharide nano-selenium suspension, and continue stirring in the dark for 24 hours to allow the polysaccharide and nano-selenium to fully combine and obtain the reaction solution.

[0075] (5) Centrifuge the reaction solution to collect the complex, wash it three times with deionized water, and freeze-dry it to obtain the polysaccharide nano-selenium complex.

[0076] Example 3

[0077] The preparation method of the composite polysaccharide nano-selenium complex in this embodiment includes the following steps:

[0078] (1) Dissolve Boletus polysaccharide, Portulaca polysaccharide and Astragalus polysaccharide in deionized water at a ratio of 1:0.8:1.2 to prepare a polysaccharide mixed solution with a concentration of 3 mg / mL;

[0079] (2) Dissolve sodium selenite (Na2SeO3) in deionized water to prepare a selenium source solution with a concentration of 70 mmol / L;

[0080] (3) Under stirring conditions, 1 mL of 70 mmol / L selenium source solution was added to 7 mL of polysaccharide mixed solution and stirred for 1 h to obtain polysaccharide nano selenium suspension;

[0081] (4) Add 5 mL of freshly prepared 70 mmol / L ascorbic acid solution dropwise to the polysaccharide nano-selenium suspension, and continue stirring in the dark for 24 hours to allow the polysaccharide and nano-selenium to fully combine and obtain the reaction solution.

[0082] (5) Centrifuge the reaction solution to collect the complex, wash it three times with deionized water, and freeze-dry it to obtain the polysaccharide nano-selenium complex.

[0083] Example 4

[0084] The preparation method of the composite polysaccharide nano-selenium complex in this embodiment includes the following steps:

[0085] (1) Dissolve Boletus polysaccharide, Portulaca polysaccharide and Astragalus polysaccharide in deionized water at a ratio of 1.2:1:0.8 to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0086] (2) Dissolve sodium selenite (Na2SeO3) in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0087] (3) Under stirring conditions, 1 mL of 60 mmol / L selenium source solution was added to 5 mL of polysaccharide mixed solution and stirred for 1 h to obtain polysaccharide nano selenium suspension;

[0088] (4) Add 4 mL of freshly prepared 60 mmol / L ascorbic acid solution dropwise to the polysaccharide nano-selenium suspension, and continue stirring in the dark for 24 hours to allow the polysaccharide and nano-selenium to fully combine and obtain the reaction solution.

[0089] (5) Centrifuge the reaction solution to collect the complex, wash it three times with deionized water, and freeze-dry it to obtain the polysaccharide nano-selenium complex.

[0090] Comparative Example 1

[0091] The preparation method of the polysaccharide nano-selenium complex in this comparative example includes the following steps:

[0092] (1) Dissolve the Boletus edulis polysaccharide in deionized water to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0093] (2) Dissolve sodium selenite (Na2SeO3) in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0094] (3) Under stirring conditions, 1 mL of 60 mmol / L selenium source solution was added to 5 mL of polysaccharide mixed solution and stirred for 1 h to obtain a polysaccharide nano-selenium suspension:

[0095] (4) Add 4 mL of freshly prepared 60 mmol / L ascorbic acid solution dropwise to the polysaccharide nano-selenium suspension, and continue stirring in the dark for 24 hours to allow the polysaccharide and nano-selenium to fully combine and obtain the reaction solution.

[0096] (5) The reaction solution was centrifuged to collect the complex, washed three times with deionized water, and freeze-dried to obtain the Boletus polysaccharide nano-selenium complex.

[0097] Comparative Example 2

[0098] The preparation method of the polysaccharide nano-selenium complex in this comparative example includes the following steps:

[0099] (1) Dissolve purslane polysaccharide in deionized water to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0100] (2) Dissolve sodium selenite (Na2SeO3) in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0101] (3) Under stirring conditions, 1 mL of 60 mmol / L selenium source solution was added to 5 mL of polysaccharide mixed solution and stirred for 1 h to obtain a polysaccharide nano-selenium suspension:

[0102] (4) Add 4 mL of freshly prepared 60 mmol / L ascorbic acid solution dropwise to the polysaccharide nano-selenium suspension, and continue stirring in the dark for 24 hours to allow the polysaccharide and nano-selenium to fully combine and obtain the reaction solution.

[0103] (5) The reaction solution was centrifuged to collect the complex, washed three times with deionized water, and freeze-dried to obtain the purslane polysaccharide nano-selenium complex.

[0104] Comparative Example 3

[0105] The preparation method of the polysaccharide nano-selenium complex in this comparative example includes the following steps:

[0106] (1) Dissolve Astragalus polysaccharide in deionized water to prepare a polysaccharide mixed solution with a concentration of 2 mg / mL;

[0107] (2) Dissolve sodium selenite (Na2SeO3) in deionized water to prepare a selenium source solution with a concentration of 60 mmol / L;

[0108] (3) Under stirring conditions, 1 mL of 60 mmol / L selenium source solution was added to 5 mL of polysaccharide mixed solution and stirred for 1 h to obtain a polysaccharide nano-selenium suspension:

[0109] (4) Add 4 mL of freshly prepared 60 mmol / L ascorbic acid solution dropwise to the polysaccharide nano-selenium suspension, and continue stirring in the dark for 24 hours to allow the polysaccharide and nano-selenium to fully combine and obtain the reaction solution.

[0110] (5) The reaction solution was centrifuged to collect the complex, washed three times with deionized water, and freeze-dried to obtain the Astragalus polysaccharide nano-selenium complex.

[0111] Performance Test Example 1: Fourier Transform Infrared Spectroscopy

[0112] Fourier transform infrared spectroscopy was performed on the polysaccharide nano-selenium complexes prepared in Example 1 and Comparative Example 1, and the results are as follows: Figure 1 As shown, infrared spectral analysis of the *Boletus edulis* monopolysaccharide nano-selenium complex and the composite polysaccharide nano-selenium complex revealed multiple characteristic absorption peaks in the spectrum of the *Boletus edulis* monopolysaccharide nano-selenium complex prepared in Comparative Example 1, corresponding to different functional groups in the polysaccharide molecule. For example, 3419 cm⁻¹... -1 The absorption peak at 2933 cm⁻¹ is due to the stretching vibration of OH. -1The absorption peaks at the specified locations are due to the stretching vibration of CH, which are typical characteristic peaks of carbohydrates. However, the spectrum of the composite polysaccharide-selenium nanocomposite prepared in Example 1 shows absorption peaks similar to those of the single-polysaccharide-selenium nanocomposite from *Boletus edulis*, indicating that the introduction of nano-selenium did not significantly alter the basic structure of the polysaccharide. However, changes in the position or intensity of some peaks may be due to interactions between nano-selenium and polysaccharide molecules. When polysaccharide acts as a stabilizing template to modify nano-selenium, the greater the redshift of the infrared absorption peaks of its hydroxyl and carbonyl groups, the stronger the interaction between the polysaccharide and nano-selenium. For example, the change in the intensity of the OH stretching vibration absorption peak suggests that nano-selenium may interact with the hydroxyl groups of the polysaccharide. This interaction may contribute to improving the stability of nano-selenium and may affect its bioactivity.

[0113] Performance Test Example 2: Stability Test

[0114] Experimental Methods: The polysaccharide-selenium nanocomposites prepared in Example 1 and Comparative Examples 1-3 were prepared into 1 mg / mL polysaccharide-selenium nanocomposite solutions. 1 mL of the 1 mg / mL polysaccharide-selenium nanocomposite solution was added to 5 mL of PBS (pH = 5.6, 7.4), water, DMEM solution, and DMEM solution (containing 10% FBS), respectively, and incubated at room temperature for 4 days. The size changes of BLPs-SeNPs after incubation in PBS (pH = 5.6, 7.4), water, DMEM solution, and DMEM solution (containing 10% FBS) were monitored at different time points (0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h) using a Zeta potentiometer.

[0115] Experimental results are as follows Figure 2 As shown, the composite polysaccharide-selenium nanocomposite prepared by this invention can maintain a stable state for nearly 72 hours in various physiological solutions, including water, PBS solution (pH=5.6, 7.4), DMEM, and DMEM containing 10% FBS. Compared with nanocomposites composed of single polysaccharides (Boletus edulis polysaccharide, Portulaca oleracea polysaccharide, or Astragalus membranaceus polysaccharide), it has superior stability. This is because the polysaccharide composition can form a more stable three-dimensional network structure through synergistic effects, which can effectively prevent the aggregation of nano-selenium particles compared with single polysaccharides. At the same time, the functional groups of different polysaccharides (such as hydroxyl groups, carboxyl groups, etc.) form multiple coordination bonds with nano-selenium, which enhances the structural stability of the composite. As a result, the composite polysaccharide-selenium nanocomposite exhibits a longer shelf life and better dispersibility during storage and transportation.

[0116] The combination of complex polysaccharide nano-selenium complexes can be optimized according to target applications (such as pharmaceuticals, food, and cosmetics) to meet the needs of different fields; the physicochemical properties of the complex (such as particle size, surface charge, and solubility) can be precisely controlled by adjusting the polysaccharide ratio; in complex environments (such as the gastrointestinal tract and blood), complex polysaccharide nano-selenium complexes exhibit stronger adaptability and stability.

[0117] Performance Test Example 3: Antioxidant Activity Test

[0118] Free radicals are important pathogenic factors in many acute and chronic diseases, and their excessive accumulation can trigger severe oxidative stress. The body's inherent antioxidant defense system plays a crucial role in maintaining redox balance, scavenging excess free radicals, and protecting cells from oxidative damage. Excess free radicals can react with biomolecules (including membrane lipids, proteins, carbohydrates, and nucleic acids), leading to damage to cell structure and function. This oxidative damage is considered an important mechanism leading to aging and related diseases. Studies have shown that the development of many chronic diseases, such as metabolic syndrome (diabetes, hypertension), liver disease, cardiovascular disease, and malignant tumors, are closely related to free radical-mediated oxidative damage. In this test, vitamin C was used as a positive control. The in vitro antioxidant activity of three single-polysaccharide selenium nanocomplexes and a complex polysaccharide selenium nanocomplex was evaluated using DPPH free radical, ABTS cationic free radical, and hydroxyl free radical scavenging assays.

[0119] (1) Scavenging DPPH free radicals

[0120] Experimental Methods: A 0.1 mmol / L DPPH-ethanol working solution was prepared in advance. 5.0 mg of the polysaccharide-selenium nanocomposite prepared in Example 1 and Comparative Examples 1-3 was accurately weighed and dissolved in 5 mL of anhydrous ethanol to prepare a 1 mg / mL stock solution. Test solutions of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL were obtained through serial dilution. Ascorbic acid (Vc) was used as a positive control, and control solutions of corresponding concentrations were prepared.

[0121] The experimental groups are as follows:

[0122] Test group: Mix 1 mL of DPPH solution with sample solutions of different concentrations in equal volumes;

[0123] Background sample group: 1 mL of ethanol was mixed with 1 mL of sample solutions of different concentrations;

[0124] Blank control group: 1 mL of ethanol was mixed with 1 mL of DPPH solution.

[0125] After reacting the above mixture in the dark for 30 min, the absorbance was measured using a UV-Vis spectrophotometer at the maximum absorption wavelength of 517 nm. Three independent replicates were performed for each concentration. The DPPH radical scavenging rate was calculated according to formula (1):

[0126]

[0127] In the formula: A1 is the absorbance value of the test group; A2 is the absorbance value of the background sample group; A0 is the absorbance value of the blank control group.

[0128] (2) Ability to scavenge ABTS free radicals

[0129] Experimental method: Take 10 μL of polysaccharide nano-selenium complex and Vc standard solution prepared in Example 1 and Comparative Examples 1-3 with gradient concentrations respectively, and add them to the corresponding reaction system in sequence according to the operating procedure of ABTS free radical scavenging ability test kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0130] The experimental groups are as follows:

[0131] Test group: 10 μL sample + ABTS working solution (as per kit);

[0132] Background sample set: 10 μL sample + buffer (ABTS-free);

[0133] Blank control group: 10 μL solvent (such as PBS) + ABTS working solution;

[0134] Standard curve set: gradient concentration of Vc + ABTS working solution.

[0135] After thoroughly mixing the reaction solution, incubate at room temperature in the dark for 6 minutes. Measure the absorbance of each reaction system at a characteristic wavelength of 405 nm using a microplate reader, with three replicates for each concentration gradient. Calculate the clearance rate based on the standard curve and formula (1).

[0136] (3) Ability to scavenge hydroxyl radicals

[0137] Experimental Methods: The polysaccharide nano-selenium complex sample solutions and ascorbic acid (Vc) standard solutions obtained in Example 1 and Comparative Examples 1-3 were prepared using a gradient dilution method. Following the operating instructions of the hydroxyl radical scavenging ability assay kit (Beijing Solarbio Science & Technology Co., Ltd.), the appropriate reaction systems were added sequentially.

[0138] The experimental groups are as follows:

[0139] Test group: Sample + H2O2 + colorimetric reagent;

[0140] Background sample group: Sample + PBS (without H2O2 / chromogenic agent);

[0141] Blank control group: PBS + H2O2 + chromogenic agent;

[0142] Standard curve group: gradient concentrations of Vc + PBS.

[0143] After thoroughly shaking and mixing each reaction system, incubate them in a 37°C water bath for 15 min. Use a UV-Vis spectrophotometer to detect the absorbance of each reaction system at the characteristic wavelength of 510 nm. Set up 3 independent replicates for each sample. Calculate the clearance rate of the sample according to the standard curve and formula (2).

[0144]

[0145] In the formula: A1 is the absorbance value of the test group; A2 is the absorbance value of the background sample group; A0 is the absorbance value of the blank control group.

[0146] Experimental results: such as Figure 3 As shown, compared with the polysaccharide nano-selenium complex prepared by single polysaccharide, the composite polysaccharide nano-selenium complex exhibits stronger free radical scavenging ability and shows a significant dose-response relationship within a specific concentration range, proving that the synergistic effect between polysaccharide compositions can enhance the antioxidant activity of nano-selenium. In addition, the composite polysaccharide nano-selenium complex prepared by this invention shows higher bioavailability and more significant therapeutic effects in cell experiments and animal models.

[0147] Performance Test Example 4: Cytotoxicity Test

[0148] The CCK8 assay kit was used for detection. The cell density of RAW 264.7 cells was adjusted to 1×10⁻⁶. 4 Cells were seeded at a concentration of 100 μg / mL into 96-well plates and cultured for 24 h. Cells were then treated with different concentrations of polysaccharide-selenium nanocomplex (0, 12.5, 25, 50, 100, and 200 μg / mL) for 24 h. After treatment, the culture medium was changed, and 10% CCK8 solution prepared with RAW 264.7 medium was added to each well, and the cells were cultured for another 2 h. Finally, the OD value was measured at 450 nm. The experiment was repeated three times.

[0149] Experimental results are as follows Figure 4 As shown, compared with polysaccharide-selenium nanocomposites prepared from single polysaccharides, the composite polysaccharide-selenium nanocomposites exhibit higher cell survival rates, which is due to the following two reasons:

[0150] (1) Co-modification with a complex polysaccharide nano-selenium complex can reduce the surface energy of nano-selenium and reduce its toxicity to normal cells;

[0151] (2) The synergistic protective effect of polysaccharides can slow down the release rate of nano-selenium in vivo and avoid toxic reactions caused by excessive local concentration.

[0152] Therefore, in the safety evaluation, the damage to normal cells caused by the composite polysaccharide selenium nanocomposite was significantly lower than that caused by the single polysaccharide selenium nanocomposite.

[0153] In summary, the composite polysaccharide-selenium nanocomposite prepared by the present invention through synergistic modification of nano-selenium with multiple polysaccharides is significantly superior to single polysaccharide-selenium nanocomposite in terms of stability, bioactivity, safety, application adaptability and functional diversity. It is a novel functional material with greater development potential and has broader application potential in fields such as multifunctional drug delivery, functional food additives and high-end cosmetic raw materials.

[0154] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A composite polysaccharide nano-selenium complex, characterized in that: The complex includes polysaccharide-modified selenium nanoparticles, which are obtained by redox reaction of a polysaccharide composition with a selenium solution. The polysaccharide composition consists of Boletus edulis polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide. The mass ratio of Boletus edulis polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide is 0.5~3:0.5~3:0.5~3.

2. The composite polysaccharide nano-selenium complex according to claim 1, characterized in that: The mass ratio of Boletus edulis polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide is 0.8~1.2:0.8~1.2:0.8~1.

2.

3. A method for preparing a composite polysaccharide nano-selenium complex, characterized in that, The preparation method includes the following steps: S1. The polysaccharide composition is mixed with water to obtain a polysaccharide mixed solution. The polysaccharide composition is composed of Boletus edulis polysaccharide, Portulaca oleracea polysaccharide and Astragalus membranaceus polysaccharide, and the mass ratio of Boletus edulis polysaccharide, Portulaca oleracea polysaccharide and Astragalus membranaceus polysaccharide is 0.5~3:0.5~3:0.5~3. S2. Stir the selenium solution and the polysaccharide mixed solution evenly to obtain a polysaccharide nano-selenium suspension; S3. Mix the stabilizer with the polysaccharide nano-selenium suspension and stir until the reaction is complete to obtain the reaction solution; S4. Centrifuge, wash, and dry the reaction solution to obtain the composite polysaccharide nano-selenium complex.

4. The preparation method according to claim 3, characterized in that: The concentration of the polysaccharide composition in the polysaccharide mixed solution is 1-5 mg / mL.

5. The preparation method according to claim 3, characterized in that: The concentration of the selenium solution in step S2 is 30~80 mmol / L, and the volume ratio of the selenium solution to the polysaccharide mixed solution is 1:3~8.

6. The preparation method according to claim 3, characterized in that: The stabilizer is selected from sodium citrate and ascorbic acid.

7. The preparation method according to claim 3, characterized in that: The concentration of the stabilizer is 30-80 mmol / L.

8. The application of the composite polysaccharide nano-selenium complex as described in claim 1 or 2, or the composite polysaccharide nano-selenium complex prepared by any of the preparation methods described in claims 3-7, in the preparation of pharmaceuticals, food, and cosmetics.

9. The application of polysaccharide compositions in the preparation of composite polysaccharide nano-selenium complexes, characterized in that: The polysaccharide composition comprises Boletus polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide, wherein the mass ratio of Boletus polysaccharide, Portulaca oleracea polysaccharide, and Astragalus membranaceus polysaccharide is 0.5~3:0.5~3:0.5~3. The application includes at least one of the following: (1) Application in improving the stability of nano-selenium; (2) Application in improving the antioxidant capacity of composite polysaccharide nano-selenium complex; (3) Application in reducing the cytotoxicity of complex polysaccharide nano-selenium complexes; (4) Application in improving the bioavailability of complex polysaccharide nano-selenium complex.

10. The application according to claim 9, characterized in that: The antioxidant capacity includes the ability to scavenge DPPH free radicals, the ability to scavenge ABTS free radicals, or the ability to scavenge hydroxyl free radicals.

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